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Gravitational and Space Biology

Volume 19, Number 2

August 2006

Publication of the American Society for Gravitational and Space Biology ISSN 1089-988X

ASGSB EDITORIAL BOARD

Augusto Cogoli Zero-G LifeTec GmbH

Zürich, Switzerland

Luis Cubano Univ. Central del Caribe

Camuy, Puerto Rico

Emily Holton

NASA Ames Research Center

Moffett Field, CA

John Kiss Miami University

Oxford, OH

Patrick Masson University of Wisconsin

Madison, WI

Gloria Muday Wake Forest University

Winston Salem, CT

Anna-Lisa Paul University of Florida

Gainesville, FL

April Ronca Wake Forest University

Winston Salem, CT

Gerry Sonnenfeld SUNY Binghamton

Binghamton, NY

Paul Todd SHOT, Inc.

Greenville, IN

Sarah Wyatt Ohio University

Athens, OH

William Landis NE Ohio Univ College of Medicine

Rootstown, OH

PUBLISHING STAFF

Stan Roux Editor-in Chief

University of Texas

Austin, TX

Mary E. Musgrave Publishing Editor

University of Connecticut

Storrs, CT

Robert Blasiak Assistant Editor

Albert-Ludwigs-Universität Freiburg

Freiburg, Germany

John Kiss Symposium I Editor Miami University

Oxford, OH

Charles Wade Symposium II Editor US Army Inst. of Surgical Res.

Fort Sam Houston, TX

Paul Todd Symposium III Editor SHOT, Inc.

Greenville, IN

ii Gravitational and Space Biology 19(2) August 2006

GENERAL INFORMATION

Gravitational and Space Biology (ISSN 1089-988X) is a journal devoted to research in gravitational and space biology. It

is published by the American Society for Gravitational and Space Biology, a non-profit organization whose members share a

common goal of furthering the understanding of the biological effects of gravity and the use of the unique environment of

spaceflight for biological research. Gravitational and Space Biology is overseen by a steering committee consisting of the

Publications Committee, the Editor, the President, and the Secretary-Treasurer of the ASGSB.

The American Society for Gravitational and Space Biology was created in 1984 to provide an avenue for scientists

interested in gravitational and space biology to share information and join together to speak with a united voice in support of

this field of science. The biological effects of gravity have been acknowledged since Galileo’s time, but only since the 1970s

has gravitational biology begun to attract attention. With the birth of the space age, the opportunity for experimentation over

the full spectrum of gravity finally became a reality, and a new environment and research tool became available to probe

biological phenomena and expand scientific knowledge. Space and spaceflight introduced new questions about space

radiation and the physiological and psychological effects of the artificial environment of spacecraft.

The objectives of ASGSB are:

• To promote research, education, training, and development in the areas of gravitational and space biology and

to apply the knowledge gained to a better understanding of the effect of gravity and space environmental

factors on the flora and fauna of Earth.

• To disseminate information on gravitational and space biology research and the application of this research to

the solution of terrestrial and space biological problems.

• To provide a forum for communication among professionals in academia, government, business, and other

segments of society involved in gravitational and space biological research and application.

• To promote the study of concepts and the implementation of programs that can achieve these ends and further

the advancement and welfare of humankind.

A Collaborative Production: This issue of the Gravitational and Space Biology was produced through collaboration with

the Professional Writing and Technical Communication Program at the University of Massachusetts Amherst. Under the

direction of Dr. John Nelson, the PWTC Program has trained students for a variety of professions that demand excellent

technical writing and editing skills. Since its inception in 1990, the program has placed nearly 100% of its graduates. The

American Society for Gravitational and Space Biology is pleased to sponsor an editorial fellowship for students in the PWTC

program at the University of Massachusetts, and gratefully acknowledges the contributions of its students and directors to the

production of our journal.

MEMBERSHIP: The American Society for Gravitational and Space Biology welcomes individual, organizational, and

corporate members in all of the basic and applied fields of the space and gravitational life sciences. Members are active in the

fields of space medicine, plant and animal gravitational physiology, cell and developmental biology, biophysics, and space

hardware and life support system development. Membership is open to nationals of all countries. Members must have

education or research or applied experience in areas related to the Society’s purposes: i.e., Doctorate, Masters with 2 years

experience, Bachelors with 4 years experience (student members must be actively enrolled in an academic curriculum leading

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Hill, NC 27515, or at the society website (http://www.asgsb.org).

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Copyright © 2006 by the American Society for Gravitational and Space Biology

Gravitational and Space Biology 19(2) August 2006 iii

American Society for

Gravitational and Space Biology

Proceedings of the 21st Annual Meeting of the

American Society for Gravitational and Space

Biology (Reno, NV November 1-4, 2005)

Featuring:

Symposium I: Biological Advanced Life Support Systems

Symposium II: Astronaut Health: From the Bench to Flight Across the Gravity

Continuum

Symposium III: Planetary Biology and Terraforming

Additional Short Papers

iv Gravitational and Space Biology 19(2) August 2006

Table of Contents

Symposium I: Biological Advanced Life Support Systems ………………………..……...…………..1

THE BIOLOGY OF LOW ATMOSPHERIC PRESSURE – IMPLICATIONS FOR EXPLORATION MISSION

DESIGN AND ADVANCED LIFE SUPPORT - A-L. Paul and R. J. Ferl ……………...………………………….…….3

PLANT-GROWTH LIGHTING FOR SPACE LIFE SUPPORT: A REVIEW – G.D. Massa, J.C. Emmerich, R.C.

Morrow, C.M. Bourget and C.A. Mitchell…………………………………………………………………….…………….…19

INCREASED BACTERIAL RESISTANCE AND VIRULENCE IN SIMULATED MICROGRAVITY AND ITS

MOLECULAR BASIS - A. Matin, S.V. Lynch and M.R. Benoit………………………………………….………..…31

Symposium II: Astronaut Health: From the Bench to Flight Across the Gravity Continuum…. 43

EXPLORATION CLASS MISSIONS AND RETURN: EFFECTS ON THE IMMUNE SYSTEM -

G. Sonnenfeld ………………………………………………………………………………………………………………….…..45

NUTRITION, METABOLISM AND THE CRITICAL PATHS: A CRITICAL REVIEW - T.P. Stein ………..….49

TESTING EXERCISE COUNTERMEASURES DURING 30 DAYS OF SIMULATED MICROGRAVITY:

LESSONS LEARNED FROM STUDIES OF IDENTICAL TWINS – A.R. Hargens, B.R. Maclas, C.M. Echon, E.

Brzezinski, A. Hawkins, K. Hawkins, and R.S. Meyer……………………………………………….....................……...…53

TRANSLATIONAL MEDICINE: FROM GROUND-BASED STUDIES OF TRAUMATIC INJURIES TO

ASTRONAUT HEALTH AND EARTH BENEFITS. – C.E. Wade...................................................................….....65

Symposium III: Planetary Biology and Terraforming………………………………………...…….77

PLANETARY BIOLOGY AND TERRAFORMING – P. Todd.................................……................................….....79

LAST PLACE TO BOIL AWAY, FIRST PLACE TO LOOK: THE HUNT FOR WATER AND LIFE ON MARS –

L.H. Kuznetz ……………………………………………………………………………………………………………………….85

EXTREMOPHILES FOR ECOPOIESIS: DESIRABLE TRAITS FOR AND SURVIVABILITY OF PIONEER

MARTIAN ORGANISMS – D.J. Thomas, J. Boling, P.J. Boston, K.A. Campbell, T. McSpadden, L. McWilliams,

and P. Todd ……………………………………………………………………………………………………………….………91

PLANETARY ECOSYNTHESIS AS ECOLOGICAL SUCCESSION – J.M. Graham………………………...…….105

Short Papers…….…………………………………………………………………………………….121

Advanced Life Support and Biotechnology:

DEVELOPMENT OF A MICROFLUIDIC ION SENSOR ARRAY (MISA) TO MONITOR GRAVITY-

DEPENDENT CALCIUM FLUXES IN CERATOPTERIS SPORES - A.R. De Carlo, M. Rokkam, A. ul Haque, S.T.

Wereley, P.P. Irazoqui, H.W. Wells, W.T. McLamb, S.J. Roux, D.M. Porterfield…………………………..…………..123

USE OF AN INTEGRATED FLOW-CHAMBER ADHESION ASSAY FOR MEASURING LEUKOCYTE

ADHESION PROPERTIES IN SIMULATED AND ACTUAL MICROGRAVITY – D.F. Kucik, R.L. Rouleau,

L.W. Smith, X. Wu and K.B. Gupta……………………………………………………………………………………..……..125

DEVELOPMENT OF THE EMCS HARDWARE FOR MULTIGENERATIONAL GROWTH OF DROSOPHILA

MELANOGASTER IN SPACE - M. E. Sanchez, M. Shenasa, A. Maldonado, A. Kakavand, D. Leskovsky, E.

Houston, A. Howard, M. K. Steele, and S. Bhattacharya…………………………………………………….……..……...127

Gravitational and Space Biology 19(2) August 2006 v

INVESTIGATING LOCAL IMPACTS OF HEAT-PULSE SENSORS FOR MEDIA MOISTURE CONTENT -

M.A. Ask, J.J. Prenger, D. Rouzan-Wheeldon, V. Rygalov, J. Norikane and H.G. Levine………………..……………129

PERFORMANCE EVALUATION OF A LABORATORY TEST BED FOR PLANETARY BIOLOGY – N.A.

Thomas, P. Todd, G.W. Metz, M.A. Kurk, D.J. Thomas……………………………………………………….…….…….131

Animal Development, Physiology and Gravity Response:

A STUDY OF THE EFFECTS OF SPACE FLIGHT ON THE IMMUNE RESPONSE IN DROSOPHILA

MELANOGASTER – T.F. Fahlen, M. Sanchez, M. Lera, E. Blazevic, J. Chang, and S. Bhattacharya……..……....133

EFFECTS OF ALTERED GRAVITY ON IDENTIFIED PEPTIDERGIC NEURONS OF THE CRICKET ACHETA

DOMESTICUS – U. Kirschnick, H-J. Agricola and E.R. Horn ………………………………………………..…………135

COUNTERMEASURES TO THE EFFECTS OF GRAVITY ON THE SKULLS OF HUMAN INFANTS - R. Lee,

J. English, J. Duke , and J. Teichgraeber…………………………………………………………………………………….137

NUTRIENT DIFFUSION THROUGH ARTICULAR CARTILAGE: DEVELOPMENT AND USE OF A MODEL

SYSTEM – C. Marshall, R. Flowers, N. Goli, M. Vandromme, D. Paulsen and B. Klement ……………….………..139

HYPERGRAVITY INDUCES DAMAGE TO ROD PHOTORECEPTORS - A.J. Barnstable, A.R. Tink, S. Viviano,

L. Baer, C. Wade, C.J. Barnstable and J. Tombran-Tink……………………………………………………….………….141

Cell Biology:

A GLOBAL TRANSCRIPTIONAL ANALYSIS OF STREPTOCOCCUS PNEUMONIAE IN RESPONSE TO

LOW-SHEAR MODELED MICROGRAVITY – C. Allen, C. Galindo, N. Williams, U. Pandya, A. Chopra, and D.

Niesel ……………………………………………………………………………………………………………..……………….143

PROTEOMIC RETRIEVAL FROM NUCLEIC ACID DEPLETED SPACE-FLOWN HUMAN CELLS - D.K.

Hammond, T.F. Elliott, K. Holubec, T.L. Baker, P.L. Allen, T.G. Hammond and J.E. Love…………….……….……145

THE EFFECT OF CHAGES OF GRAVITY ON HUMAN MONOCYTE CELL (TUR) PHAGOCYTOSIS – C.B.

Johnson, L.S. Waldbeser ………………………………………………………………………………………………….....…147

MICROTUBULE DISRUPTIONS AND REPAIR PHENOMENA IN CULTURED GLIAL CELLS UNDER

MICROGRAVITY – M.A. Masini, F. Strollo, F. Ricci, M. Pastorino, B. M. Uva, ………………………..…………..149

MICROGRAVITY-INDUCED CHANGES IN GENE EXPRESSION IN ACTIVATED T-LYMPHOCYTES

INVOLVE MULTIPLE REGULATORY PATHWAYS – N.E. Ward, N.R. Pellis, S.A. Risin and D. Risin...……..151

POSSIBLE INVOLVEMENT OF FLOW DETECTION IN THE ACTIVATON OF OSTEOBLASTS – M. Takaoki,

H. Park, N. Murakami, D. Shiba, and J-I. Gyotoku ……………………………………………………………………...…153

Plant Development and Gravity Response:

TRANSCRIPTIONAL PROFILING OF THE gps1 MUTANT OF ARABIDOPSIS – V. Nadella, C.D. Hildenbrand

and S.E. Wyatt………………………………………………………………………………..…………………………………..155

NITRIC OXIDE AND CGMP DEPENDENT SIGNALING IN ARABIDOPSIS ROOT GROWTH – J. Jacobi, J.

Elmer, K. Russell, R. Soundur, and D.M. Porterfield.………………………………………………………………………157

REGULATION OF TRANSCRIPTION IN ROOTS OF ARABIDOPSIS GRAVITY MUTANTS – J.W. Yester,

J.M. Kimbrough, R. Salinas-Mondragón, P.H. Masson, C.S. Brown, and H. Winter Sederoff………………………..159

MODIFICATION OF RESERVE DEPOSITION IN WHEAT AND BRASSICA SEEDS BY SYNTHETIC

ATMOSPHERES AND MICROGRAVITY - A. Kuang, J. Blasiak, S. Chen, G. Bingham, M.E. Musgrave..……..161

Index ………………………..……...………………………………………………………………….A-1

vi Gravitational and Space Biology 19(2) August 2006

Gravitational and Space Biology 19(2) August 2006 1

Symposium I: Biological Advanced Life

Support Systems

John Kiss, Editor

2 Gravitational and Space Biology 19(2) August 2006


THE BIOLOGY OF LOW ATMOSPHERIC PRESSURE – IMPLICATIONS FOR EXPLORATION

MISSION DESIGN AND ADVANCED LIFE SUPPORT

Anna-Lisa Paul and Robert J. Ferl

University of Florida, Gainesville, FL

ABSTRACT

Atmospheric pressure is a variable that has been often

manipulated in the trade space surrounding the design and

engineering of space exploration vehicles and extraterrestrial

habitats. Low pressures were used to reduce structural

engineering and launch mass throughout the early human space

program; moreover, low pressures will certainly be considered

in future concepts for the same reasons. Fundamental

understanding of the biological impact of low pressure

environments is therefore critical for the successful

consideration of this variable, being particularly important when

considering future, potentially complex bioregenerative life

support systems. However, low pressure biological effects are

also critical considerations that should be incorporated into near

term vehicle designs, designs that may set hardware and

operations criteria that would carry over into far-term future

designs.

In order to begin to define the fundamental biological responses

to low atmospheric pressure, we have identified the molecular

genetic responses central to the initial exposure of the model

plant Arabidopsis to hypobaric stress. Less than half of the

genes induced by hypobaria are induced by hypoxia,

establishing that response to hypobaria is a unique biological

response and is more complex than just an adaptation to low

partial pressures of oxygen. In addition, the suites of genes

induced by hypobaria confirm that water movement is a

paramount issue in plants. Current experiments examine gene

expression profiles in response to a wide variety of pressures,

ranging from slight to extreme hypobaria. Results indicate that

even small changes in atmospheric pressure have attendant

biological consequences deserving consideration during the

concept and design of vehicles and habitats. Moreover, the range

of pressures to which plants can adapt suggests that very low

pressures can be considered for plant-specific habitats.

The choices of atmospheric pressure within spaceflight and

extraterrestrial habitats are not merely engineering

considerations but are biological considerations of the highest

order, and modern molecular tools can be employed to increase

understanding of the biological consequences of pressure

engineering decisions.

INTRODUCTION

An almost bewildering myriad of environmental

parameters have been presented to Terran life forms

during the process of evolutionary change. Life has

therefore adapted and colonized a vast variety of

environments and many of those environments contain

extremes of one parameter or another; however, extreme

terrestrial altitude has escaped colonization by higher life

forms because of a suite of parameters that together

prevent habitability. Atmospheric pressure is one of the

major parameters that limit life to lower earth altitudes.

Figure 1. The relationship between altitude and atmospheric

pressure. As the elevation increases from sea level the

atmospheric pressure decreases. The range of the earth’s

atmosphere is 101 kPa at sea level to near 0 kPa at 30,000 m.

The current atmospheric attributes of the Earth’s surface

are the culmination of the physics of our planet and the

impact of over a billion years of biology and geology.

Biology has evolved and expanded into a wide range of

environments, including those that press the limits of

terrestrial altitudes, where physiology is limited by the

extremes of temperature, moisture and atmospheric

pressure that are the intrinsic components of terrestrial

high altitudes (Figure 1). Indeed, it is only where all three

of these extremes converge that we see an absence of life

on our planet. On tropical mountains mammals and large

plants (e.g. hyraxes and giant lobelias of Kilimanjaro) are

found no higher than altitudes of 5000 m (Njiro, 2005)

and herbaceous plants no higher than 5600 m (Körner,

2003). Yet even these altitude limits are dependent upon

other environmental variables. For example, the upper

limit of forests in tropical mountains may be 4000 m, yet

that same limit can be less than 2000 m in more temperate

latitudes (Körner and Paulsen, 2004). In addition, for

mammals and birds, the limits of typical habitation appear

to be delimitated as much by easy access to food and

available oxygen as by temperature, moisture and

atmospheric pressure. For humans, this habitation limit is

around 4200 m in the latitude of the Himalayas, where the

village of Kibber, India, is located (Table 1). In general,

human excursions to higher altitudes and lower pressures

____________________

* Correspondence to: Robert J. Ferl Horticultural Sciences Department

University of Florida

Gainesville, FL 32611-0690

Email: [email protected]

Phone: 352-192-1928x301; Fax: 352-392-4072

A-L. Paul and R. J. Ferl — Biology in Low Atmospheric Pressure


requires supplemental oxygen – though oxygen alone

cannot alleviate all difficulties for humans at low

pressures and high altitudes (Maggiorini et al., 2001;

Bartsch et al., 2005).

In natural environments, plant growth at high altitudes is

more limited by temperature than pressure; the 5600 m

limit on Kilimanjaro is not imposed by the atmospheric

pressure but rather by the fact the ground freezes every

night. Laboratory experiments indicate that if plants are

kept from freezing and are provided with adequate water,

they can be maintained at pressures far less than that

present at the summit of Kilimanjaro (e.g. Mansell et al.,

1968; Gale, 1973; Boston, 1981; Rule and Staby, 1981;

Andre and Richaux, 1986; Musgrave et al., 1988a; Andre

and Massimino, 1992; Daunicht and Brinkjans, 1992;

Ohta et al., 1993; Corey et al., 1996; Iwabuchi et al.,

1996; Corey et al., 2000; Ferl et al., 2002; Goto et al.,

2002; He et al., 2003; Paul et al., 2004). It is only in

artificial environments, such as those of the human

spaceflight program, that atmospheric pressure becomes a

variable independent of the temperature, moisture and gas

composition concerns that accompany terrestrial altitudes.

In the contained, closed, and engineered volumes of

extraterrestrial habitats and vehicles, challenges are

created by the need to contain atmospheric pressure

against the vacuum of space. Hence, atmospheric pressure

has been independently manipulated to levels well beyond

the limits imposed by altitude conditions on Earth.

The idea that plants can be successfully cultured in very

low atmospheric pressures for the purposes of advanced

life support in non-terrestrial environments has serious

implications for attaining the goal of taking humans to

new planetary surfaces. A primary and long-term goal of

sustaining life in remote space locations is to minimize

the amount of mass, and therefore energy, required to

launch and maintain life support systems. Furthermore, if

one considers maximizing the use of local resources, then

it would be desirable to make use of ambient light, which

makes it necessary to have a structure with maximum

transparency. This then leads to the question of what

materials would be both sufficiently transparent and

sufficiently strong to contain a plant growth atmosphere

that would sustain a higher pressure than the near vacuum

present on the Moon or the low pressure atmosphere on

the Martian surface. At present there are no materials that

would be generally accepted as sufficiently transparent,

lightweight, and strong enough to meet all of these criteria

at a full earth normal pressure. However, reducing the

pressure within a plant habitat would consequently reduce

the intrinsic strength required for such structures and

materials, reducing the mass of material that must be

lifted from the Earth’s surface, and potentially allowing

the capture of ambient light as a resource.

In this review we present a brief history of the various

atmospheric pressures and gas compositions that have

been used within the human-habitable vehicles of the

space programs, with an eye toward the possible

atmospheric configurations that might be used in future

vehicles and habitats. This narration is followed by a

general discussion of the uses of low pressure

atmospheres in plant biology applications. These two

threads will be integrated with a discussion of

experiments focused specifically on low pressure

atmospheres in plant space biology applications. We will

then develop an argument that low atmospheric pressures

present a serious environmental challenge to plants, a

challenge that requires an adaptive response and

redirection of metabolic resources. Understanding of this

response is enhanced by analysis of the gene expression

Table 1. Atmospheric pressure relative to altitude – a biological perspective. These data provide a perspective of the habitation of humans,

flora and fauna at increasing altitude and reduced atmospheric pressure.



changes that take place as plants respond and adapt. Much

like response and adaptation to other environmental

stresses, such understanding can lead to both a definition

of the current limits of terrestrial plants as well as a path

for producing plants with enhanced capacity for growth

and production at low pressures. Such information is

critical for the space program, as atmospheric pressure

directly impacts the designs and operational procedures

that are being considered for future space vehicles and

extraterrestrial habitats, including greenhouses.

ATMOSPHERIC PRESSURES IN SPACE

EXPLORATION

It is a generally accepted fact that reduction in the

pressure differential between an internal and external

environment correlates with reduction in engineering

costs, especially in the form of structural mass. Given that

mass is such a crucial cost variable in launch

considerations, low atmospheric pressure environments

have been utilized throughout the human space programs.

Lower atmospheric pressures simply reduce the masses of

structural components of space vehicles and have the

associated effect of reducing the amount of atmospheric

consumables required for the mission. Historically such

reductions in mass allowed for increased mission lengths

and increased payload masses.

The Mercury, Gemini, and Apollo environments were

operated at 34 kPa. In order to compensate for the

hypoxia attendant with such a low pressure, the gas

composition of the atmosphere was maintained at 100%

oxygen (Baker, 1981; Martin and McCormick, 1992)

(Table 2). While the pure oxygen environment provided a

suitable mitigation of hypoxia for the astronauts, that

atmosphere carried with it the risk of fire. It is worth

noting that recent studies have also begun to elucidate the

negative effects of prolonged exposure to pure oxygen

environments; however, hyperoxia appears to be toxic

only at partial pressures above 30 kPa. It should also be

noted that all Extravehicular Vehicle Activity (EVA)

space suit environments, beginning in the Gemini

program, have been maintained at 26 kPa in order to

minimize the physical effort required for manipulations of

the suit components. This need to operate EVAs at

reduced atmospheric pressure imposes constraints on

atmospheric management that continue to the present day

(see Table 2). The Skylab environment was also operated

at 34 kPa; however, the composition of the atmosphere

was maintained at 70% O2 : 30% N2. This pressure and

composition was a compromise that reduced the

engineering costs as well as the fire risk, yet maintained

astronaut health with regard to hypoxia. EVAs during

Skylab were conducted with minimal transition to the 26

kPa, pure oxygen environment of the EVA suits, so that

no pressure changes occurred within Skylab during EVA

activities or EVA preparations. Anecdotal evidence

indicates that the Skylab astronauts noticed no obvious ill

effects of living for prolonged periods at hypobaric

pressures, save for the attendant cooling effects of rapid

evaporation of water and sweat and the difficulty of

hearing due to poor sound propagation.

Table 2. US Space vehicles and atmospheric pressure. These data provide descriptions of various NASA orbital and transit vehicles with

respect to their internal cabin atmospheres.



The Space Shuttle, Mir and the International Space

Station environments were and are operated at Earth-

normal pressures near 101 kPa with an Earth-normal gas

mixture of 21% oxygen. Operation at 101 kPa solves

many of the issues of atmospheric pressure and

composition effects on biology. However, there remain

two pressure issues in recent and current operations;

unintentional excursions to low pressures during leaks,

and intentional reductions in pressure to accommodate

EVA activities. Mir suffered several leak events that

seriously impacted operations (Holliman and Aaron,

1997; Zak, 2000), most notably after collision with a

Progress resupply vehicle. While the ISS maintains

pressure in preparation for and during EVAs (requiring

astronauts to do pressure / atmosphere accommodation in

the air lock), the pressure in the Shuttle is lowered to 70

kPa for 24 hrs or more (Figure 2) to accommodate the

acclimation-deacclimation activities that are necessary for

the human transition between 101 kPa / 21% oxygen and

26 kPa / 100% oxygen (Winkler, 1992; Wieland, 1998).

The reduced pressures are employed to facilitate both the

pre-breathing requirements as well as the actual EVA

events.

Figure 2. Pressure profile from the orbiter Endeavour during

the day of the spacewalk EVA on December 10, 2001. The

actual time of the EVA itself was 4 hr 12 min, yet the shuttle

pressure was altered for essentially a full day to accommodate

all of the acclimation for the astronauts to move to and from the

lower pressure, pure oxygen environment of the EVA suits. Data

courtesy of Joe Benjamin, Dynamac, KSC.

PLANTS IN SPACE VEHICLES

Plants have experienced virtually all of the spaceflight

pressure scenarios mentioned above. Seeds were carried

aboard Gemini and Apollo missions, most notably in the

Biostack series of experiments of Apollo and in the Moon

Trees, which were personal effects of astronaut Stu Russa

on Apollo 14. Apparently seeds were also carried by

Astronaut Ed White in his space suit during the first EVA

space walk on Gemini 4. However, in these instances the

exposure was of quiescent seeds, with no studies of those

seeds performed until their return to earth and subsequent

growth at earth normal pressures. Nonetheless, the corn

and Arabidopsis seeds of Biostack germinated and grew,

though with abnormalities generally correlated with hits

of heavy ion radiation. On Skylab, rice plants were

sprouted and grown in a study of tropisms, inadvertently

demonstrating plant germination and development at

reduced atmospheric pressure, within the enhanced

oxygen levels of Skylab (see Table 2 and see also

http://history.nasa.gov/SP-401/ch5.htm). On Mir, plants

were being grown during the Progress collision and

subsequent loss of atmospheric pressure. Perhaps most

importantly, plant experiments have been conducted on

the Space Shuttle on flights where EVA activities

occurred. While many shuttle environmental variables are

replicated in simulation chambers at KSC, atmospheric

pressure is simply not addressed in the simulator ground

controls. Therefore plants have experienced a number of

exposures to spaceflight relevant atmospheric pressures,

but not always in situations where the effects of that

atmospheric pressure might have been noted or

accounted. This concept of unrecognized consequences of

changes in atmospheric pressure extend to several

relevant but non-spaceflight environments, such as the

KC-135 parabolic flight aircraft, which experiences

pressure deltas during each parabola as the plane’s

pressurization system attempts to maintain cabin pressure

during the extreme changes in altitude associated with

parabolic flight (Figure 3).

Figure 3. Pressure profile of a typical KC-135 parabolic flight.

These data were collected by a HOBO datalogger during a

typical life sciences KC-135 flight in January of 2000. These

data are not calibrated and are presented for demonstration

only. Note the pressure drop as the plane takes off and the

pressure stabilizes at a lower pressure characteristic of airliners

at cruise altitude. Note also that each parabola is characterized

by a further transient drop in pressure as the aircraft cabin

pressure system struggles to maintain a constant pressure. The

parabolas occur usually in groups of ten, characterized by the

closely spaced deviations in cabin pressure. The stable areas of

the graph between the groups of parabola pressure deviations

denote the times of constant altitude turns during which the KC-

135 changes direction to orient for the next series of parabolas.



PLANT PHYSIOLOGY IN HYPOBARIC

ENVIRONMENTS

Although plants are able to survive and even thrive at

pressures well below the threshold of typical human

habitation, it is very likely that most plants would

perceive such hypobaria as a stressful environment

requiring response and adaptation. The likelihood of a

hypobaria stress response is derived from knowledge of

plant responses to other forms of environmental stress,

including hypoxia, cold and dehydration (Ferl et al.,

2002). Yet, since hypobaria, alone, is not a native

terrestrial environment, the nature of the hypobaria stress

response could be entirely unique. Thus, fundamental

understanding of plant responses and adaptability to low

atmospheric pressures is a requisite part of developing

insights into pressure effects on terrestrial life-forms in

general as well as maximizing plant growth under

hypobaric advanced life support conditions. There is a

rich history of low atmospheric pressure research within

the plant biology community, a history that explores the

responses of plants to hypobaria and the adaptations of

plants to related environmental stresses such as hypoxia.

Most early and many recent hypobaria observations were

focused on direct physiological changes and metabolic

impact of low atmospheric pressures on plants (reviewed:

Daunicht and Brinkjans, 1996; Salisbury, 1999; Wheeler

et al., 2001; Corey et al., 2002; Ferl et al., 2002). While

many of the early investigations had a connection to the

international Advanced Life Support (ALS) community,

others explored the effects of low pressure environments

on the post-harvest physiology of fruits, vegetables and

flowers in commercial applications (e.g. Burg, 2004 and

references therein).

One of the earliest hypobaria studies attempted to recreate

a Mars-like atmosphere in bell jars and observed the

effect of that environment on rye seed germination.

Although seeds would germinate in an approximation of

martian atmospheric composition (0.24% CO2, 0.09% O2,

1.39% argon, balance of N2) at normal and slightly

reduced atmospheric pressures (101 kPa and 50 kPa),

lower ranges of pressure (10 kPa and 3 kPa) would not

support germination (Siegel et al., 1962, 1963). Another

early study was conducted by the Air force in 1968 to

explore the utility of using higher plants for food and

atmospheric regeneration in extended human space

missions (Mansell et al., 1968). Since the atmospheres of

the Mercury, Gemini and Apollo spacecraft were kept at

lower atmospheric pressures with elevated partial

pressures of oxygen, it was important to determine

whether suitable plants could also thrive in similar and

related hypobaric conditions. The growth and

development of turnip plants (Brassica rapa) were

evaluated at 50 kPa compared against a control of 93 kPa.

In these experiments, the partial pressure of oxygen was

kept at normoxic conditions of 21 kPa. After 21 days of

growth, it was concluded that there were no adverse

effects seen in the 50 kPa plants, although it was noted

that transpiration rates were elevated compared to the 93

kPa control (Mansell et al., 1968). It is interesting to note

in a historical context how early the spaceflight

community was actively considering the utility of plants

as part of advanced life support systems, under space-

vehicle relevant pressures and gas compositions. These

plant experiments were performed shortly after a series of

animal experiments were conducted in 1965 to determine

the toxicology of the 34 kPa low pressure, pure oxygen

environment being employed for the space capsule

atmosphere (Thomas, 1965). A few years later plants

were also subjected to germination and growth

experiments in a simulated space capsule environment

(Lind, 1971). It can be concluded that these early studies

clearly established the viability of both plants and animals

in low atmospheric pressures and within gas compositions

relevant to space exploration.

In the 1960’s it was also discovered that hypobaric

pressures may have an impact on plant hormone related

physiology, and this realization expanded the interest in

hypobaric research to the commercial fruit industry.

Holding fruit at sub-atmospheric pressures delayed

ripening (Burg and Burg, 1965) and subsequent studies

related this phenomenon to the depletion of ethylene from

fruit tissues that was accelerated by the hypobaric

conditions. Tomatoes, bananas, mangos, cherries, limes

and guavas were incubated in atmospheric pressures

ranging from 48 kPa to 20 kPa and compared to similar

treatments in normal atmospheric pressure (Burg and

Burg, 1966a, 1966b). Later studies used hypobaric

conditions to enable the discrimination between the

effects of ethylene and abscissic acid in the formation of

abscission zones. Bell jars with atmospheres reduced to

20 kPa were used to determine that ethylene is the

primary effector of abscission in citrus fruits (Cooper and

Horanic, 1973). Since these early studies there has been a

wide application of these principles (e.g. Dilley et al.,

1975; Spalding and Reeder, 1976; Lougheed et al., 1978;

Nilsen and Hodges, 1983; Jardine et al., 1984; Kirk et al.,

1986) and this field has been reviewed recently (Burg,

2004).

Investigations into the underlying physiology of growing

plants in hypobaric environments continued to be

explored with both basic physiology and spaceflight

applications in mind, although early experiments

sometimes produced mixed results. For instance, in one

study tomato seedlings grown at 17 kPa were stunted in

growth, while plants grown at 33 kPa were more robust

when compared to the 100 kPa control (Rule and Staby,

1981). Yet in another, tomato plants were uniformly

stunted in reduced atmospheric environments of 40 and

70 kPa compared to 100 kPa control (Daunicht and

Brinkjans, 1992) while the negative effects of extreme

hypobaria (3 kPa) on rye seed germination could be

mitigated with added oxygen (Andre and Richaux, 1986).

Thus, as plant growth experiments explored low pressure

environments, it was found that the composition of the

air, especially with respect to O2 and CO2 can have a

profound effect on the physiological response of the

plants growing in a reduced overall pressure,

complicating the interpretation of the underlying effects



of low pressure that are independent of gas composition.

The two atmospheric components that contribute most to

complicating a purely hypobaric response are CO2 and O2.

For CO2 related processes, the increase in molecular

diffusion rates of CO2 at lower atmospheric pressures

enhances the ability of plants to take up the gas in

hypobaric environments (Gale, 1972). Thus the decrease

in absolute CO2 concentrations at lower pressures is

counterbalanced by the increase in CO2 diffusion rates. In

natural environments this positive effect could in turn be

counteracted by the decreases in temperature that

accompany reduced atmospheric pressures at high

altitudes (Gale, 1973), as well as by the concomitant

decreases in stomatal aperture, increases in transpiration

rates, and the increases in water exchange demands that

accompany higher elevations and lower pressures (Smith

and Geller, 1979; Kirk et al., 1986; Mott and Parkhurst,

1991; Gale, 2004). It has been suggested that such

demands might impact stomatal distribution and density

in plants adapted to high altitudes (Körner et al., 1986)

and this correlation has even been used to predict

probable attitudes of habitation for fossilized leaves

(McElwain, 2004). However, there is some dispute in this

latter application (Johnson et al., 2005) as stomatal

distribution can be associated with changes in CO2

concentrations independent of altitude (Lake et al., 2001).

In experimental scenarios where temperature and pressure

can be controlled, it appears that, at least for hypobaric

pressures of about 50 kPa and above, the increase in CO2

uptake facilitated by increased diffusion rates of CO2

counterbalances the relative scarcity of the gas (Smith and

Donahue, 1991).

As mentioned in earlier sections, the amendment of

oxygen to hypoxic atmospheres can have a profound

effect on the ability of animals to cope with hypoxic

environments. Oxygen also ameliorates some of the

effects of hypobaric stress in plants. One such study

demonstrated that wheat was capable of germinating and

growing at total atmospheric pressures of 10 kPa, and that

pressures of 20 kPa even enhanced growth when the

partial pressure of nitrogen was kept low by using oxygen

to displace much of the nitrogen in the maintenance of the

total atmospheric pressure. The effect of this replacement

created an atmosphere with a total pressure of 20 kPa, and

partial pressures of O2 at 14 kPa, N2 at 3.4 kPa and CO2 at

3.4 kPa. Interestingly, the positive effects of this

atmosphere on growth were couched in terms of a

reduction of nitrogen rather than an increase of oxygen

(Andre and Massimino, 1992). Indeed, if a pure oxygen

atmosphere is used, rye seeds are capable of germinating

at pressures of 3 kPa (Andre and Richaux, 1986).

However, oxygen does not ameliorate all issues of growth

and development at low pressures. In a study with mung

bean seedlings that compared mitochondrial respiration

rates with overall growth, it was found that while

respiration responded to oxygen concentration

independently of the overall atmospheric pressure down

to 21 kPa, quite the opposite was found for seedling

growth. In this case, growth (as assayed by mass

accumulation and length of seedlings) was negatively

correlated with a decrease in pressure and was

independent of the partial pressure of oxygen (Musgrave

et al., 1988a). Arabidopsis and rice also appear to be

impacted by oxygen concentrations in their ability to cope

with hypobaric conditions. Although both species could

grow at atmospheric conditions of 25 or 50 kPa of total

pressures, the addition of oxygen to a partial pressure of

at least 10 kPa appeared to compensate for any

disadvantageous of growing in a hypobaric environment

(Goto et al., 2002).

Independent of the gas composition in a hypobaric

environment is the more direct effect that low

atmospheric pressures have physically on gas relations in

plant physiology; all other things remaining constant, if

the atmospheric pressure is reduced, the rate of gas

exchange will increase. Evaporation, for example,

increases as pressure is reduced, an effect that can account

at least in part for increased plant transpiration at low

pressures (Rygalov et al., 2002). However, it is not clear

whether all things do remain constant in a plant adapting

to hypobaric conditions. In a short term experiment CO2

assimilation rates and transpiration rates were enhanced

for spinach in 25 kPa environments compared to 101 kPa

controls. However, in long term experiments it was

demonstrated that the gas exchange rates for plants in 25

kPa did not vary from those grown at normal pressures.

One apparent reason for this result was that over time, the

stomatal openings in these plants became correspondingly

smaller, thereby reducing the rate of gas exchange

through these pores (Iwabuchi and Kurata, 2003). Long

term experiments with lettuce at slightly reduced

atmospheric pressure (70 kPa) demonstrated that plants

could adapt with no adverse effects, and even tended to be

slightly more robust than their 101 kPa counterparts

(Spanarkel and Drew, 2002).

These findings suggest that plants appear to respond to

low atmospheric pressures through a fairly complex set of

adaptations, and further, there are situations where the

benefits of hypobaric environments may outweigh the

metabolic cost, in part through effects on the gaseous

plant hormone ethylene. Where plants are cultivated in

closed containers, hypobaric environments of 30 kPa total

pressure appear to offset the detrimental effects of

ethylene and other volatile biological compounds that

normally accumulate in closed systems (He et al., 2003).

Some of these effects appeared to be due to an inhibition

of ethylene production by the low partial pressure of

oxygen, as hypoxia alone was not as uniformly effective

in ethylene management. Although ethylene production

could be inhibited in lettuce grown at 101 kPa with a

partial pressure of oxygen equivalent to that found at 30

kPa of total pressure, the same was not true of wheat.

Ethylene production in wheat could only be controlled

when the overall atmospheric pressure was reduced to 30

kPa (He et al., 2003).



LOW ATMOSPHERIC PRESSURE HABITATS

From a physiological point of view, plants cope well with

hypobaric atmospheres, and conditions can be engineered

for closed system environments where plants may even

benefit from atmospheric pressures as low as 30 kPa. This

set of circumstances lends itself very well to the physical

engineering needs of vehicles and habitats that

exploration may take to other planetary surfaces (Wheeler

et al., 2001; Corey et al., 2002) and sets the stage for the

genetic engineering to further enhance the production of

plants that may populate such habitats (Ferl et al., 2002).

Low atmospheric environments were proposed for

Martian greenhouses in early concepts that examined

human exploration on Mars (Boston, 1981). It was

thought that low pressure greenhouses would be the most

effective means to grow plants in support of a mission on

a planet that had a vast differential between the external

atmospheres (ca. 0.7 kPa) and the internal environment.

Early experiments to support the concept employed plants

grown in largely CO2 atmospheres at a total pressure of 5

kPa. Radish, alfalfa and mung beans were germinated and

grown for 2 to 4 days with only minimal mortality (10%)

and much of the mortality was due to secondary effects,

such as fungal contamination (Boston, 1981). The idea of

an inflatable structure within a man-made or natural (e.g.

lava tube) rigid structure captured many imaginations and

has endured as a valid model for more than 20 years. The

inflatable greenhouse concept supports the notion that on

a long duration mission to Mars resupply is not an easy

operational option, and any decrease in initial supply

mass enhances launch capabilities (McKay and Toon,

1991; Schwartzkopf and Mancinelli, 1991; Mitchell,

1994; Schwartzkopf, 1997; Kennedy, 1999; Salisbury,

1999; Clawson, 2000; Fowler et al., 2000; Alling et al.,

2002; Corey et al., 2002; Sadler and Giacomelli, 2002;

Hublitz et al., 2004). Habitats have been envisioned that

range from significantly reduced atmospheric pressure

that would require pressure suits and robotic interventions

(Boston, 1981; Clawson, 2000; Corey et al., 2002) to

habitats that are on the edge of human comfort (50 kPa)

that would permit short excursions by crew with minimal

support (Hublitz et al., 2004). Many of these designs

employ transparent inflatable structures to make maximal

use of ambient light (Clawson et al., 2005).

PLANT GENE EXPRESSION IN HYPOBARIC

ENVIRONMENTS

Plants are able to cope with a vast variety of

environmental conditions that differ from a well hydrated

temperate meadow. Plants can engage metabolisms that

enable them to thrive in niches characterized by extremes

in temperature, humidity and oxygen availability. The

responses associated with these stress environments (cold,

desiccation, flooding, heat shock) have been well

characterized; yet until recently, it was not known

whether the response strategies elicited by hypobaria

engaged unique pathways or similar pathways as the

known environmental stresses (Ferl et al., 2002). To

address this question, experiments were conducted with

Arabidopsis to examine the patterns of gene expression on

a genome wide scale as plants were introduced to

hypobaric and comparable hypoxic environments (Paul et

al 2004). Affymetrix GeneChip

8K arrays were used to

characterize the effects of 24 hour exposure of

Arabidopsis seedlings to an atmospheric environment of

10 kPa. The gene expression patterns were then compared

to those of plants exposed to a normal pressure hypoxic

environment of 2% oxygen (the partial pressure of O2 at

10 kPa) and both patterns compared to 24 hour exposure

to Earth-normal air at 101 kPa.

The hypobaric and hypoxic environments were created

within a Low Pressure Growth Chamber (LPGC). The

LPGC controlled lighting, temperature, CO2 partial

pressure and humidity across all experiments such that the

only variables were either atmospheric pressure or oxygen

content. The experimental plants were nine day old

Arabidopsis seedlings grown on nutrient agar plates, a

configuration that ensured that both roots and shoot

received identical atmospheric conditions throughout the

treatment, and that the entire surface of the plant received

the full impact of the environment in which it was placed.

It is also important to note that the relative humidity of

the plant’s microenvironment inside the culture plate

remained at or above 95% in every treatment. Further, all

plants remained turgid and showed no signs of wilting or

desiccation at the end of the 24 hours of treatment.

Of the 8,000 genes represented on the array, more than

200 were differentially expressed in the shoots of plants

exposed to a hypobaric environment of 10 kPa for 24

hours. A comparable number of genes were similarly

differentially expressed in hypoxic treatment - yet only a

fraction of the two sets of differentially expressed genes

overlapped in patterns of expression (Figure 4). Many of

the hallmarks of hypoxic stress were found in both the 10

kPa and 2% O2 treatment, as oxygen is limiting in both

scenarios (groups 3 and 4, as well as members of groups 2

and 5, Figure 4). Genes required to support fermentative

pathways and those involving oxygen transport are

required under low oxygen conditions (e.g. Klok et al.,

2002) so it is not surprising to find representatives of

these metabolisms in both treatments. The genes

repressed by both hypoxia and hypobaria included

examples from a wide range of metabolic pathways that

are globally down-regulated in response to hypoxic stress

(Sachs et al., 1980; Sachs et al., 1996; Vartapetian and

Jackson, 1997; Klok et al., 2002). The hypobaria response

therefore included many of the gene activity changes

typical for a hypoxic response, but the hypobaric response

was not limited to genes involved in hypoxia.

The group of genes that was most highly induced by

hypobaria, yet unaffected by hypoxia, were ones involved

with desiccation-related metabolic pathways. Many of the

genes represented in this group displayed as much as a

30-fold difference in expression between 2% O2 and 10

kPa (see group 1, containing 30 genes, Figure 4). Among

the most abundant genes in this category were cold



induced genes (e.g. Cor28), dehydrins, which are

typically associated with ABA regulated seed storage

proteins (e.g. LEA) as well as many other examples of

desiccation and ABA regulated genes (Yamaguchi-

Shinozaki and Shinozaki, 1993a; Siddiqui et al., 1998;

Thomashow, 1999; Finkelstein et al., 2002; Soulages et

al., 2003). Also found in this category were genes that are

associated with the regulation and distribution of stomates

in Arabidopsis leaves (Berger and Altmann, 2000) and

many genes associated with the mediation of signal

transduction, especially those related to kinases (Guo et

al., 2002; Yoshida et al., 2002), calcium binding

(Takahashi et al., 2000; Sadiqov et al., 2002) and a

cytochrome p450 (Reddy et al., 2002). There were two

smaller groups of genes that exhibited interestingly

unique patterns of gene expression among the hypoxic

and hypobaric treatments. First, there were a few genes

that were repressed in hypoxia, but were unaffected by

hypobaria. Representatives from this group encoded

proteins similar to those involved in oxygen sensing

processes that are mediated through a heme- or iron-based

sensor (Aravind and Koonin, 2001; Zhu and Bunn, 2001;

Quinn et al., 2002). Second, there were those that were

induced in hypoxic environments, while remaining

unaffected by hypobaria (some of the genes in group 6 of

Figure 4). Genes encoding heme-related proteins were

also found in this group, as well as genes typical of a

hypoxic stress response. The dissimilarity in gene

expression patterns in what might be considered related

oxygen sensing systems, suggests that that hypobaria and

hypoxia may differentially activate and repress certain

alternative oxygen sensing and transport pathways.

Figure 4. Differential expression of Arabidopsis genes in response to hypobaria (10 kPa) and hypoxia (2% O2). Over 200 genes of the

8,000 of the Affymetrix Arabidopsis Gene Chip© were differentially expressed in the shoots of plants exposed to a hypobaric environment of

10 kPa or a hypoxic treatment of 2% O2 for 24 hours. The panel on the left contains three columns of stacked, colored lines. Each line

represents one gene that exhibits differential expression with regard to the Earth Normal (101 kPa / 21% O2) control. When a gene is

induced relative to the control it is indicated in red, when it is repressed it is indicated in green. The reference column of normal sea level

pressure looks entirely black as all expression is calculated relative to the values therein. The two columns to the right (hypoxia and

hypobaria) display patterns of differential gene expression and are clustered into 7 groups of gene exhibiting similar response. The cluster

number and the number of genes represented in that cluster are indicated between the two panels. The graph on the right displays the

relative normalized gene expression averages (Paul et al., 2004) for the groups of genes within the group for hypoxic (open bars) and

hypobaric (closed bars) treatments. The color version of this figure can be found on line: http://asgsb.org/publications.html ). In the

grayscale print version, induced appears as a darker gray and repressed as a lighter gray.



The specificity of the hypobaric response was also

evaluated in transgenic Arabidopsis plants transformed

with Adh/GFP or Cor78/GFP transcriptional promoter

fusions. Induction of the alcohol dehydrogenase gene

(Adh) is a well characterized component of a hypoxic

stress response while the cold response 78 gene (Cor78)

is typically associated with cold and drought stress, and in

these experiments, hypobaria. The sensing portions of the

transgenes were composed of the promoter regions of

these genes. The structural portion of each transgene

encodes Green Fluorescent Protein (GFP), which can be

visualized non-destructively in vivo when illuminated

with short wave blue light (488 nm). The plants were

exposed to hypobaric and hypoxic environments for 24

hours and then illuminated with 488 nm light to determine

the extent of tissue specific reporter gene expression. The

Adh/GFP plants showed abundant reporter gene

expression in the meristematic regions of the shoots and

roots with both hypoxic and hypobaric treatment,

however, the Cor78/GFP plants only expressed the

reporter gene in response to hypobaric treatment

(Figure 5). In addition, although Adh/GFP and

Cor78/GFP both showed reporter gene expression in

hypobaria, the tissue-specific patterns of expression were

different for each plant. Adh/GFP expression was

concentrated in the apical meristem and newly emerged

leaves, particularly in trichome cells. Very little

expression of the reporter was seen in more mature tissues

in Adh/GFP plants. Cor78/GFP on the other hand, was

expressed throughout the leaves, although again

predominated in the newly emerged leaves and trichome

cells (the latter partly owing to the large, clear nature of

these cells). In addition, GFP expression extended from

the aerial portion of Cor78/GFP plants along the stem to

the root/shoot junction (Paul et al., 2004). Thus, the

response to hypobaria is not limited to those tissues

responding to hypoxia, and that the signaling mechanisms

for the hypobaria-specific response are fundamentally

different from those of the hypoxia response with regard

to the distribution of those cells undergoing the

response.The extensive changes in gene expression

patterns clearly indicate that adaptation to low pressure

environments requires a robust and dynamic adaptive

response. Some of the altered gene expression patterns are

similar to those involved in the hypoxic stress response,

as would be expected from the low oxygen partial

pressures present in hypobaria; however, many of the

changes in gene expression patterns are unique to

hypobaria. These unique differential gene expression

patterns demonstrate that hypobaria is not at all equivalent

to hypoxia as an abiotic stress and clearly suggest that, for

plants, the response to hypobaria is much more complex

than the acclimation to the reduced partial pressures of

oxygen inherent to low atmospheric pressures. These

conclusions regarding complexity based on molecular

data begin to explain why the physiological data in

previous experiments do not necessarily yield easy

explanations.

So, if hypobaria is a more complex stress than hypoxia,

what other metabolic processes does it impact? As

indicated by the induction of the Cor78 gene discussed

above, one of the largest group of genes that showed over

expression in hypobaria while being unaffected by

hypoxia is characterized by genes associated with

desiccation and ABA signaling related processes (e.g.

Ozturk et al., 2002). Examples include ABI2 (Chak et al.,

2000), the APL3 subunit of ADP-glucose

pyrophosphorylase (Weber et al., 1995; Rook et al.,

2001), LEAs, (e.g. Siddiqui et al., 1998; Kim et al., 2002)

Figure 5. Tissue specific transgene expression in response to hypobaria (10 kPa) and hypoxia (2% O2). Transgenic plants containing

either the Adh/GFP or Cor78/GFP transgene were photographed in white and 488nm fluorescent light. The top row shows Adh/GFP

plants after exposure to 24 hours of 10 kPa hypobaria (left) and after 24 hours of 2% O2 hypoxia (right). The apical meristems regions and

the roots (where visible) are expressing the GFP reporter, and these regions glow green in the right hand picture of each pair. The bottom

row shows Cor78/GFP plants after exposure to 24 hours of hypobaria or hypoxia. In the case of the Cor78/GFP plants, transgene

expression was induced solely by the hypobaric treatment (left hand panels), plants exposed to hypoxic environment did not express the

transgene (right hand panels). The color version of this figure can be found on line: http://asgsb.org/publications.html ). In the grayscale

print version, GFP expression appears as white or light gray on the black background of the photographs.



dehydrins and rab-like proteins, (e.g. Mantyla et al., 1995;

Nylander et al., 2001; Kizis and Pages, 2002) rd29B and

cor15B (Wilhelm and Thomashow, 1993; Yamaguchi-

Shinozaki and Shinozaki, 1993b) and aldehyde

dehydrogenase (Kirch et al., 2001). Thus, it appears that a

primary impact of a hypobaric environment, independent

of oxygen stress, is on the mechanisms by which a plant

perceives and responds to water movement or desiccation.

It is important to emphasize that it is the perception

mechanism that is being impacted, not the actual

desiccation process. The plants in these experiments were

grown in a humid environment (>95% rh within the

plates), showed no loss of fresh weight or turgor, and yet

still responded to 10 kPa as if they were dehydrated or in

the process of dehydration. This pivotal result indicates

that the desiccation response is likely due to the

perception of increased water flux caused by the low

pressure environment rather than actual loss of water

content within the plants.

Thus the analysis of the differential gene expression

patterns from these experiments lead to two fundamental

conclusions: first, that hypobaria does not equal hypoxia,

and second, that the primary metabolic pathways that are

engaged by hypobaric stress include those that encompass

the desiccation response or otherwise touch ABA

mediated metabolisms. The response to hypobaria is

much more complex than the acclimation to low partial

pressures of oxygen. Therefore, it would be predicted that

complete compensation for hypobaria in plants would not

be accomplished by increasing the oxygen content, as was

done for low pressure environments of the Mercury,

Gemini and Apollo vehicles. The desiccation-related

response of Arabidopsis plants at 10 kPa in the absence of

real desiccation or wilting also raises the question as to

whether adaptation to a low atmospheric pressure actually

requires the activation of desiccation related pathways, or

if the induction of desiccation-related metabolism may

not be necessary for survival. If the desiccation response

is not necessary for adaptation to low pressure, then the

inappropriate induction of these pathways could represent

costs to production and genetic engineering to remove the

response may be beneficial. If the desiccation response is

indeed required, then enhancing the desiccation response

may increase production at low atmospheric pressure.

CONCLUSION

The experiments and conclusions discussed here expose

the need for continued evaluation of the effects of low

pressure on biological systems. In some ways, the gene

expression data suggest that we actually have only a small

fraction of the data necessary to make informed choices

on the gas composition and pressures within vessels that

support plant growth. By extension, these data also beg

the questions of what sort of undetected responses and

adaptations to low pressures occur in other organisms,

including humans, and whether low pressure

environments have contributed to any of the existing

spaceflight data on biology and biological responses in

spaceflight environments. There is little data available on

the response of any animal system to hypobaric

environments at the level of gene expression, but there are

studies that explore the physiological responses of

animals to hypobaric environments. Of particular interest

are those that indicate hypobaric environments could

contribute to compromised health and resistance to

disease. Early ground-based studies in mice demonstrated

that hypobaric environments, even when supplemented

with oxygen, can lead to stimulated growth of

gastrointestinal bacteria (Gillmore and Gordon, 1975),

retard healing of staphylococcus skin infections (Schmidt

et al., 1967), increase mortality in animals with

intraperitoneal infections (Ball and Schmidt, 1968) and

depress interferon levels (Huang and Gordon, 1968).

More recently, studies have linked hypobaric

environments with an impaired immune response in rats

(SaiRam et al., 1998) mice (Biselli et al., 1991) and

humans (Meehan et al., 1988; Facco et al., 2005). Rarely

has hypobaria been studied independently from hypoxia,

but a recent study examining the effects of mild hypobaria

on fluid balance issues conducted hypobaric treatments in

normoxic atmospheres to control for the effects of

hypoxia. They concluded that although there is a

synergistic effect of hypoxic and hypobaric stress at

altitude, hypobaria alone can adversely impact the fluid

and ionic balance of humans (Loeppky et al., 2005).

So the short term questions surrounding low pressure

spaceflight and extraterrestrial environments involve

deepening our understanding of responses and adaptation

to hypobaria for all organisms and biological systems that

may be a part of spaceflight and extraterrestrial habitats.

Indeed, unpublished molecular data indicate that even the

relatively small pressure drops to 75 kPa that occur in the

space shuttle effect the expression of hundreds of genes in

Arabidopsis. That being the case, even seemingly small

choices about operations and procedures conducted within

habitat environments can have measurable and dramatic

impacts on the biology of those habitats. Therefore very

near term decisions involving, for example, the choices of

atmospheric composition and pressure in the Crew

Exploration Vehicle will have long term implications for

biology. In the longer term, the use of low pressure

environments may enable life support systems that

otherwise would be impossible to construct or maintain

under higher atmospheric pressures, keeping viable the

concepts of inflatable greenhouses. For example,

transparent greenhouses could be erected on Mars using

currently available materials, but only if the internal

pressure of the greenhouse could be maintained below 7.5

kPa (Boston, 1981). The rather dramatic changes in gene

expression that occur at 10 kPa suggest that response and

adaptation to lower pressures will be even more

extensive, but the study of such pressures are well within

the capacity of current experiment chamber design and

molecular expression technology.



ACKNOWLEDGEMENTS

The spaceflight and low pressure experiments of the Ferl

laboratory were supported by NASA grants NAG 10-316,

NAG 10-291 and AO-99-HEDS-01-032. The authors

wish to recognize the tremendous contributions of the

many scientists whose work is summarized in this review.

In particular, we thank Andrew Schuerger, Jeff Richards,

Ken Corey, Ray Bucklin, Ray Wheeler, Vadim Rygolov,

Phil Fowler and other folks involved in developing the

low pressure facilities at KSC. We thank Mike Dixon and

his associates at the University of Guelph and the

Controlled Environment Systems Research Facility

(CESRF). We thank Bill Wells, Joe Benjamin and Kelly

Norwood from KSC for providing source material

regarding shuttle and KC-135 atmospheric pressures. We

also thank our colleagues at UF who participated in low

pressure and recent KC-135 experiments, including

Jordan Barney, Matt Reyes, Bill Gurley, Mike Manak and

John Mayfield.

The authors also wish to acknowledge the difficulty of

deriving original references for some of the discussions

presented in this paper. Any mistakes or

misrepresentations or misattributions are regretted and we

would welcome any comments, corrections or any

additional references at [email protected] or [email protected].

This paper is dedicated to the memory of Guy Etheridge.

His appreciation of science and love of space research had

such a wonderful influence on all.

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PLANT-GROWTH LIGHTING FOR SPACE LIFE SUPPORT: A REVIEW Gioia D. Massa

1, Jeffrey C. Emmerich

2, Robert C. Morrow

2, C. Mike Bourget

2, and Cary A. Mitchell

1

1 Department of Horticulture and Landscape Architecture and NASA Specialized Center of Research and Training

in Advanced Life Support, Purdue University, West Lafayette, IN 47907 2 Orbital Technologies Corporation, Madison, WI 53717

ABSTRACT

The lighting system is one of the most important components of

a greenhouse or chamber that will be used for plant growth in an

Advanced Life-support System (ALSS). Several designs of

such lighting systems have been proposed including the use of

natural sunlight with supplemental electric lighting. Although

electric lighting is energy intensive, it is necessary when

balanced against the hazards and limitations of plant growth

under natural sunlight on the surface of the moon or Mars. The

characteristics of different electric light types are reviewed, and

functionality for space application is compared. Different

lighting systems used in Earth-based advanced life-support

(ALS) simulations are highlighted. A new design using light-

emitting diodes (LEDs) for intracanopy lighting, i.e. lighting

entire crop canopies for energy savings and crop enhancement,

is discussed. When the benefits of LEDs are combined with the

advantages of intracanopy lighting, electric lighting for ALS

becomes increasingly feasible.

INTRODUCTION

Food crops will have to be grown when it is necessary to

support human life off Earth for an extended time period.

NASA’s (former) Advanced Life Support (ALS) program

included research associated with water purification, air

revitalization, waste processing, food production and

preparation, and system integration using a combination

of bioregenerative and physico-chemical technologies.

The ultimate goal of life-support research is to support

human habitation off Earth for an indefinite period by

creating a sustainable life-support system that is open

with respect to energy but closed with respect to mass.

NASA’s ALS program evolved from an earlier Closed

Ecological Life-Support System, or CELSS, program.

Several other countries or groups have worked on ALS-

related topics, including Russia, Japan, Canada, the

European Space Agency, and most recently, China. Early

work on human life support emphasized synergisms

between plants and humans in closed systems, with

particular focus on future crop-production systems in

space.

The early ALS work in the US, the USSR, and elsewhere

focused on use of microalgae such as Chlorella sp. for

food production as well as for oxygen and water

revitalization in an ALSS. The lighting systems used for

algae will not be discussed here, as most research moved

early on to higher plants, but for an excellent review of

those experiments, refer to Gitelson, Lisovsky, and

MacElroy (2003).

More recent ALS research includes the design and testing

of habitats for plant growth that will be used on the

surface or subsurface of the moon or Mars. One such

design proposes the use of an inflatable greenhouse that

relies on solar photosynthetically active radiation (PAR)

to directly irradiate crops. The design of such

greenhouses requires transparent films that would have to

withstand low external atmospheric pressure, large

external temperature gradients and swings, strong UV-C

fluxes, micrometeorite impacts, solar particle events,

cosmic-galactic radiation, and must address issues

associated with the widely varying intensity of solar PAR

available to support plant growth (Cockell and Andrady,

1999; Cockell, 2001; Rontó et al., 2003; Horneck et al.,

2003). On the moon, the latter relates to extended periods

of darkness; on Mars, to global dust storms of

unpredictable duration, to diurnal and seasonal light

cycles, and to the planet’s variable distance from the sun.

To deliver solar PAR to plants growing underground or in

containment on the moon or Mars, there not only will be

the same solar-availability constraints, but additional

losses of light as it travels through fiber optics or light

pipes (Cuello et al., 1998; Jack et al., 2002). Jack et al.

(2002) recently evaluated the efficiency of Fresnel-lens

Himawari and parabolic-mirror optical waveguide solar

collection and distribution systems for plant growth. The

first of these devices consists of a collection of Fresnel

lenses that track the sun and focus collected irradiance

onto a cable of fiber optics that can be directed into the

plant-growth area (Jack et al., 2002). The second device

has primary concentrators in the form of rotating

parabolic mirrors that focus the light onto a solid quartz

secondary concentrator that is then linked to a fiber-optic

cable (Jack et al., 2002). Losses of 2.5% - 6.7% per meter

were seen as the light was transmitted through the optical

fibers (Jack et al., 2002; Nakamura et al., 1998).

Although intense light levels over 800 µmol m-2

s-1

could

be obtained in areas of the plant-growth chamber with the

optical waveguide concentrator, lack of uniformity and

variability of light levels was extremely high (Jack et al.,

2002). Cloudy or rainy days reduce the available light

and can cause crop loss (Nakamura et al., 1998). On

Mars, dust storms of great intensity and long duration

occur and limit the already reduced solar radiation that is

available. On the moon, night length is about 2 weeks in

length at most locations except for the south polar region.

Direct or indirect, solar radiation is not reliable enough to

____________________

* Correspondence to: Gioia D. Massa

Department of Horticulture and Landscape Architecture

Purdue University

625 Agriculture Mall Drive

West Lafayette, IN 47907-2010


Phone: 765-496-2124; Fax: 765-494-0391

G.D. Massa— Plant-growth Lighting for Space Life Support


be the primary source of photosynthetic energy for human

life support on the moon or Mars.

Since solar energy is not a viable primary option to

support crop production, electric lighting must be

considered. Characteristics to consider when adapting an

electric lighting source for a life-support system away

from Earth include the following:

● Conversion efficiency of electrical power to

photons of photosynthetically active radiation

(PAR)

● Thermal burden (amount and location relative to

crop canopy)

● Lifetime of light source

● Durability of light source

● Mass of light source and associated hardware

● Volume (both storage and usage)

● Light output

-- Quantity

-- Quality

--Tunability/dimmability (versatility for a variety

of crops)

Electrical conversion efficiency refers to the wattage of

electric power that is converted to wattage of light

formed, specifically PAR (400 - 700 nm, Sager and

McFarlane, 1997). Electrical energy that is not converted

to light energy is given off as sensible heat as well as

long-wave radiation, both from ballasts (transformers) as

well as from light sources (lamps) themselves. That part

of the thermal burden generated by lamp surfaces needs to

be removed from the plant-growth area and either

redirected to some heat-requiring region of an ALSS or be

rejected from the habitat. The electrical power required to

run the lamps and to remove the heat burden becomes the

single most expensive cost factor for crop production in

an ALSS. Thus, a primary goal of plant research for ALS

is to generate the largest amount of edible biomass

possible for the least amount of electrical energy used

(i.e., optimize for energy-use efficiency).

Another consideration in adapting an electric lighting

source for ALS is the lifetime of the source, which

impacts the number, volume, and mass of replacement

lamps that must be launched as well as the crew time

needed to maintain a lighting system. Durability of the

source refers to its structural integrity when subjected to

the rigors of launch from Earth to space, including routine

handling, and the conditions encountered in microgravity

for a duration that might be fairly short (Lunar transit,

ISS) or long (Mars Transit). Improved launch, transit,

and landing technologies may reduce such stresses, but

the nature and composition of the light source will

continue to be a factor both for functionality as well as for

issues of astronaut safety.

The mass and volume of a light source, including all

necessary mounting infrastructure, and accessory

equipment, become important in terms of the lift capacity

needed to send those materials to the desired location in

space. Current estimates of launch costs from the Earth’s

surface vary depending on vehicle, but can be

approximated at $10,000 per Kg to low earth orbit and

$300,000 per Kg to the surface of Mars in 2000/2002

dollars, with costs to the lunar surface falling between the

two (Futron Corporation, 2002; A. Drysdale, 2005 Pers.

Comm.). Related issues include the transport and storage

volume of replacement lamps, both in the spacecraft and

at the outpost. Another issue in the design is volume of

space that the lighting infrastructure will occupy within

and above a plant-growth compartment. Larger lighting

systems will limit the space available to accommodate

plants. Therefore, a design with smaller, less massive

lighting systems is desirable.

Other design considerations include a need for flexibility

in the output of light quality, quantity, intensity, and

positioning of the lighting system. Specifically, design

questions to be addressed regarding light quantity include

how many plants each light source will support, and how

flexible a particular light source is with respect to planting

designs? Also, how will photon flux influence lighting

configuration and uniformity? The quality factor of light

output can be very important as well. Some species have

special wavelength requirements for flower initiation in

long-day plants, such as barley requiring far-red

wavelengths (Deitzer et al., 1979). Intumescence in

tomato is eliminated by the presence of UV (Lang and

Tibbitts, 1983) or far-red light (Morrow and Tibbitts,

1988). Certain crops, like lettuce and wheat, grow under

red light alone (Hoenecke et al., 1992, Goins et al., 1997),

but these and other crops have improved biomass

production with the addition of small amounts of blue

light (Hoenecke et al., 1992, Goins et al., 1997; Brown et

al., 1995). Is it better to have one standard white-light

source including all wavelengths, allowing increased

cropping flexibility? Is it better to have custom light

sources designed for each crop, allowing energy

conservation? Or, is some middle ground preferable? The

issues of tunability (e.g., altering color output by shifting

red : blue ratio) and dimmability (altering intensity of

emission) also are light-output factors. Some types of

crops thrive under light with high blue fluxes, while

others grow better under red-enriched light with minimum

blue. Certain crops prefer dim light, while others require

bright light, or may require it only during certain periods

of development. Fluorescent lamp dimming is linear, with

little efficiency drop-off as input power is reduced to a

lower limit (Osram Sylvania, 2004). High-intensity

discharge (HID) lamps have an issue of spectral shift as

input power is decreased (Bubenheim et al., 1995).

Additionally, minimum power levels required for an arc

to fire are still high for both metal halide (MH) and high-

pressure sodium (HPS) lamps (Bubenheim et al., 1995).

For LEDs, which are current controlled, dimming is linear

and continuous, with a correlation between PAR and

current that can be greater than 0.99 (Massa et al., 2005b).

It would be best to maximize edible biomass output per

unit energy input by tuning the light source to suit the

crop at different stages of its development, but features



such as dimming typically come with associated energy,

mass, and volume costs for traditional lamp types.

Another consideration in reducing the energy requirement

for crop production in a closed system involves reducing

the transpiration rate of crops and thus the amount of

energy that will be required to condense transpired water

vapor. If HID lamps are used, the area of a closed-canopy

crop stand necessary to continuously support the food

requirement of one person in an ALSS (~50m2) will

transpire approximately 4 L/ m2/dayof water vapor, which

amounts to 200 L of water transpired per person per day

(Wheeler et al., 2003). While this at first may seem like a

benefit, in terms of water reclamation, an average person

requires only 2.5 L per day of water to satisfy their

drinking-water requirement (Hopkins, 1993), and at most

40 L of water per day in an ALSS for all purposes

(Mitchell et al., 1996). This leaves an excess of 160 L of

water per crewmember, which adds substantially to the

energy burden of crop production. The change of phase

from liquid to vapor requires 582 cal/g of water at 25˚C,

and the reverse phase change back to liquid is similar.

Each latent heat draw will add 0.68 KW-h for every liter

of water condensed from transpiration (Nobel, 1974),

adding to the thermal burden of crop production in an

ALS system. If a crop-lighting technology can be

developed resulting in a lower thermal load on plants,

then this transpiration rate and the concomitant water-

condensation burden also can be reduced.

Several excellent articles have been written reviewing the

efficiency and usefulness of different types of electric

light sources for plant-growth applications. Bickford and

Dunn (1972) wrote a definitive reference book on this

subject characterizing not only available light sources, but

also addressed the specialized lighting requirements of

numerous crops. An international workshop organized in

1994 compiled a number of papers on plant and animal

lighting in controlled environments with intent for use in

a life-support system (Tibbitts et al., 1994a). Sager and

McFarlane (1997) reviewed radiation impacts on plant

growth and evaluated a wide range of lighting sources for

use in controlled environments. The light sources used in

terrestrial plant growth chambers, however, are not

necessarily a good indicator of what would work best in

an ALSS. Traditional light sources, for example, can be

hot, fragile, and require large, heavy ballasts. Table 1

compares some characteristics important to an ALSS

between different electric light sources.

Table 1. A comparison of several characteristics of light sources important for an ALSS. These values are averages and may not reflect

recent advances in some lamp types.

High Intensity Discharge (HID) Lamps

Parameter

Cool White

Fluorescent

High Pressure

Sodium

Metal

Halide

Microwave Light-

Emitting

Diodes

% Power to

PAR

22%1 35%

1 29%

1 38%

2 Red, 21.5%

Blue, 11%3

Lifetime 20,000 hr1 70%

or less output at

6,000 hr4

24,000 hr1

12,000-

20,000hr4

10,000 hr or

more1

~100,000 hr5

Composition Glass tubes, Hg

vapor, phosphor

coating4

Ceramic

alumina arc

tube contains

Hg, Xe, and

Na, glass outer

bulb1, 6

Quartz tube

in glass bulb,

metal halides

+ pressurized

Hg4

Electrodeless

quartz bulb

filled with S1

Solid state,

materials vary

with LED

color, discrete,

or multiple3, 5

Light quality 400-700nm4

Red shifted,

Peak 550-

650nm4

Blue-biased

400-700nm4

400-700nm1

Various, use

multiple LEDs

for desired

spectrum

Availability available available available not available available

1. MacLennan et al., 1994, 2. Ciolkosz et al., 1998, 3. Mike Bourget, Personal Communication, 2005, 4. Sager and MacFarlane, 1997, 5.

Barta et al., 1992, 6. Bickford and Dunn, 1972



LIGHTING IN ALS RESEARCH

Fluorescent light was a standard for controlled plant

growth research for many years, and numerous baseline

studies were done using this lighting system. With

fluorescent light, especially cool-white fluorescent, and

often with additional incandescent light, plants grow

“normally” and in approximate proportion to growth of

plants outdoors (Downs and Hellmers, 1978). However,

fluorescent lamps decline gradually in output, do not

typically emit high intensities, and have a limited lifetime

(Sager and McFarlane, 1997). For physiological studies

this may not be an issue, but for crop production,

fluorescent lamps lack the sustained photosynthetic

photon flux (PPF) capability necessary for high

productivity. Most of the lighting sources previously

considered for ALS are in the (extremely fragile) HID

category of lamps. In the Russian Bios–2 facility, a 4.5

m2 phytotron was irradiated with four water-cooled 6-kW

xenon lamps with an average irradiance of 90-115 W/m2

of PAR (Gitelson et al, 2003). Bios-3 was an impressive

closed life-support system containing two plant-growth

compartments, an algae cultivation compartment, and a

habitation unit for three crewmembers. Bios-3 was

unique in that it was designed for control entirely by

crewmembers within the system, as in an off-Earth life-

support scenario, and had teams of people enclosed for up

to 6 months. In the plant chamber, Bios-3 used the same

lamp type as Bios-2, with 20 lamps placed in each of two

31.5 m2 compartments (Gitelson et al, 2003). To reduce

the IR radiation coming from the xenon lamps, the light

sources were installed in a quartz jacket. This was then

enclosed by a glass jacket, and water for cooling flowed

between the two layers to remove heat. In Bios-3, lamp-

cooling water was pumped directly from the nearby

Siberian Yenisei River since it is both cold and pure

(Gitelson et al, 2003). In Bios-3, plant productivity

produced oxygen levels greater than required for a three-

person crew. Food production from crops ranged from

30% of their food requirements in the first three-person

trial to 77.5% in the final two-person, five-month closed

trial. Other foods, predominantly animal products, were

stored in lyophilized or canned forms. Roughly 21% of

crew time for a three-person crew and 19% of crew time

for a two-person crew was spent maintaining the

operation of plant and algal growth areas. Significant

amounts of time were also spent in food preparation,

water purification, and other life support-related

maintenance tasks. Overall, Bios-3 achieved 78-95%

closure of the system and reduction of pre-stored

substances that would need to be supplied for human life

support (Gitelson et al., 2003).

At Kennedy Space Center (KSC), a different kind of HID

lighting was utilized within the closed Biomass

Production Chamber (BPC). This large, closed plant-

growth chamber was a converted hypobaric test chamber

for the Mercury program. Generally, this chamber was

lighted by ninety-six 400-W HPS lamps with remote,

dimmable ballasts, but occasionally MH lamps were used

when crops required it (Wheeler, 1992; Wheeler et al.,

2003). The BPC had 20 m2 of plant growth area within a

volume of 113 m3, and more than twenty tests were

performed. Several crops were grown, including

potatoes, wheat, soybean, lettuce, tomatoes, and rice, and

one test even ran for longer than a year with four

successive potato generations grown in the same nutrient

solution. Numerous important conclusions were reached

during the course of those experiments, and research

continues using conventional growth chambers (Wheeler

et al., 2003). Such studies demonstrated that, if crops

required wide spacing at maturity, productivity and

radiation-conversion efficiency could be improved if they

are transplanted from smaller areas. Also, a nearly linear

response was found between daily PAR and productivity

across a range from 15-60 mol m-2

day-1

for a variety of

unrelated crops (Wheeler et al., 2003). Another key

result that complements other published research indicates

that, in nearly all plant species tested, those grown in a

controlled environment have higher protein, and ash

levels and lower carbohydrate levels than crops grown in

the field (Wheeler et al., 2003).

The Lunar-Mars Life Support Test Project (LMLSTP) at

Johnson Space Center (JSC) incorporated an 11.2 m2

Variable Pressure Growth Chamber (VPGC) to grow a

wheat crop for air revitalization (Gitelson et al., 2003).

Eight banks of 400 W HPS lamps were used in this

chamber and it was demonstrated that wheat growth in

this area revitalized 25% of the carbon dioxide produced

by a crew of four over the 91 days of Phase III testing

(Lawson, 2004). Physico-chemical systems regenerated

the remaining 75%. In addition, some food was provided

from the wheat as bread, and from a separate small plant

growth chamber that used LEDs to produce lettuce

(Gitelson et al., 2003; Lawson, 2004). Following these

tests, the BIO-Plex facility at JSC was designed to use

water-jacketed HPS lamps for crop growth (Tri, 1999).

However, the current status and plans for this integrated

plant growth and human habitation facility remain unclear

(Gitelson et al, 2003).

Lastly, the Closed Ecology Experimental facility, or

CEEF, on the northern island of Honshu, Japan, utilizes

HPS lamps in four plantation chambers, as the sole PAR

source in three of those chambers, and as a supplement to

sunlight in the other (Masuda et al., 2005; Nitta, 2005).

CEEF has a total cultivation area of 150 m2, and early

experimental results indicated that, without increased

productivity, they would need almost 255 m2 crop growth

area per person for a balanced diet. Peanut cultivation for

oil accounted for 78% of this estimate, due to the low

level of fats available in a typical vegetarian diet (Masuda

et al., 2005). Although rice and soybean showed high

productivity within the facility, several other crops require

further optimization to produce optimum biomass.

Currently, only short-term closure experiments have been

performed in CEEF, although one-month habitation

experiments are scheduled to start in the summer of 2006

(Nitta, 2005).



LED RESEARCH

Light-emitting diodes, LEDs, are a comparatively new

light source for plant growth and are being actively

investigated for numerous applications. Every week new

articles appear in the popular press about advances in

LED technology and the potential of this solid-state light

source for automotive and home lighting, computing,

public works light sources, etc. Red LEDs originally had

15-18% efficiency, but now are up to almost 22%,

whereas blue LEDs were only 3-4% efficient and are now

at 11%. This increase in efficiency makes LEDs

competitive with other sources for plant-growth lighting

(Tennessen and Ciolkosz, 1998; M. Bourget, 2005 Pers.

Comm.). Another important advance in LED research is

the commercial availability of “chip-on-board” LED light

engines. Unlike discrete LEDs with plastic lenses, these

light engines are small printed-circuit wafers that pack

large numbers of small LEDs of selectable emission

colors into close proximity. For example, the ORBITEC

light engine can array 132 LEDs of five colors in a 6.25

cm2 square (Massa et al., 2005a). This allows for

unprecedented color blending and very bright light levels.

LED emissions are current-controlled, and the light output

is directly proportional to input current within their

operating range, so unlike other types of dimming

systems for lighting, dimming of LEDs directly reduces

power usage. LEDs have solid-state construction, are

extremely durable, and resistant to shock. Transparent

coatings on the chips protect them against high humidity

and allow for cleaning without reducing light levels. LED

chips, like discrete LEDs, have low mass and volume.

LEDs generally emit light in a narrow region of the color

spectrum. The number of available colors is extremely

large, with one of the most efficient being red LEDs

emitting at 640 nm, where light has a relative quantum

efficiency for photosynthesis of ~96% (Sager and

McFarlane, 1997). Experimentation has demonstrated

that different species can be grown successfully under

LEDs, including spinach (Goins and Yorio, 2000), lettuce

(Goins et al., 2001; Kim et al., 2004), radish (Goins et al.,

2001), wheat (Goins et al., 1997), and micropropagated

potato plantlets (Miyashuta et al., 1995). Generally, about

15% blue light is required for normal growth, and yields

have been achieved that are comparable to growth under

white light (Yorio et al., 1998). Research has

demonstrated that green light also can have beneficial

effects for growth and plant assessment, especially within

dense foliar canopies (Kim et al., 2004; 2005).

INTRACANOPY LIGHTING FOR CROP GROWTH

Intracanopy (IC) lighting aims to improve lighting

efficiency by providing light distribution throughout the

canopy of a crop. In planophile crops, where leaves

present themselves perpendicular to overhead light and

eventually close off their inner canopy to light, mutual

shading of lower leaves by those above leads to net

carbon loss via respiration, premature leaf drop, and often

flower bud and fruit abortion inside the canopy (Ohler et

al., 1996). Thus, unshaded top and side leaves end up

doing all photosynthetic work for the entire crop stand. If

the light sources could instead irradiate from within the

canopy, a much greater percentage of available leaf

surface could be utilized for photosynthetic work. This

should increase biomass output per energy input

efficiency. Additionally, light intensity drops off

exponentially from a point irradiation source according to

the inverse square law, where

I = E / d 2

with I being the irradiation on a surface at a distance d

from the light source emitting radiant energy E (Bickford

and Dunn, 1972). Thus, light levels drop off rapidly with

increasing increments of distance between lamp and plant,

so that with the necessary separation of hot light sources

above a crop stand the amount of light incident upon the

leaves is highly attenuated, further requiring that the hot

source be high-emitting and high power. If a much cooler

light source can be maintained in close proximity to or

even touching leaves, more light will be available at leaf

level for lower power cost. This will lead to a greater

energy-use efficiency of the biomass-production system.

IC lighting has been previously examined, either as a

supplement to traditional overhead lighting, or as a sole

lighting source. Stasiak and colleagues tested soybean

grown under microwave lamps and supplemented with

side-mounted lighting that was piped into the canopy via

glass tubes lined with optical lighting film to levels of at

least 150 µmol m-2

s-1

PAR at 100 mm from the tube

surface. When overhead light of 400-1200 µmol m-2

s-1

PAR was supplemented with inner canopy lighting,

productivity increased 23-87% (Stasiak et el., 1998).

Also, Tibbitts and Wheeler found that using fluorescent

side lights or MH light pipes with overhead-lighted potato

crops gave increases in tuber dry weights of 12-16%

(Tibbitts et al., 1994b). Sideward lighting systems for

production of plants from cuttings was developed to

reduce the vertical PAR gradient found in overhead-

lighted propagation chambers (Hayashi et al., 1992; Kozai

et al.,1992). One system used fluorescent lamps and it

was demonstrated that sideward lighting reduced the

electricity cost per potato plantlet produced from cuttings

(Hayashi et al., 1992). Fluorescent lamps, however, take

up a large volume of space, and they release heat that then

has to be removed. To counteract these issues, Kozai and

others (1992) used diffusive optical fibers as a light

source for side lighting. This allows plant containers to

be stacked, and also allows placement of containers near

the light source, thereby increasing the efficiency of light

capture and the vigor of biomass accumulation by

plantlets (Kozai et al, 1992).

If low-intensity IC lighting is used as a sole source of

PAR starting from the seedling stage, Frantz and others

demonstrated that expanding cowpea leaves adapted

physiologically to become shade leaves, with lower light-

saturation levels and light-compensation points than

plants lit with more intense light from above (Frantz et al.,

1998). They used short, 15-watt fluorescent tubes



suspended within the crop canopy by monofilament and

surrounded by transparent Mylar sleeves to prevent leaf

scorch (Frantz et al. 1998; 2000; 2001). With IC lighting

as a sole source, they found twice as much edible biomass

production per unit energy input as in overhead-lit

canopies (Frantz et al., 2000). The two lighting

architectures combined, however, did not increase overall

yield relative to input wattage, probably because the

fixed-position overhead lights were underutilized until the

plants grew to sufficient height (Frantz et al., 2000).

Frantz and colleagues demonstrated that increasing lamp

number within the canopy by 38% raised stand

productivity by 45%, and that the highest energy-use

efficiencies could be obtained by switching lights on

higher up in a canopy as the plants increased in height

(Frantz et al., 2001). When the data were normalized,

plants grown under low-intensity IC lighting produced

50% of the edible biomass of those grown under high-

intensity overhead lighting but with only 10% of the total

electrical energy input (Frantz et al., 1998). Further

increases could not be accomplished, however, due to the

volume occupied by the heat-shielded lamps – if more

lamps were added to the canopy, the available planting

space decreased. Those proof-of-concept studies with

fluorescent lamps illustrated the need for a cool, small-

volume light source that will allow switching on of lights

to keep pace with plant growth. Vertical, linear-arrayed

LEDs were found to fit those requirements.

LED RECONFIGURABLE LIGHTING ARRAYS

The NASA Specialized Center of Research and Training

in Advanced Life Support (ALS NSCORT) was created to

develop technologies to lower the equivalent system mass

(ESM) of an advanced life-support system (Drysdale,

1997). The crops focus area of the ALS NSCORT has

entered into a collaboration with Orbital Technologies

Corporation (ORBITEC, Madison, WI) to develop a

reconfigurable LED lighting array that will significantly

reduce the power and energy required to grow plants

using electric lights. The development and preliminary

testing of this lighting-array system has been described in

previous publications (Massa et al., 2005a; 2005b).

Briefly, the prototype system uses ORBITEC’s

proprietary light engine, consisting of 100 chip-on-board

LEDs set into a 6.25 cm2 square chip. There are sixty-

four 640-nm-emitting (red) LEDs, sixteen 440-nm-

emitting (blue) LEDs, and twenty 540-nm-emitting

(green) LEDs on each chip. Additionally, there are two

photodiodes. The green LEDs and the photodiodes are in

place to accommodate future system-upgrade capabilities.

The small size and close proximity of the LEDs allows for

uniform spectral blending of photon emissions. Since the

LEDs are current controlled, with the colors controlled

separately, both the red-blue ratio and the light intensity

output can be adjusted continuously. Twenty each of

these light engines are mounted along a hollow linear

support (approximately 3 cm wide x 1.5 cm thick x 65 cm

long) that is attached to an electronics enclosure

(approximately 5 cm x 12 cm x 10 cm) also containing

two fans. Figure 1 shows such a “lightsicle”. The hollow

design of the lightsicles allows air to be drawn through

them from the bottom of the canopy past the circuitry

controlling the LEDs and out the top of the enclosure,

thus removing electrically generated heat from the

vicinity of the plants. Each lighting array presently

consists of 16 such lightsicles sized to light a growth area

~0.25 m2 and a growth volume ~0.15 m

3. These arrays

are currently configured to energize light engines from the

bottom up, so that the lights can be switched on

incrementally to keep pace with changing plant height.

Fig. 1 A lightsicle for the reconfigurable lighting array with the major external components labeled. Each lightsicle consists of 20 LED

light engines mounted to a tubular support, with associated electronics. Cooling air is pulled through the support past the internal

electronics by fans mounted on the electronics enclosure.



The electronics systems within the lightsicles

communicate with a control enclosure via a Controller

Area Network (CAN) communication system. The

control enclosure allows the user to select LED power

levels as well as the number of light engines energized to

allow manual control that keep pace with plant growth.

Red and blue LEDs have independent controls.

Photoperiod is controlled by a programmable timer. The

control enclosure also houses the system power supply

and plugs directly into grounded 110 V power sources.

Each lightsicle can be hung independently in a variety of

configurations, allowing for flexibility in the intracanopy

plant-growth arrangement. In addition, the lightsicle

array can be reconfigured into a rectangular planar array

consisting of 20 x 16 light engines for close-canopy,

overhead lighting. Figure 2A and B shows lightsicles in

intracanopy and overhead configurations, respectively.

This close-canopy configuration is ideal for lighting

rosette (e.g., lettuce) or erectophile (e.g. dwarf wheat)

crops. The light array can be brought in close proximity

to crop surfaces without scorching them, and after the

development and integration of automated switching

protocols, the energized engines will be able to track and

mirror plant growth. Light engines positioned directly

above each seedling will switch on automatically, and

then adjacent engines will illuminate as the leaves of the

seedlings are produced and expand until all engines are

on. As with the IC lighting, greater efficiency will be

achieved by not lighting empty space, but rather targeting

lighting only where photosynthesis can occur.

Fig. 2 A. Intracanopy LED lightsicle array with LEDs off. B. Overhead LED array with LEDs energized. C. Intracanopy array with closed

canopy of cowpea plants. LEDs are not energized. D. Overhead array with closed canopy of cowpea while LEDs are energized. Note

senescence of lower leaves. Plants in C. and D. are both 32 days old.



Five hardware tests were performed with the first

prototype lighting array using cowpea crop stands.

Modifications to the experimental design were made

between trials 1 through 4 with incremental

improvements in crop productivity achieved at each

successive trial (Massa et al., 2005a). Figure 2C shows

an example of an intracanopy-lighted plant canopy prior

to harvest. Following intracanopy trial 4, the lighting

system was reconfigured into a planar array, and a fifth

trial was run using conditions identical to trial 4 but

mounting the lights overhead (Fig. 2B.). In the overhead

trial, all light engines were energized throughout the trial,

while in the intracanopy trials, lights were switched on

incrementally to keep pace with plant growth. To

normalize the total power usage, the overhead lights were

run at a current that was identical to the average daily

current of intracanopy trial 4 so that the same total

amount of electrical energy (99kW-h) was used during the

month-long trial. In the overhead trial, we observed

mutual shading and drop of the lowermost leaves, so that

11% of the total biomass senesced prior to the end of the

trial. Figure 2D shows the overhead-lighted canopy prior

to harvest. Overall, plants grown under overhead lights

produced less biomass and had a reduced energy

conversion rate than plants grown with intracanopy lights,

with overhead-lighted plants averaging 75% of the

productivity of intracanopy-lighted plants (data not

shown).

These trials were conducted prior to the correction of

certain electronic anomalies reported by Massa et al.

(2005a). In the first hardware tests, PAR output from the

light engines was very low when only lower engines were

energized, with a maximum of 100 µmol m-2

s-1

emitted

from a lower engine with 5 engines energized. As more

engines on each lightsicle were energized, the light output

from each engine increased until more than 700 µmol m-2

s-1

of PAR were detected from the same lower engine

when all 20 engines were energized at the same power

level. Thus, when plants were young and only a few light

engines were energized, the light emission from that

engine was still very low, causing elongate growth and

spindly stems of seedlings. This light-output issue has

been rectified through a modification of the software

controlling the light-engine drivers, and now we are able

to obtain uniform irradiation from a given engine

regardless of the number of light engines energized along

the array. A second set of lightsicles has been constructed

by ORBITEC, and tests are underway to examine IC vs.

OH irradiation in a side-by-side experiment. An

additional feature added to the second prototype array is

an extension so that the control box is raised on 8 of the

16 lightsicles. This allows the shorter lightsicle

electronics enclosures to nestle under the longer

lightsicles, giving a much wider range of possible lighting

configurations. Light-engine positions in the longer

lightsicles are the same as in the shorter ones.

FUTURE DIRECTIONS

A canopy gas-exchange-measurement system is being

developed specifically for IC LED lighting. A custom-

made, whole-canopy cuvette will allow real-time

photosynthesis and transpiration rate measurements of an

entire crop stand growing among the IC lights. Gas

exchange will be measured as a function of environmental

parameters such as light level, red-blue ratio, CO2, and

temperature. This powerful tool will permit rapid

optimization of IC lighting and growth conditions for a

variety of ALS candidate crop species.

A second research focus is being developed at ORBITEC

under a Phase II SBIR from NASA for “High Efficient

Lighting with Integrated Adaptive Control (HELIAC)”.

This project focuses on the development of automated

plant detection and light-engine switching using green

LEDs and photodiodes embedded on individual light

engines. Automation of the switching system to energize

LEDs only when leaves are in front of light engines will

conserve considerable energy by not lighting empty

space, will maximize biomass production by keeping pace

with plant growth, and will significantly reduce the

personnel time involved with light operation.

Additionally, these added capabilities will allow

development of a close-canopy lighting system for

targeted overhead lighting of erectophile and rosette

crops.

CONCLUSIONS When considering a light source for ALS, several

important characteristics must be kept in mind: A variety

of light sources have been evaluated from this

perspective. LEDs, especially the relatively new chip-on-

board LED light engines, appear to be optimal lighting

systems for ALS crop growth for a variety of reasons. As

a rapidly developing technology, electrical efficiency of

these light sources continues to increase. In addition, the

ability to precisely select a spectrum that is efficient for

photosynthesis, growth, and flowering, the durable solid-

state nature of LEDs, the relatively cool emitter surface,

their long lifetime, tunability of the spectrum and

irradiation levels, and ability to easily remove heat all

combine to make this lighting type the best contender for

ALS crop production. When the benefits of LEDs are

coupled with techniques that apply light only where there

is photosynthetic capability, the increased lighting

efficiency will result in a significant reduction in the

power required to maintain desired levels of biomass

production, reducing the cost of growing plants in an

ALSS, and bringing crop growth on Luna and Mars that

much closer to reality.

ACKNOWLEDGEMENTS

The authors wish to acknowledge Mercedes Mick for help

with lighting installation, plant growth, and data

collection and analysis. We also wish to thank Bruce

Bugbee and Ray Wheeler for helpful discussions, and

Thomas Crabb and Charles Barnes for support and



encouragement. This work was supported in part by

NASA grant NAG5-12686.

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INCREASED BACTERIAL RESISTANCE AND VIRULENCE IN SIMULATED MICROGRAVITY

AND ITS MOLECULAR BASIS

A. Matin1, S.V. Lynch

2 and M.R. Benoit

1

1 Department of Microbiology & Immunology, Stanford University School of Medicine

2 Department of Anesthesia and Perioperative care, UCSF Medical School

ABSTRACT

Experiments conducted during space flights showed that space

conditions affect basic aspects of bacterial physiology, such as

growth, conjugation efficiency, and secondary metabolite

production. Since logistical constraints hamper research during

space flights, use of ground based systems has greatly stimulated

progress in this field. A commonly used system is the rotating

bioreactor which simulates some aspects of microgravity.

Studies using this system have shown that bacteria grown under

diminished gravity become more virulent and resistant to a

variety of antimicrobial treatments [referred to as

“comprehensive cellular resistance (CCR)]. In this respect, the

diminished gravity effect resembles the well studied bacterial

general stress response. The latter is centrally controlled by an

alternate sigma factor, called σs. However, the diminished

gravity-conferred CCR is not controlled by this sigma factor in

rapidly growing cells. Moreover, the proteins known to confer

CCR under the Earth conditions do not appear to have a role in

this resistance under the diminished gravity conditions. Growth

in the HARV system altered the of σs regulation pattern at

transcriptional, translational and posttranslational levels. Since

macromolecular folding patterns play a role in this regulation,

the findings raise the possibility that diminished gravity may

alter the folding pattern of macromolecules. The effect of

diminished gravity on bacterial biofilms, which are responsible

for serious and recalcitrant diseases, has recently become

possible on earth by the designing of a novel HARV adaptation.

The results show that diminished gravity stimulates biofilm

formation and makes them highly resistant to antimicrobial

agents. Given that the human immune response is compromised

in space, these results highlight the serious danger that microbes

can potentially pose in space.

INTRODUCTION

Space travel, although tantalizing, is fraught with hazards.

There are first the purely human factors, such as the

psychological stress originating from the fear of the

unknown, isolation, and the claustrophobic atmosphere of

a confined environment; and upsetting of the normal life

rhythm that results in sleep deprivation and

malnourishment. Then there are the peculiarities of space:

unusually strong radiation and diminished gravity. Life on

Earth evolved in the presence of gravity and thus the latter

aspect is an unprecedented experience for the earthlings.

A good deal of research in recent years has concerned

with determining the effect of diminished gravity on

human physiology as well as on bacterial characteristics.

And the answer seems to be that diminished gravity or

‘microgravity’ as it is termed, weakens humans and

makes bacteria stronger, more difficult to kill, and more

accomplished at causing disease. In this review, we will

discuss the findings underlying these conclusions.

Bone decalcification and loss are well documented in

astronauts during space travel and residence. This

predisposes them to bone fracture as well as kidney stones

from resorbed bone material. In microgravity, muscles

atrophy and blood production decreases. The latter results

in diminished pumping by the heart and, combined with

the concomitant blood shift to the upper torso, can

damage heart muscles. In addition, microgravity

compromises the human immune response. This appears

to result from several factors: the proportion and number

of circulating lymphocytes and their cytokine production

are detrimentally altered, lymphocytes produce lower

levels of human leukocyte antigen, there is increased

apoptosis of peripheral blood mononuclear cells, and

dendritic cells become defective in phagocytosis

(Sonnenfeld and Shearer, 2002). Additionally,

pharmacodynamics and pharmacokinetics are expected to

be altered during spaceflight (Graebe et al., 2004).

The study of spaceflight effects on bacteria originated

during the advent of manned spaceflight programs in the

1960’s. Since that time, many bacterial studies have been

conducted onboard spacecraft, and several published

reviews of these studies exist (Cioletti, 1991; Dickson,

1991; Moore, 1996; Pierson and Mishra, 2000; Klaus,

2002). In general, these studies have shown that

spaceflight causes increased final cell populations,

shortened lag phase duration, increased conjugation

efficiency, and increased production of secondary

metabolites. Other findings, which pose a more direct

threat to human health, include decreased antibiotic

effectiveness during spaceflight experiments (Tixador et

al., 1985; Lapchine et al., 1988; Tixador et al., 1994) and

increased spacecraft contamination. Observations of

spacecraft contamination include the discovery of

biofilms in the Space Shuttle water system (Koenig and

Pierson, 1997), free-floating condensate containing

microbes onboard the International Space Station (Ott et

al., 2004), and microbial contamination onboard the Mir

Space Station (Novikova, 2004). The combination of

potentially less effective antibiotic treatment, increased

____________________

* Correspondence to: A. Matin

Department of Microbiology & Immunology

Stanford University School of Medicine

Stanford, CA 94305


Phone: 650-725-4745; Fax: 650-725-6757

A. Matin — Increased Bacterial Resistance and Virulence in Simulated Microgravity


Fig. 1A HARV system used to simulate conditions of microgravity in Earth-based investigations. B. HARV vessel components. (Synthecon,

Inc.)

bacterial growth, contaminated spacecraft, and

suppressed astronaut immune systems presents a serious

threat to crew health. This threat will become

exacerbated with increased mission duration (Pierson,

2001), and future missions planned for travel outside of

low Earth orbit will not be capable of a swift return home

in the case of an untreatable infectious disease.

Studies on the effect of diminished gravity on bacteria as

well as mammalian cells have been greatly facilitated by

the invention of earth based systems that reproduce some

aspects of microgravity. The clinostat, which was

developed over a century ago for studying gravitropic

plant responses (Dedolph and Dipert, 1971), rotates a

container of suspension cultures about an axis that is

perpendicular to the gravity vector. Thorough descriptions

of this device and its limitations have been published

(Albrecht-Buehler, 1992; Klaus, 2001). More recently,

rotating systems such as the high aspect ratio vessel

(HARV) bioreactor (Fig. 1a), have been designed for

studying mammalian tissue culture (Wolf and Schwarz,

1991; Schwarz et al., 1992; Wolf and Schwarz, 1992).

The HARV has front and back faces that are separable;

the front face contains two sampling ports, and the back

face is equipped with a semi-permeable membrane for

aeration (Fig. 1b). The vessel is completely filled with

the medium and inoculum so that no head space remains.

Air bubbles are removed, to prevent fluid mixing. Two

such vessels are used per experiment, of which one is

rotated about a vertical axis, perpendicular to the

gravitational vector, and the other about a horizontal axis,

parallel to this vector.

In both the clinostat and the HARV, virtually no relative

fluid motion exists and after an initial start-up time, solid

body rotation of the fluid ensues. Since mixing occurs by

gentle rotation in the absence of stirring vanes, and

aeration is affected by dissolved gasses with no headspace

or bubbles, there is no shear-field inhomogeneity;vessels

rotated around either axis thus possess a low-shear

environment. In the vessel rotated about the horizontal

axis, particles quickly reach a terminal settling velocity,

but cumulative sedimentation is avoided due to

hydrodynamic forces, viz., shear, centrifugal and Coriolis

(Hammond and Hammond, 2001). The gravity vector is

continually re-oriented as the cells and fluid rotate

together, which dramatically influences large cells, e.g.,

those of plants with gravity sensing mechanisms

(Dedolph and Dipert, 1971).

Both the clinostat, and more recently the HARV, have

been used to study bacteria. Although planned

experiments to demonstrate the effectiveness of the

HARV as a spaceflight analog have yet to be conducted

(Niesel et al., 2005), ground-based clinostat experiments

have been correlated with spaceflight experiments

yielding similar results, at least in terms of increased final

cell populations (Kacena et al., 1999; Brown et al., 2002).

Due to the small size of bacteria, rotating culture systems

operated at the correct rotation rate can reduce

gravitational cell motion to less than Brownian motion

(random diffusion) for the duration of the experiment

(Todd and Klaus, 1996), thereby making a suitable

simulation of the reduced fluid motion provided by

weightlessness. This condition has been referred to as

Low Shear Modeled MicroGravity (LSMMG) (Nickerson

et al., 2003; Nickerson et al., 2004) or simply Simulated



MicroGravity (SMG) (Lynch and Matin, 2005; Matin and

Lynch, 2005).

An interesting alternate method of simulating

weightlessness was devised by Benoit and Klaus (2005).

A genetically engineered, gas vesicle-forming strain of

Escherichia coli was used. These vesicles are organelles

with a protein coat which is permeable to ambient gases,

and certain bacteria produce them to stay afloat in the

water column in nutritionally favorable regions. The

engineered E. coli strain remained suspended without the

need for rotation and exhibited growth characteristics

similar to the isogenic wild type (not producing gas

vesicles), when the latter was grown in a rotated clinostat.

EFFECT OF SMG ON BACTERIAL VIRULENCE

Nickerson and co-workers demonstrated that when mice

were inoculated with SMG grown Salmonella enteritica

serovar Typhimurium, they died faster than mice

inoculated with an equal number of bacteria (2x106 CFU)

grown under normal gravity conditions (Fig. 2) On the

10th

day of inoculation, while only 40% of the mice

inoculated with the NG grown cells had died, the number

of mice that had died following inoculation with SMG

grown bacteria was 100% (Nickerson et al., 2000).

Autopsy showed a more extensive colonization by the

latter; thus, for instance, 10,000 vs. 300 cells were

recovered from the spleens of mice infected with SMG

and NG-grown bacteria, respectively.

Fig. 2. Infection of mice with S. enteritica serovar Typhimurium

cultured in SMG (solid symbols) results in shortened time to

death compared to S. enteritica cultured under normal gravity

conditions (open symbols). Reproduced with permission from

Nickerson et al. (2000).

EFFECT OF SMG ON BACTERIAL RESISTANCE

TO ANTIMICROBIAL STRESSES

A large body of work conducted with bacteria grown

under conventional normal gravity conditions over the

last two or so decades has shown that increased bacterial

virulence is accompanied with increased resistance to

antimicrobial stresses. To test if this was true of the SMG

effect, Lynch et al. (2004) determined the effect of SMG

growth on the resistance of the bacterium E. coli to two

treatments that are detrimental to bacteria, exposure to

high salt or to high acidity. In these experiments, two

growth phases were examined, the exponential phase of

rapid growth and the stationary phase, in which little or

no net growth is seen and bacteria exist in a resting state.

While both phases occur in nature, it is the stationary

phase or condition resembling this phase that is most

commonly experienced by bacteria in nature (Matin,

2001).

Fig. 3. SMG-cultured E. coli demonstrates increased resistance

to both osmotic and acid stresses during both exponential (A)

and stationary (B) phases of growth. Reproduced with

permission from Lynch et al. (2004).

Growth under SMG conditions increased exponential

phase E. coli resistance to each of the separately tested

stresses (Fig. 3A); in the case of the salt treatment, for

instance, nearly all the NG-grown cells but only some

40% of the SMG-grown cells, respectively, were killed. It

is well established that stationary phase bacteria grown

under conventional flask cultures under normal gravity

exhibit increased resistance compared to their exponential

phase counterparts (Jenkins et al., 1990; Matin, 1991). As

shown in Fig. 3B, this is also true of the HARV NG

cultures. The striking thing, however, is that growth under

SMG caused the already resistant stationary phase cells to

become even more resistant, becoming virtually

invincible to antimicrobial treatments. That E. coli grown

under SMG (compared to shake flask) conditions were

less susceptible also to ethanol exposure, a common

method to disinfect, was shown by Gao et al. (2001).

RESEMBLANCE OF THE SMG EFFECT TO THE

GENERAL STRESS RESPONSE

The three antimicrobial treatments named above against

which growth under SMG made E. coli more resistant

harm bacteria in different ways. Exposure to low pH is



detrimental because it acidifies the cytoplasm and leads to

decomposition or denaturation of vital cell constituents.

High salt dehydrates the cell, and exposure to ethanol

damages the cell envelope. That SMG made cells resistant

to these three disparate stresses, which kill bacteria by

causing different kinds of injuries, indicates that the

mechanism of resistance activated by SMG encompasses

preventing and/or repairing injury to many different cell

constituents.

Fig. 4. Comparison of the H2O2 resistance of exponential phase

(○), or glucose-starved (□) E. coli cultures to growing cultures

stressed by heat (∆), H2O2 (▲), or ethanol (●). Reproduced

with permission from Matin (2001).

In this respect, the SMG effect resembles the general

stress response (GSR) that has been extensively studied

under conventional culture conditions of normal gravity

(Matin, 2001). This phenomenon is illustrated in Fig. 4.

When bacteria are exposed to small doses of H2O2, they

acquire the capacity to resist much higher and normally

lethal doses of this toxic agent. But the striking thing is

that protection against lethal doses of H2O2 results from

prior exposure of cells to a large number of separate

unrelated antimicrobial treatments, such as starvation,

heat, or ethanol. This sort of cross protection operates

against almost any stress so that, for instance, H2O2-

treated cells become more resistant to the other

deleterious agents as demonstrated in Fig. 4; and heat or

starvation-stressed cells acquire cross protection to

unrelated insults such as osmotic shock, or exposure to

low pH. Thus, the essence of GSR is that exposure of

bacteria to small sub-lethal dose of an antimicrobial

treatment makes them resistant not only to subsequent

exposure of lethal doses of the same agent but also to

others that injure bacteria by different mechanisms.

MOLECULAR AND BIOCHEMICAL BASIS OF

GSR AND SMG EFFECTS

What is the molecular basis that renders cells

comprehensively resistant after exposure to small doses of

a deleterious agent? Bacterial cells exposed to a large

variety of harmful agents respond by increasing the

cellular concentration of a “stress” sigma factor, σs. This

sigma factor replaces the “house keeping” sigma factor

(σ70

) on the RNA polymerase enzyme, thereby changing

its regulatory properties. σs-RNA polymerase recognizes

a different promoter sequence compared to σ70

-RNA

polymerase; promoter regions consist of sequences

present in front of the coding region of genes. The σs-

RNA polymerase recognized promoters are present in

front of genes that encode proteins, which can protect

against different types of cell injuries. Examples of

proteins thus induced are molecular chaperones, such as

DnaK which prevents damage to and can repair denatured

proteins; the SOS response proteins, e.g., PexB that can

insulate the cell against DNA damage, and proteins such

as D-alanine carboxypeptidase and PexAB that are

concerned with cell envelope integrity (Matin, 2001).

Consequently, bacteria subjected to sub-lethal dose of one

harmful treatment become capable of withstanding

multiple types of damage to their cell constituents.

Fig. 5. Quantification of σs protein levels in exponential (A) and

stationary (B) phase NG and SMG cultures. Reproduced with

permission from Matin and Lynch (2005).

As we have seen (Fig. 3), growth under SMG also makes

cells resistant to mechanistically different killing agents,

and since it resembles GSR in this respect, it was logical

to suspect that the underlying molecular and biochemical

mechanisms may be similar, and that SMG acted in the

same way as do other deleterious agents. However,

exponential phase cells grown under SMG conditions



possessed lower σs levels than their NG-grown

counterparts (Fig. 5A; (Lynch et al., 2004)). This is the

first instance in which increased cellular resistance to

deleterious treatments is found accompanied with lowered

levels of σs. In contrast, the stationary phase SMG-grown

cells that exhibit super resistance (Fig. 3), conformed to

the normal paradigm: their increased resistance was

accompanied by increased σs levels compared to the NG-

grown cells (Fig. 5B). The conclusion suggested by these

findings, namely, that the SMG-conferred resistance is

independent of σs in exponential phase, but dependent on

it in the stationary phase was confirmed by repeating

these experiments with a mutant of E. coli missing this

sigma factor. The mutant developed resistance normally

in the exponential phase (Fig. 6A), when grown under

SMG conditions, but was severely weakened under both

NG and SMG conditions in stationary phase (Fig. 6B).

Fig. 6. Stress resistance of an rpoS mutant strain grown under

NG and SMG conditions in exponential (A) and stationary (B)

phases of growth. Reproduced with permission from Lynch et al.

(2004).

The Nickerson group has examined the genes that are

influenced in exponential phase-cells grown under SMG

conditions (Wilson et al., 2002). Expression of over 160

genes was affected by growth under SMG conditions in S.

enterica typhimurium serovar Typhimurium. Surprisingly,

none of the up-regulated genes included those known to

encode protective proteins of the type mentioned above.

Furthermore, many of the genes involved in determining

virulence under the conventional flask culture conditions

were in fact down-regulated by SMG growth. The ferric

iron uptake protein, Fur, which has been shown to have a

role in SMG acid resistance, could have a larger role in

regulating gene expression under these conditions, since

many of the SMG-induced genes apparently possess Fur-

binding sites (Wilson et al., 2002). How gene expression

is regulated by SMG growth in the stationary phase has

not yet been investigated. It is clear however, that the

SMG and conventional normal gravity stress resistance

and virulence mechanisms differ in at least two respects:

in the exponential phase, it is independent of σs and does

not involve induction of genes known to be responsible

for conferring protection on the bacterial cell; and in that

the relationship of this mechanism to σs, and possibly also

to the known stress genes, reverses during transition from

exponential to stationary growth phase.

MOLECULAR REGULATION OF σσσσS

These unexpected findings led Lynch et al. (2004) to

explore if fundamental cell processes, such as regulation

of protein synthesis, were affected by growth under SMG.

They addressed this question by looking at σs for two

reasons, one that under SMG it is up or down regulated

depending on the growth phase; and two that its synthesis

has been thoroughly studied in conventional gravity

conditions, providing guidelines on aspects to study.

These prior conventional investigations have established

that σs synthesis is regulated by a combination of

regulatory mechanisms that operate at transcriptional,

translational efficiency, and protein stability levels.

Fig. 7. Western analysis of σs protein degradation over time in

exponential (A) and stationary (B) phases of growth under NG

and SMG conditions. N, Normal gravity time point; S, Simulated

microgravity time point. Reproduced with permission from

Lynch et al. (2004).

σs is encoded by the rpoS gene. To study the effect of

SMG on the transcription rate of this gene, the steady-

state levels of the rpoS messenger RNA (mRNA) were

quantified as well as the half-life of this mRNA in cells

grown under NG and SMG conditions, using quantitative

PCR. The transcription rate was not altered in exponential

phase but was decreased by one-half in the stationary

phase in SMG-grown cells (Table 1). A much more

pronounced effect of SMG was seen on the stability of σs

protein (Fig. 7). In the exponential phase, growth under

SMG rendered this protein over three-fold less stable. A

similar, less pronounced, but reproducible effect occurred

also in the stationary phase cells. Translational efficiency

of the rpoS mRNA, i.e., the amount of σs protein

synthesized per molecule of the rpoS mRNA, was

calculated from the rate of σs synthesis (calculated in turn

from steady state σs

levels and from the half-life of this

protein) and that of rpoS mRNA synthesis (Table 2). It

increased over two-fold under SMG regardless of the

growth phase.



Table 1. Copies of rpoS mRNA synthesized per minute under NG and SMG conditions in exponential or stationary phases of growth

(Lynch et al., 2004).

NG SMG

Exponential phase 1.6 x 107

1.6 x 107

Stationary phase 2.8 x 105 1.2 x 10

5

Table 2. Translational efficiency of σs protein per copy of rpoS mRNA per minute (Lynch et al., 2004).

NG SMG

Exponential phase 3.6 x 1011

8.4 x 1011

Stationary phase 3.3 x 1011

8.4 x 1011

It is not known how SMG affects these parameters of σs

synthesis. It is possible that the levels of molecules that

regulate these processes are affected by growth under

SMG. Another possibility is suggested by the fact that

regulation at all the levels mentioned above involves

higher order structures of the concerned molecules.

Stability of the σs protein depends on whether it is subject

to proteolysis by a specific protease, called the ClpXP

protease (Schweder et al., 1996; Becker et al., 1999;

Hengge-Aronis, 2002). Why it is attacked more readily by

the ClpXP protease under certain conditions and not

others is not fully understood. Inactivation of another

protein, called RssB (or SprE), appears to have a role

under carbon starvation conditions. In addition, the

folding pattern of σs could also be involved. The activity

of ClpXP protease is markedly affected by the folding

pattern of its substrate (Kenniston et al., 2004).

Experiments conducted in space have shown that proteins

form crystals more readily under these conditions. Taking

these findings together, it has been hypothesized that

diminished gravity and low shear may influence protein

folding pattern (Fig. 8; (Lynch et al., 2004)). In the

present case, this may mean that growth under SMG

causes the σs protein to acquire a configuration that is

more amenable to attack by the ClpXP protease.

Fig. 8. Schematic representation of post-translational degradation of σs protein involving RssB, the degradation tag (termed SsrA) and the

ClpXP protease complex.



Fig. 9. rprA a small non-translated regulatory RNA possesses stretches of sequence homology with the untranslated region of rpoS,

permitting binding and relief of translation inhibition of the rpoS mRNA. Reproduced with permission from Matin and Lynch (2005).

Secondary structure formation also plays a central role in

the translational efficiency of rpoS mRNA. This

messenger molecule possesses internal homologies in its

untranslated region that result in the formation of a

hairpin structure, which block its translational start site,

making it unavailable to the ribosomes. Studies under

conventional conditions of normal gravity strongly

suggest that this structure is relaxed when cells are

exposed to antimicrobial treatments. This is brought about

by interaction between the rpoS mRNA, a protein called

Hfq, and one of several small RNAs, for example, rprA.

The latter possesses homologies to the interfering

sequence responsible for the hairpin structure formation

in the rpoS mRNA and, in association with Hfq and

perhaps other factors, can disrupt this structure. As a

result the translational start site becomes untangled and

available to the ribosomes, promoting synthesis of σs

(Fig. 9). SMG could act in stimulating translation by

affecting any of the participants in this mechanism,

including a direct effect in minimizing secondary

structure formation within the rpoS mRNA. Only future

work will reveal what factors are involved in these

phenomena, but if diminished gravity and low shear can

influence folding patterns of molecules, implications of

space conditions on life processes would be wide ranging

indeed.

EFFECT OF SMG ON BACTERIAL BIOFILM

RESISTANCE

Biofilms are bacterial communities that are surrounded by

a polysaccharide matrix. Different cells within such

communities may perform different functions, and

biofilms may have a primitive circulatory system to

ensure efficient distribution of nutrients and removal of

waste products. Bacterial biofilms have a considerable

medical importance because they exhibit much greater

resistance to antibiotics and other antimicrobial treatments

than their constituent individual cells when they exist

outside the community. Consequently, diseases in which

bacterial biofilms have a major role, such as cystitis,

endocarditis, and cystic fibrosis, are chronic and difficult

to treat (Fux et al., 2005).

During experiments onboard the Space Shuttle,

Burkholderia cepacia formed biofilms (Pyle et al., 1999)

and Pseudomonas aeruginosa biofilms developed on

polycarbonate membranes (McLean et al., 2001).

Furthermore, biofilms were found in the Space Shuttle

water system (Koenig and Pierson, 1997) and on surfaces

and equipment onboard the Mir Space Station (Novikova,

2004). These findings point to increased danger to

astronaut health. Conversely, however, biofilms are

commonly used for nitrification and organic carbon

removal of wastewater, making them suitable for



wastewater treatment devices onboard future long-

duration space missions (Sharvelle et al., 2002). Because

biofilms will be present in the spacecraft environment,

understanding the effects of microgravity on biofilms will

be important for their control and exploitation.

How SMG growth affects biofilm resistance is therefore

of interest. However, this question has not been

adequately addressed because of a lack of earth-based

system to generate SMG-grown biofilms. Recently, a

modification of the HARV apparatus, involving the use of

appropriate beads, permitted facile bacterial biofilm

cultivation under SMG conditions on Earth (Lynch,

Mukundakrishnan, Ayyaswami and Matin, submitted).

SMG stimulated more copious biofilm formation and

these were much more resistant than their NG grown

counterparts to two antibiotics, penicillin and

chloramphenicol, as well as to the antimicrobial

treatments, such as exposure to high salt and ethanol. The

SMG-mediated enhanced resistance to salt and ethanol,

but not to the antibiotics, required the presence of σs in

the cells since an rpoS mutant failed to exhibit this

phenomenon for the former but not for the latter agents.

What aspect of the biofilms is controlled by σs that

enhances their resistance to various agents in a differential

manner has not as yet been determined. Lynch, Dixon and

Matin. (submitted) have, however, recently identified

another gene in uropathogenic E. coli which controls

biofilm resistance and whose mode of action has become

partially known. Comparison between biofilms of wild

type uropathogenic E. coli and a rapA mutant shows that

the rapA gene contributes to the increased resistance of

biofilms by promoting matrix exopolysaccharide

formation. When penetration of a fluorescent probe of

penicillin was visualized, it became clear that the

antibiotic penetrates the biofilm of the mutant much more

rapidly than that of the wild type. In addition, the rapA

gene is required for the expression of two genes that

appear to encode multidrug resistance pumps. Thus, this

gene appears to enable biofilms to utilize a dual strategy

for resistance to antimicrobials, retarded penetration and

more effective means of effluxing the antimicrobial that

does manage to finds its way into the biofilm cells. The

studies on the rapA gene have so far been done only

under the conventional conditions of normal gravity. How

SMG may affect these parameters is under investigation.

SMG AND SPACEFLIGHT EFFECTS ON

SECONDARY METABOLITE AND SPORE

PRODUCTION

In addition to the general stress response and increased

virulence, other survival strategies of bacteria include the

production of secondary metabolites, such as antibiotics,

which kill off competing organisms, and spore

production, which allows cells to wait out poor

environmental conditions. A few reports exist of

increased antibiotic production by bacteria on spacecrafts

(Lam et al., 1998; Luo et al., 1998; Lam et al., 2002;

Benoit et al., 2005). Following 15 days of Streptomyces

ansochromogenus growth onboard a Chinese satellite,

Luo et al. (1998) found that some mutants produced

increased concentrations of Nikomycin compared to

ground controls. A 17-day experiment conducted on the

Space Shuttle (STS-80) showed increased production of

Actinomycin D by Streptomyces plicatus. Due to

spaceflight hardware constraints, however, the absolute

production was low compared to conventional culture

methods. In a follow-up study, a newly designed space

bioreactor was used allowing antibiotic production at

similar concentrations to conventional methods. This

study involved a 72-day experiment conducted onboard

the International Space Station. Samples taken at days

eight and twelve showed increased spaceflight

production, but subsequent production was lower than the

matched ground controls.

Several ground-based studies of bacterial secondary

metabolite production have been conducted with HARV

bioreactors. In four of these experiments, secondary

metabolite production (of β-lactam antibiotics and

rapamycin) was inhibited by SMG compared to normal

gravity mode of the HARV (Fang et al., 1997a; Fang et

al., 1997c; Fang et al., 2000; Gao et al., 2001). In the fifth

experiment, gramicidin synthesis by Bacillus brevis was

unaffected by SMG (Fang et al., 1997b). However, the

site of antibiotic accumulation shifted from inside to the

outside of the cell, which is beneficial as it facilitates

purification. In addition, secondary metabolite production

in the RWV bioreactors was much lower than production

in shake flasks for all five studies. Demain and Fang

(2001) hypothesized that reduced shear stress in the RWV

bioreactors caused the reduced secondary metabolite

production. In support of this hypothesis, beads were

added to RWV bioreactors to increase shear stress,

resulting in increased production of secondary metabolites

(Fang et al., 2000; Gao et al., 2001). However, bacterial

production of a polymer has been shown to increase for

bacteria grown in a HARV compared to shake flask

cultures (Thiruvenkatam and Scholz, 2000).

In terms of spore production, Mennigmann and Lange

(1986) reported a lower number of spores from B. subtilis

cultures in spaceflight (5×104 spores ml

-1) compared to

ground controls (8×105 spores ml

-1). In a follow on

experiment, Mennigmann and Heise (1994) reported the

opposite trend: the ratio of spores from spaceflight to

ground cultures was 12.64 and the ratio of spores per cell

was higher for flight cultures (0.64) compared to ground

cultures (0.17). A centrifuge was used onboard the Space

Shuttle during this experiment to provide 1 g flight

control, which showed no significant difference in spore

production from 1 g ground control. Launch

accelerations and space radiation were therefore ruled out

as possible causes of altered spore production. Lam et al.

(2002) reported that cultures of Streptomyces plicatus

flown in space for 17 days sporulated profusely when

plated post-flight, but the ground controls lost their ability

to sporulate when similarly plated. Finally, Benoit et al.

(2005) reported that significantly less spores were

recovered from the spaceflight residual viable cultures.



However, the post-flight sporulating ability of the flight

cultures was found to be approximately 8 times that of the

corresponding ground controls. In addition, scanning

electron micrograph images showed that spore

morphology was altered by spaceflight.

CONCLUSION

Spaceflight alters bacteria in several ways, but the

potential threat to humans is not well understood.

Rotating bioreactors such as the clinostat and HARV are

effective at simulating some aspects of microgravity for

bacterial cell cultures. While much remains to be learned,

it is clear already that SMG growth affects bacterial

resistance and virulence both in planktonic and biofilm

mode of growth in ways as to constitute a serious

potential threat to astronaut health. The molecular

mechanisms underlying this phenomenon remain largely

unexplored but initial indications are that novel, as yet

unknown, strategies may be involved. Besides being

potentially harmful, bacteria and their biofilms also have

beneficial uses, for example, in microbial-based

regenerative systems of waste water treatment. These

systems, commonly used on Earth, represent a viable

alternative to physical and chemical methods in use today

on the International Space Station. Long duration

missions planned for travel outside of low Earth orbit may

demand regenerative systems, which are intended to

minimize consumable mass. Thus, continued

investigation of molecular physiology of bacteria under

SMG is necessary to better control their harmful effects as

well as exploit their beneficial roles.

ACKNOWLEDMENT

Work reported from this laboratory was

supported by NASA grant NNA04CC51G.

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Symposium II: Astronaut Health: From

the Bench to Flight Across the Gravity

Continuum

Charles Wade, Editor


EXPLORATION CLASS MISSIONS AND RETURN: EFFECTS ON THE IMMUNE SYSTEM Gerald Sonnenfeld

Division of Research and Department of Biological Sciences

Binghamton University, State University of New York

Binghamton, NY 13902-6000, USA

ABSTRACT

Immune responses have been shown to be altered when humans,

animals and cell cultures are exposed to space flight conditions.

It is clear that, although there have been some issues regarding

infection on short-term space flights, alterations in the immune

system have not been a “show-stopper” for space flight missions

to date. However, as very long-term multi-year exploration

class missions are contemplated, the potential for space flight-

induced alterations of the immune system to have major impact

on crew health and success of the mission increases. The

increased concern is due to exposure to new conditions such as

novel forms of radiation, and the isolation of the crew from the

ability to obtain rapid relief and rescue due to health problems.

If, during space flight, immune capacity is diminished while

bacterial growth and virulence is enhanced, and antibody

efficacy is altered, a difficult situation could arise. If an

ineffective antibiotic is transported for use by the crew, there

will be no replacement readily available during an exploration

class mission. Additionally, radiation exposure combined with a

suppressed immune system could facilitate tumor development

after return to earth. In order to protect crews and assure the

success of exploration class missions, studies must be

undertaken in space, as well as on earth using ground-based

analog models, to fully establish the risk and to develop

countermeasures to prevent or minimized unwanted outcomes.

INTRODUCTION

It is clear that immune responses are dramatically altered

after space flight (Space Studies Board, 1998; Sonnenfeld

et al., 2003). Multiple experiments, including human,

animal and tissues cultures studies, have demonstrated

that a variety of immune responses are altered after

exposure to space flight conditions during actual flight

experiments or during experiments using ground-based

analog models that re-create some of the conditions of

space flight (Space Studies Board, 1998; Sonnenfeld et

al., 2003). Among the immune responses that have been

shown to be affected by space flight conditions are:

leukocyte blastogenesis (division of white blood cells

required to start immune responses), cytokine production,

leukocyte subset analysis, natural killer cell activity and

delayed hypersensitivity skin tests (Space Studies Board,

1998; Sonnenfeld et al., 2003).

Although it is clear that the immune response changes

during and after space flight, the biomedical significance

of these changes is still not fully established (Space

Studies Board, 1998; Sonnenfeld et al., 2003). There

have been reports of changes in resistance to infection

during space flight, with the development of a urinary

tract infection in one case (Space Studies Board, 1998;

Sonnenfeld et al., 2003). In the Apollo program, problems

with development of respiratory tract infections in crews

led to the development of the Crew Health Stabilization

program countermeasure of decreased interaction of

crews with other people on the ground still in use today

(Space Studies Board, 1998; Sonnenfeld et al., 2003).

Additionally, it is now clear that latent viruses, such as

Epstein-Barr virus, are reactivated in space flight crews

(Pierson et al, 2005; Shearer et al., 2005; Stowe et al.,

2001). Studies using the hindlimb unloading model of

rodents some space flight conditions have shown that

hindlimb unloading of mice has resulted in decreased

resistance to infection with bacteria and viruses

(Sonnenfeld et al., 2003). Therefore, the potential for

space flight conditions inducing changes in the immune

systems yielding altered resistance to infection and/or

tumors certainly exists. However, it is also clear that

decreased resistance to infection or tumors has not been a

“show-stopper” to date for the space flight program

(Space Studies Board, 1998; Sonnenfeld et al., 2003). In

almost every case, crews have been able to function

normally without serious incident.

However, we are now facing a new era of space flight, i.e.

the development of exploration class missions.

Exploration of space beyond low earth orbit has the

possibility of changing the conditions of the flight

environment to create greater risk.

THE EXPLORATION CLASS MISSION ISSUES

As we now move towards exploration class missions

beyond low earth orbit, we face new scenarios then what

has been seen before during space flight missions (Table

1). We will face new types of radiation that have never b

been encountered before by humans in space or, if they

have been encountered, humans have never been exposed

to them in space for the length of time they will face in

the future (Space Studies Board, 1996).

Additionally, crews will be away from earth for a more

extended period of time, with limited chances for very

rapid rescue, return or re-supply (Board on Health

Science Policy, 2001). This means a scenario could

develop where if an infection developed and an antibiotic

____________________

* Correspondence to: Gerald Sonnenfeld, PhD

Vice President for Research

Binghamton University

State University of New York

Post Office Box 6000

Binghamton, NY 13902-6000


Phone: 607-777-4818; Fax: 607-777-2188

G. Sonnenfeld — Exploration Class Missions and Return: Effects on the Immune System


was taken to which the organisms was resistant, there

would be limited opportunity to provide a different

antibiotic or to rapidly return the individual to earth for

additional treatment. Additionally there have been

unconfirmed reports of antibiotics not working as

effectively in space as on the ground (Space Studies

Board, 1998).

An additional challenge that may be faced was made clear

by some modeling experiments on earth (Nickerson et al.,

2001, 2004; Wilson et al., 2002). In these experiments,

bacteria were grown in clinostat-derived tissue culture

models that replicated some space flight conditions such

as low shear. These experiments clearly demonstrated

that growth in those conditions altered both the growth

rates of the bacteria and the expression of virulence

factors (Nickerson et al., 2001, 2004; Wilson et al., 2002).

Therefore, a possible scenario could develop where a

bacteria or virus that is part of the normal microbial

components of human microbial flora (that normally does

not cause a problem for the host) could have enhanced

growth and virulence in space, not respond well to the

available antibiotics, and the astronaut host for this

bacteria could have a space-flight impaired immune

response (that would not have a negative effects under

normal conditions). The possibility for a very serious

infectious disease problem that could threaten both the

health of the astronaut and the success of the mission is

obvious!

We must be prepared to both detect and deal with this

problem. There are several steps that should be taken

(Table 2). We must carry out experimentation in

appropriate space flight models on the ground and in

space to fully identify the risk. We must know what

changes happen to infectious agents and to the host

immune system under the conditions we will encounter in

exploration class missions.

We must know how to test whether the host is

compromised. This involves development of standards

for immunological parameters that need to be tested for

astronauts on a routine basis. This testing needs to be

carried out on the ground before flight to establish base

lines and on a regular basis before and after the mission.

We must assure that the standard testing can actually be

carried out in space both on the exploration vehicles and

on the ground on the Moon or Mars. Only then can we

see if the host’s immune defenses are compromised.

We must also assure that we are prepared to deal with

violations of standards induced by space flight conditions.

This would involve understanding the growth and

virulence characteristics of microorganisms that could be

potential pathogens under space flight conditions.

Additionally, there is a great need for testing of antibiotics

and antivirals to ensure that they actually function as they

are designed under space flight conditions. We must also

assure that countermeasure are developed that will

ameliorate or eliminate any negative effects of the space

flight environment on the immune system. In case of

antimicrobials or lack of provision of the appropriate

antimicrobials, be must be sure that the crews are as

immuncompetent as possible throughout their space flight

missions.



CONCLUSION

Only by vigilance and preparedness can we avoid

unexpected deleterious effects of exploration class space

flights on the immune system and, therefore, the health of

the crews. A high level of preparedness will be required

to assure the health and survivals of exploration class

mission crews and the successful completion of

exploration class missions.

AKNOWLEDGEMENTS

Studies funded in the author’s laboratory were funded, in

part, by the National Aeronautics and Space

Administration through NASA Cooperative Agreement

NCC9-58 with the National Space Biomedical Research

Institute.

REFERENCES

Board on Health Sciences Policy, Institute of Medicine.

(2001). Safe passage:Astronaut care for exploratory

missions. National Academies Press, Washington, DC.

Nickerson, C.A., Goodwin, T.J., Terlonge, J., Ott, C.M.,

Buchanan, K.L., Uicker, W.C., Emami, K., LeBlanc,

C.L., Ramamurthy, R., Clarke, M.S., Vanderburg, C.R.,

Hammond, T., Pierson, D.L (2001)Three-dimensional

tissue assemblies: novel models for the study of

Salmonella enterica serovar Typhimurium pathogenesis.

Infect. Immun. 11:7106-7120.

Nickerson, C.A., Ott ,C.M., Wilson, J.W., Ramamurthy,

R., Pierson, D.L. (2004).

Microbial responses to microgravity and other low-shear

environments.

Microbiol. Mol. Biol. Rev. 68:345-361.

Pierson, D.L., Stowe, R.P., Phillips, T.M., Lugg, D.J.,

Mehta, S.K. (2005) Epstein-Barr virus shedding by

astronauts during space flight. Brain Behav. Immun.

19:235-242.

Shearer, W.T., Zhang, S., Reuben, J.M., Lee, B.N., Butel,

J.S.. (2005). Effects of radiation and latent virus on

immune responses in a space flight model.

J. Allergy Clin. Immunol.. 115:1297-1303.

Sonnenfeld, G., Butel, J.S., Shearer, W.T. (2003). Effect

of the space flight environment on the immune system.

Rev. Environ. Health 18:1-18.

Space Studies Board, National Research Council. (1996).

Radiation hazards to crews of interplanetary missions:

Biological issues and Research strategies. National

Academies Press, Washington, DC.

Space Studies Board, National Research Council. (1998).

A strategy for research in space biology and medicine in

the new century. National Academis Press, Washington,

DC.

Stowe, R.P., Mehta, S.K., Ferrando, A.A., Feeback, D.L.,

Pierson, D.L.. (2001). Immune responses and latent

herpesvirus reactivation in spaceflight. Aviat. Space

Environ. Med..72:884-891.

Wilson, J.W., Ott, C.M., Ramamurthy, R., Porwollik, S.,

McClelland, M., Pierson ,D.L., Nickerson, C.A. (2002).

Low-Shear modeled microgravity alters the Salmonella

enterica serovar typhimurium stress response in an RpoS-

independent manner. Appl. Environ. Microbiol. 68:5408-

5416.


NUTRITION, METABOLISM AND THE CRITICAL PATHS: A CRITICAL REVIEW T.P. Stein

Department of Surgery, University of Medicine and Dentistry of New Jersey - SOM, Stratford, NJ, 08084

ABSTRACT

A mission to Mars is estimated to take ~2.5 years. However

there remain some problems which until they are resolved will

limit how long humans can remain away from earth. The

biomedical counter-measures part of the critical paths has

evaluated these risks. They include, bone loss, cardiovascular

effects, muscle loss, nutrition and radiation exposure. Three of

the risks are related to metabolism, namely bone loss, muscle

atrophy and nutrition. Probably the most encouraging aspect of

these problems is that they are known, and because they are

important for human health on the ground there are reasonably

effective treatments available for the ground analogs. However

it still remains to confirm that the actual proposed flight

protocols are effective, first on the ground (with analogs of

flight hardware) and then inflight.

INTRODUCTION

Forty years have elapsed since the first humans first

ventured into space. By any standards the venture has

been successful; humans survive well enough that

manned missions to Mars are being planned. A mission

to Mars is estimated to take ~2.5 years. However there

remain some problems which until they are resolved will

limit how long humans can remain away from earth.

Probably the most encouraging aspect of these problems

is that they are known, and because they are important for

human health on the ground there are reasonably effective

treatments available for the ground analogs.

During the last few years NASA has systematized its

approach to developing a counter-measures program. The

process is the bioastronautics critical paths road map. The

road map lists 50 risks. The document is a living object,

undergoing frequent updates. (For the latest update, see

www.criticalpath.jsc.nasa.gov). The risks have been

divided into three color coded categories; potential show

stoppers (red, R), serious risk of mission impairment

(yellow, Y), and probably controllable (green, G). The

risks have been further sub-divided into 551 ‘critical

questions’. As with the risks, some questions are more

critical than others.

Risks in the biomedical counter-measures part of the

critical paths include, bone loss, cardiovascular effects,

muscle loss, nutrition and radiation exposure. Three of

the risks are related to metabolism, namely bone loss,

muscle atrophy and nutrition. They are the topic of this

review. Table 1 shows an abbreviated version of the

critical paths for the bone, muscle and nutrition

disciplines.

Table 1: Critical paths with metabolic/nutritional aspects.

The objectives of the critical paths road map is to assess

risks for human space exploration and to use this

information for prioritizing research and technology

programs. Defining the questions and targets provides a

means assessing progress towards the reduction and

deciding when the level of risk is low enough to be

acceptable.

BONE The losses are primarily from the weight bearing bones.

Figure 1 shows a summary of some of the available bone

data compiled by A.D. LeBlanc. Note the high degree of

individual variation. There is also much variation in loss

sites. Bone loss is unevenly distributed.

There are two counter measures for bone loss. Firstly

the use of anti-resorptive drugs (e.g. bisphosphonates).

Millions of people already take bisphosphonates as a

treatment for osteoporosis. Bisphosphonates have also

been found to be successful in long duration bed rest

studies (Shackelford et al., 2004). A second treatment for

bone loss is to place load on bone by resistance exercise.

This too has been found to be effective in attenuating the

bone loss during bed rest (Cavanagh, Licate and Rice,

2005; Shackelford et al., 2004). Thus two independent

counter-measures for the bone loss have been found to be

effective in the best ground based model for space flight

(Alkner and Tesch, 2004; Iwase et al., 2004; Shackelford

et al., 2004).

DISCIPLINE ISS RISK (1 yr) ISS

(1 yr)

MOON

(30d)

MARS

(2.5 yr)

Bone Bbone loss and

fracture risk

Y G Y

Bone Impaired fracture

healing

G G R

Bone Injury to joints and

interveterbral

structures

Y Y Y

Bone Renal stone

formation

G G G

Muscle Skeletal muscle

atrophy with

reduced strength

and endurance

G G Y

Muscle Increased

susceptibility to

muscle damage

G G Y

Nutrition Inadequate

nutritional

requirements

G G Y

____________________

* Correspondence to: T.P. Stein, PhD

Department of Surgery

University of Medicine and Dentistry of New Jersey - SOM

Stratford, NJ, 08084


Phone: 609-566-6036

T.P. Stein — Nutrition, Metabolism and the Critical Paths: A Critical Review


% C

HA

NG

E I

N B

ON

E D

EN

SIT

Y

-20

-15

-10

-5

0

5

10

COSMONAUTS ASTRONAUTS BEDREST ALENDRONATE RES. EXERCISE

Figure 1: Bone loss with space flight, bed rest and treatments.

Data supplied by Dr. A.D. LeBlanc, USRA)

The reduction of the bone loss may not be complete or

equally effective for all bones, but judging by the

published data, it is better than 80%. Reducing the bone

loss by a minimum of 80% makes the problem

manageable. Consider a worst case scenario. According

to recent flight data from the ISS, the highest bone loss

rate observed for an astronaut/cosmonaut was ~2% per

month (Lang et al., 2004). Reducing this by 80% reduces

the rate to 0.4% per month. For a 2.5 year round trip this

translates into a maximum loss of 12%. For 6 months ISS

stay the corresponding value is 2.4% which for a worst

case is acceptable. NASA’s management seems to have

bought into this argument. They have decided to give US

astronauts a bisphosphonate (Alendronate) starting in

2007.

Since the number of future US astronauts is going to be

somewhere between 20 and 40 before the shuttle is retired

means there will be little further opportunities to evaluate

any other in-flight measures for long duration missions

between now and 2010. It is not likely that there will be

other long duration opportunities between 2010 and the

start of the Mars expeditions 30 years from now.

Between shuttle and Mars there will be lunar missions.

The lunar missions will be only about 30 days in duration.

It would therefore seem that NASA has made the decision

that the bone ‘counter-measure’ problem is well on the

way to being solved. However it still remains to confirm

that the actual proposed flight protocols are effective, first

on the ground (with analogs of flight hardware) and then

in-flight. If the studies are successful, it would be

appropriate for NASA to descope ‘bone’ and its

associated questions from the critical paths and redirect

resources to other high priority risks.

MUSCLE

Skeletal muscle is plastic, responding to external

workloads. Less work is required during space flight and

bed rest from the anti-gravity muscles because there is no

longer the need to support the body mass. The result is a

reductive remodeling of the anti-gravity muscles to adapt

to the reduced work-load (Grigoriev and Egorov, 1992;

Thornton and Rummel, 1977).

In addition to a net loss of protein, there is a shift in

myosin isoforms from slow to fast isoforms (Fitts, Riley

and Widrick, 2000). Fast twitch fibers are primarily

glycolytic and prone to fatigue with endurance. This

metabolic shift towards increased reliance on glycolysis is

found with space flight (Baldwin, Herrick, and McCue,

1993), the rat hind limb suspension model (Fitts, Riley

and Widrick, 2000; Henriksen and Tischler, 1988;

Langfort et al., 1997) and bed rest (Acheson, et al., 1995).

Glycolysis is very effective for high intensity short

duration acute activities, but if sustained output is needed,

an energy profile where fat use is favored is desirable. An

inability to sustain work output is a major cause for

concern. Astronauts need to maintain as much functional

capacity as possible during space flight for extra-

vehicular activities (space station construction and

maintenance, emergency egress and eventually exploring

Mars. These are all functions that require sustained work

output. Less protein to do the work and increased

fatigability in this context are counter-productive.

A recent Soyuz landing clearly illustrated the seriousness

of this problem. After a 5½-month flight, the Soyuz

landed somewhere in Kazakhstan. It took more than 5 h

to recover the crew. For safety reasons the crew had to

get out of the landing capsule unassisted. It took them

five hours to accomplish what should have been a half

hour task. The misadventure provides strong evidence for

impaired muscle functionality after Mars-like transit,

demonstrating serious weaknesses in crew performance.

All three crew members exhibited reduced capability, up

to voluntary immobility. More work is needed on the

muscle atrophy problem. Because of the limitations of

the space craft environment it may not be possible to

totally prevent the reductive remodeling of skeletal

muscle, but certain functional capacities must be retained.

NASA has defined what these capabilities are for a

successful Mars mission. They include the need for

dexterity, essential for manipulating tools and performing

complex actions; examples given by NASA are deploying

solar arrays and erecting a habit; good hand eye

coordination is needed for driving the mission exploration

vehicle and for teleoperating robotic aides. There is of

course a need for maintaining strength, flexibility, agility

for such essential tasks as putting on and taking off the

pressure suits and most importantly the ability to move

away from danger should the situation merit. All of these

functions are well met by maintaining a similar level of

physical fitness as pre-flight.

There is no longer any doubt that exercise is effective in

ground based models for preventing disuse muscle

atrophy (Bamman et al., 1998; Ferrando, et al., 1997;

Shackelford et al., 2004). However the results have not

been replicated in flight – beginning with Skylab and

continuing through shuttle, MIR and the ISS. Indeed,



exercise has been part of US and Russian missions for

more than 30 years; yet the problem persists (Cena,

Sculati and Roggi, 2003; Smith, et al., 2005; Stein, 2001).

In the first 10 ISS missions full functionality of the

exercise equipment has been less than 50% and this

together with the inadequate dietary intake accounts for

the unsatisfactory results with the current in-flight

exercise regimens (Smith, et al., 2005, Stein, 2001).

ENERGY BALANCE

Energy deficits have been found on most, but not all short

duration shuttle missions and has been a consistent

finding with long duration missions (Rambaut, Leach and

Leonard, 1977; Rambaut, et al., 1977). The negative

energy balance is a mission related effect on dietary

intake and not a specific response to the absence of

gravity. Dietary intake is mission dependent rather than

subject dependent (figure 2, (Stein, 2001, Stein, 2000))

Astronauts on the same mission appear to eat about the

same amount of food (figure 2).

DAYS

EN

ER

GY

IN

TA

KE

(kca

l. k

g-1

.d-1

)

0

10

20

30

40

50

LMS

SLS1/2

SKYLAB 2

SKYLAB 3

SKYLAB 4

5 10 15

Figure 2: Comparison of energy intake during the first two

weeks of space flight for the 3 Skylab missions, shuttle SLS1/2

and shuttle LMS (Stein, 2001).

Elsewhere, we have suggested that the inverse

relationship between exercise and energy intake is due to

problems in disposing of the metabolic by-products from

exercise, namely heat and CO2 (Stein, 2000). Thermo-

regulatory mechanisms are less efficient during space

flight and this persists into the immediate post flight

period (Acheson, et al., 1995; Fortney et al., 1998). Heat

disposal has been shown to depress intake in both rats

(Llamas-Lamas and Combs, 1990) and humans (Edholn,

Fox, Goldsmith et al., 1964).

A negative energy balance is not an appropriate

physiologic state when the need is to counter-act a net

muscle catabolic state. Proteins are the 'machinery' of the

body. All of the metabolic functions in the body, from

cell division to obtaining energy from foodstuffs to host

defense mechanisms to doing muscle work involve

proteins. So it is important to prevent the breakdown of

body protein for use as an energy source.

A similar degree of weight loss together with poor intake

has been found with ISS astronauts. The same degree of

poor intake and weight loss (4.4 + 0.1 kg, n=6) that was

found on MIR (Stein et al., 1999) is now being found with

ISS crews (3.8 + 0.1 kg, n=24, personal communication,

S.M. Smith, NASA and (Smith, et al., 2005)).

DAYS IN ORBIT

0 50 100 150 200%

BO

DY

WT

LO

SS

-14

-12

-10

-8

-6

-4

-2

0

2

4

6

Figure 3: Weight change with flight on the ISS (Smith, 2005).

There is great variation on the magnitude of the weight

loss. It is not known whether the weight loss is

continuous or whether an adaptation/accommodative state

is achieved. Figure 3 shows data from the ISS (Smith, et

al., 2005). The lines represent individual weight changes.

The two heavy black lines represent least squares

regression analysis of the data with either a linear or

curvilinear fit. The curve fits suggests that a steady state

is reached after about 100 days. The linear regression

suggests that the weight loss is continuous. Both

regressions are statistically significant. Which is correct?

If the weight loss is continuous, then the problem is very

serious, chronic weight loss is unsustainable over the 2.5

yr period it will take to go to Mars. On the other hand,

establishment of an adapted state after ~100 days may be

acceptable. This question needs to be addressed.

As yet there is no counter-measure being evaluated for the

inadequate intake. We would like to suggest one.

Weekly measurement of body mass with dietary

consultation with the ground support team. In contrast to

dietary records, measurement of body mass is a real

measurement and can be directly related to energy

balance status. Astronauts losing weight inflight should

be encouraged to eat more. Body mass devices have been

flown on many prior missions beginning with Skylab and

including shuttle.



REFERENCES

Acheson KJ, Decombaz J, Piguet-Welsch C, Montigon F,

Decarli B, Bartholdi I, and Fern EB. Energy, protein, and

substrate metabolism in simu-lated microgravity. Am J

Physiol (Regul and Integ) 269: R252-260, 1995.

Alkner BA and Tesch PA. Efficacy of a gravity-

independent resistance exercise device as a

countermeasure to muscle atrophy during 29-day bed rest.

Acta Physiol Scand 181: 345-357, 2004.

Baldwin KM, Herrick RE, and McCue SA. Substrate

oxidation capacity in rodent skeletal muscle: effects of

exposure to zero gravity. J Appl Physiol 75: 2466-2470,

1993.

Bamman MM, Clarke MS, Feeback DL, Talmadge RJ,

Stevens BR, Lieberman SA, and Greenisen MC. Impact

of resistance exercise during bed rest on skeletal muscle

sarcopenia and myosin isoform distribution. J Appl

Physiol 84: 157-163., 1998.

Cavanagh PR, Licate AA, and Rice AJ. Exercise and

pharmacological countermeasures for bone loss during

long duration space flight. Gravitational and Space

Biology Bulletin 18: 39-58, 2005.

Cena H, Sculati M, and Roggi C. Nutritional concerns and

possible countermeasures to nutritional issues related to

space flight. Eur J Nutr 42: 99-110, 2003.

Edholn O, Fox GRH, Goldsmith IFG, and al. e. Report to

the MRC, No. APRc64/C65. London, Army personnel

research Committee, 1964. London: HMSO, 1964.

Ferrando AA, Tipton KD, Bamman MM, and Wolfe RR.

Resistance exercise maintains skeletal muscle protein

synthesis during bed rest. J Appl Physiol 82: 807-810,

1997.

Fitts RH, Riley DR, and Widrick JJ. Physiology of a

microgravity environment. J Appl Physiol 89: 823-839.,

2000.

Fortney SM, Mikhaylov V, Lee SM, Kobzev Y, Gonzalez

RR, and Greenleaf JE. Body temperature and

thermoregulation during submaximal exercise after 115-

day spaceflight. Aviat Space Environ Med 69: 137-141,

1998.

Grigoriev AI and Egorov AD. Physiological aspects of

adaptation of main human body systems during and after

spaceflights. Advances in Space Biology & Medicine 2:

43-82, 1992.

Henriksen EJ and Tischler ME. Glucose uptake in rat

soleus: effect of acute unloading and subsequent

reloading. J Appl Physiol 64: 1428-1432., 1988.

Iwase S, Takada H, Watanabe Y, Ishida K, Akima H,

Katayama K, Iwase M, Hirayanagi K, Shiozawa T,

Hamaoka T, Masuo Y, and Custaud MA. Effect of

centrifuge-induced artificial gravity and ergo-metric

exercise on cardiovascular deconditioning, myatrophy, &

osteoporosis induced by a -6 degrees head-down bedrest.

J. Grav. Physiol: 11: P243-244, 2004.

Lang T, LeBlanc A, Evans H, Lu Y, Genant H, and Yu A.

Cortical and trabecular bone mineral loss from the spine

and hip in long-duration spaceflight. Journal of Bone &

Mineral Research 19: 1006-1012, 2004.

Langfort J, Zernicka E, Mayet-Sornay MH, Dubaniewicz

A, and Desplanches D. Effects of acute and chronic

hindlimb suspension on sensitivity and responsiveness to

insulin in the rat soleus muscle. Biochem Cell Biol 75:

41-44, 1997.

Llamas-Lamas G and Combs DK. Effects of

environmental temperature and ammoniation on

utilization of straw by sheep. J Anim Sci 68: 1719-1725,

1990.

Rambaut PC, Leach CS, and Leonard JI. Observations in

energy balance in man during spaceflight. Am J Physiol

(Endo and Metab) 233: R208-212, 1977.

Rambaut PC, Smith MC, Jr., Leach CS, Whedon GD, and

Reid J. Nutrition and responses to zero gravity. Fed Proc

36: 1678-1682, 1977.

Shackelford LC, LeBlanc AD, Driscoll TB, Evans HJ,

Rianon NJ, Smith SM, Spector E, Feeback DL, and Lai

D. Resistance exercise as a countermeasure to disuse-

induced bone loss. J Appl Physiol 97: 119-129, 2004.

Smith SM, Zwart SR, Block G, Rice BL, and Davis-Street

JE. The nutritional status of astronauts is altered after

long-term space flight aboard the International Space

Station. J Nutr 135: 437-443, 2005.

Stein TP. Nutrition in the space station era. Nutr Res Rev

14: 87-114, 2001.

Stein TP. The relationship between dietary intake,

exercise, energy balance and the space craft environment.

Pflugers Arch 441: R21-31., 2000.

Stein TP, Leskiw MJ, Schluter MD, Donaldson MR, and

Larina I. Protein kinetics during and after long term space

flight on MIR. Am J Physiol (Endo and Metab) 276:

E1014-1012, 1999.

Thornton W and Rummel J. Muscular deconditioning and

its prevention in space flight. Biomedical results from

Skylab (NASA SP-377) US Government Printing Office,

Washington, DC: 191-197, 1977.


TESTING EXERCISE COUNTERMEASURES DURING 30 DAYS OF SIMULATED

MICROGRAVITY: LESSONS LEARNED FROM STUDIES OF IDENTICAL TWINS

Alan R. Hargens, Brandon R. Macias, Cece M. Echon, Eva Brzezinski, Andrea Hawkins, Kristina Hawkins, and

R. Scott Meyer

Department of Orthopaedic Surgery, University of California, San Diego, San Diego, CA 92103-8894

ABSTRACT

This paper reviews our experience with 30-day head-down tilt

(HDT) bed rest studies of supine lower body negative pressure

(LBNP) treadmill exercise countermeasure in identical twins.

We review our experience with 16 sets of identical male and

females twins in order to improve future countermeasure studies

for long-duration space flight. All male sets of twins and all but

one female set tolerated the study well and no medical

complications occurred. Our experimental design and use of

identical twins may represent an innovative method for

evaluating the physiologic efficacy of a treatment when

comparing an exercise versus control group. Also, our

nutritional procedures maintained body weight in both groups.

Pre- and post-bed rest tests were scheduled to avoid

confounding effects due to test order and test volume. As

reported elsewhere, our treadmill exercise protocol within LBNP

maintained plasma volume, orthostatic responses, upright

exercise capacity, muscle strength and endurance during bed

rest. Future bed rest studies should focus on the recruitment of

healthy, non-sedentary subjects and may want to follow our

recommendations in terms of screening, testing sequence,

nutrition, medical care and subject support. Identical twins offer

unique insights into countermeasure development and

heritability of physiologic traits.

KEY WORDS

Bed rest, recruitment, testing sequence, nutrition, identical

twins

INTRODUCTION

Microgravity leads to cardiovascular deconditioning in

humans, which is manifested by post-flight reduction of

orthostatic tolerance and upright exercise capacity

(Watenpaugh and Hargens, 1996). During upright posture

on Earth, blood pressures are greater in the feet than at

heart or head levels due to gravity’s effects on columns of

blood in the body (Hargens et al., 1992). Upon exposure

to microgravity, all gravitational blood pressures

disappear. Presently, there is no exercise hardware

available for space flight to provide this gravitational

blood pressure to tissues of the lower body.

Lower-body musculoskeletal loss is experienced by crew

exposed to long-term space flight (Arnaud and Morey-

Holton, 1990; LeBlanc et al., 1996; Morey-Holton et al.,

1996; Schneider et al., 1989; Thornton and Rummel,

1977; Whedon et al., 1977). Although physical activity is

considered the best way to protect bones, muscles and the

cardiovascular system, exercise protocols and equipment

for simulated and actual microgravity are still unresolved

(Convertino, 1990). For example, studies of simulated

microgravity by Vernikos and associates (1996) indicate

that standing for four hours completely prevents and

standing for two hours partially prevents decreases in

post-bed rest orthostatic intolerance. Walking for four

hours prevents and walking for two hours attenuates

decreases in peak oxygen uptake compared to non-

exercise controls. They conclude that various

physiological systems benefit differentially from passive

gravity/activity in one g. Moreover, in addition to the

duration of the stimulus, the number of exposures to

upright posture are important as well (Vernikos et al.,

1996).

Recent calculations suggest that all exercise in space to

date has lacked sufficient loads to maintain pre-flight

bone mass (Cavanaugh et al., 1992, Whalen et al., 1988).

Although treadmill exercise with bungee cords (about 2 hr

per day) was the most common exercise for cosmonauts

during long-duration Mir missions, biomechanical loads

on musculoskeletal tissues of the lower body are only

about 60-70% of those present on Earth (Whalen, 1993).

One example of the seeming lack of exercise

effectiveness is that three Mir 18 crewmembers varied

widely in their amounts of exercise performed, while their

post-flight muscle volumes (LeBlanc et al., 1996) and

bone resorption (Smith et al., 1999) were nearly identical.

Theoretically, an integrated countermeasure for extended

exposure to microgravity should combine high loads on

the musculoskeletal system (Whalen et al., 1988) with

normal regional distributions of transmural pressure

across blood vessels (Hargens et al., 1992) and

stimulation of normal neuromuscular locomotor patterns.

We have postulated that LBNP treadmill exercise may

prevent bed rest- and microgravity-induced

deconditioning by simulating gravity effects. Static

ground reaction force (GRF) in a LBNP chamber is a

product of the body cross-sectional area at the waist seal

(Axy) and the pressure differential between the external

ambient and internal chamber environments (∆P), where

∆P = LBNP: GRF = Axy × ∆P. For the average male

subject, an additional GRF of about one equivalent body

weight (BW) is generated for each 100 mmHg of LBNP

when the negative pressure acts only through the cross-

sectional area of the subject’s waist (Hargens et al., 1991).

____________________

* Correspondence to: Alan R. Hargens, PhD

Department of Orthopaedic Surgery

University of California, San Diego

350 Dickinson St. San Diego, CA 92103-8894


Phone: 619-543-6805; Fax: 619-543-2540

A. Hargens — Testing Exercise Countermeasures during 30 Days of Simulated Microgravity


The LBNP exercise concept avoids the discomfort of

localized high pressures typical of bungee cord harness

systems by distributing the net force of the air pressure

uniformly over the entire upper surface of the body. By

expanding the area through which the pressure produces

force, we found that the amount of negative pressure

required to generate one BW could be decreased. If the

waist seal area equals twice the subject’s waist cross-

sectional area, the negative pressure necessary to produce

one BW decreases from 100 mmHg to 50-55 mm Hg

(Watenpaugh et al., 1994). The reduced negative pressure

required to generate one (BW) of force lowers the risk of

excessive heart rate, footward fluid redistribution,

syncope, hernia, and petechiae associated with higher

levels of LBNP (Wolthius et al., 1974).

In our recent studies of identical twins exposed to 30 days

of bed rest and reported elsewhere (Cao et al., 2005;

Hargens et al., 2003; Macias et al., 2005; Monga et al.,

2006a,b; Smith et al., 2003), we hypothesized that 40

minutes of supine treadmill running per day in a LBNP

chamber at 1.0 to 1.2 BW (approximately 50-60 mmHg

LBNP) with a 5-minute resting, non-exercise LBNP

exposure at 50 mmHg after the exercise session maintains

aerobic fitness, orthostatic tolerance, and musculoskeletal

structure and function during 30 days of bed rest

(simulated microgravity). The purpose of the present

paper is to review our experience with 15 sets of identical

male and females twins in order to improve future

countermeasure studies for long-duration space flight.

EXPERIMENTAL DESIGN

To initiate the project, we proposed to study 16 sets of

identical, female and male twins following a thorough

medical examination to ensure their suitability for a safe

and well-controlled study. Acceptable subjects were

thoroughly briefed by the investigating team and provided

informed, written consent before participation in this

study.

Identical twin sets were randomly divided into two groups

to investigate the mechanism of action and efficacy of our

partial vacuum exerciser concept. These 30-day bed rest

studies were chosen to approximate longer-term

microgravity exposures. Our previous shorter-term bed

rest studies (5 and 15 days) achieved significant results

with seven subjects (Lee et al., 1997, Watenpaugh et al.,

2000). By coin toss selection, one twin exercised while

their sibling was a non-exercise control subject. The

exercising twin ran in the supine LBNP chamber for 40

minutes plus a 5-minute static LBNP period. They

exercised once a day at 1.0 to 1.2 BW of footward force,

while their sibling was a non-exercise “control” subject.

The interval exercise protocol was similar to that

employed by us in our previous two week studies: 7 min

warm-up at 40% peak oxygen uptake, followed by 3 min

at 60%, 2 min at 40%, 3 min at 70%, 2 min at 50%, 3 min

at 80%, 2 min at 60%, 3 min at 80%, 2 min at 50%, 3 min

at 70%, 2 min at 40%, 3 min at 60%, and 5 min cool-

down at 40% peak oxygen uptake (40 min total) with an

additional 5 min of supine, stationary exposure to 50

mmHg LBNP (Greenleaf et al., 1989; Lee et al., 1997).

Exercise bouts occurred at the same time of day for each

subject in the exercise group. During bed rest, the LBNP

treadmill exercise was undertaken six days per week with

Sundays allowed for rest. All exercise subjects were able

to carry out this program unless exercise was too

uncomfortable due to menstrual cramps, illness, muscle

soreness, knee pain, or initial adaptation to the suspension

system. In these cases, slower treadmill speeds or as a last

resort, lower levels of LBNP, were instituted.

A 3-day orientation period occurred prior to the start of

bed rest to acquaint subjects with our facilities, personnel,

procedures and experiments, including orthostatic

tolerance, muscle strength and exercise capacity tests. The

first phase of the bed rest study (including 6 days of

baseline testing, orientation, familiarization, 30 days of

bed rest, and 3 days of recovery) began in mid-1999. All

subjects reported to the UCSD General Clinical Research

Center (GCRC) on their designated start day. Subjects

were usually discharged from the GCRC 39 days later.

In the baseline control period, activity levels were

maintained by each subject, and ambulatory levels of

plasma and urinary markers of bone loss were measured

on the three days prior to bed rest. All physiologic tests

took place at the same time of day for a given subject.

These tests were staggered so that sufficient time was

allowed to complete each procedure and to prevent

confounding effects of one test on another test. While the

subjects lived at the GCRC, their daily sodium intake was

controlled at approximately 170 mEq (3.5 g per day) and

their diet also was controlled (approximately 2500-3000

kcal per day, depending on exercise level). Their body

weight, fluid intake, and urine output were monitored on a

daily basis. We expected that subjects would maintain a

neutral or positive fluid balance and neutral energy

balance so that they would not lose weight during 30 days

HDT. During the entire period of bed rest, all subjects

were required to remain in 6° HDT except during periods

for bath/shower and exercise (0.5-1.5 h/day), when they

were horizontal (0°). Compliance to this strict bed rest

protocol was required of all subjects so that the scientific

integrity of the results could be maintained.

In the baseline control period, the Hamilton Depression

Rating Scale and Beck Depression Inventory were

administered to each subject to assess any pre-study

depressive symptoms. These measures were re-

administered during the last week of the bed rest period.

Prior to initiation of HDT, plasma volume, leg and spinal

muscle strength, Dual Energy X-ray Absorptiometry

(DXA), spine Magnetic Resonance Imaging (MRI), heart

muscle mass, arm venous pressure, and plasma and

urinary bone markers were measured in all subjects

(Table 1). On the day before initiation of HDT, all

subjects were tested chronologically for orthostatic

tolerance, sprint speed, postural stability, gait parameters,

and upright peak oxygen uptake. By using this order of



tests, we think we were able to avoid possible

confounding effects of one test on a subsequent test. For

example, the plasma volume and orthostatic tolerance

tests were scheduled before peak upright exercise

capacity to eliminate the effect of elevated plasma volume

due to exercise on peak upright exercise capacity. During

the recovery period these tests were repeated in the exact

same sequence within 4 hr of return to upright posture to

maintain comparability. We were usually able to test

subjects at a rate of four per day in alphabetic order with

tests on one set of twins undertaken in the morning and on

a second set of twins in the afternoon. This was important

so that subjects had time to rest, eat and digest their

meals, adequate time for setup and troubleshooting

equipment was allowed and we could finish testing of

subjects before dinner.

An attempt was made to stagger the subjects with respect

to time of HDT initiation and return to upright posture to

allow investigators to perform all tests without delays.

Subjects were active and took controlled walks during

pre-bed rest and recovery days to avoid the well-known

deconditioning due to confinement and inactivity.

Experimental stations for pre- and post-bed rest tests

included: 1) bedside plasma volume early in morning, 2)

orthostatic tolerance, 3) sprint speed, 4) posture and

balance time, and 5) posture/gait analysis and peak

upright exercise capacity. There also was a room

specifically set aside for LBNP exercise. We provided

TVs, VCR players, DVD players, a computer room with

internet access, bed-side phone, and we allowed cell

phones in order to help prevent boredom. We found the

volume and frequency of tests represented an optimized

compromise between an insufficient number of

physiologic tests and too many tests that would

overburden and psychologically stress our volunteers. For

example, none of our subjects quit before or after any of

our tests, even though our testing protocol was time

consuming and physically demanding over the three day

periods before and after bed rest.

Typically, three full days were needed to set-up

equipment. We had three separate testing rooms: an

exercise room, an orthostatic tolerance testing room and

the upright treadmill testing room for peak V02. We

performed sprint testing in a hallway of the hospital that

was manned by many research and GCRC personnel to

maintain safety. However, some twins fell down during

their sprint test but none suffered injury. All subjects were

able to sprint a second time.

Two staff members were needed to operate the LBNP

exerciser. It took about 15-20 minutes to setup the subject

for exercise. Each exercising subject was transferred from

their bed to an HDT gurney and from the gurney to the

LBNP exercise apparatus. Subjects brought along their

drinking bottles and urinals to the exercise session. One

staff member applied leg straps to the subject, while the

other staff member prepared the waist seal, shoulder

straps, treadmill and heart rate monitor. One staff member

controlled the negative pressure in the chamber and

treadmill speed, while the second staff member recorded

time, HR and perceived exertion data. The second staff

member was always in immediate contact with the

subject. Also, we provided water in a container with a

flexible straw so they could drink in supine position. It

was also required to have two people in the room at all

times in case of an emergency, one to attend to the subject

and the other to call for help. An orthopaedic surgery

resident from our Department at UCSD was on call for

the duration of the study.

Scheduling tests and making final equipment check-ups

occurred one month in advance. Some of our testing was

offsite. We rented a UCSD vehicle to transport subjects so

that all subjects were covered by UCSD insurance. Our

bed rest project involved collaboration with many US and

international investigators and required data management

and communication to occur in a timely manner.

Frequently, for example, exercise logs of HR were

transformed into a .pdf file and transmitted electronically

to a co-investigator reviewing exercise work loads. Also,

scanning documents into .pdf files allowed for immediate

review of data by national and international collaborators,

and it allowed for a back-up system of data storage.

Scheduling of tests supported by the UCSD Medical

Center (plasma volume, MRI, echocardiography) required

an appointment at least one month in advance. This was

an issue because we needed to test four subjects

concurrently which required large blocks of clinical time

to be set aside.

LESSONS LEARNED FROM RECRUITMENT OF

TWINS

Twinsworld.com, an internet newsletter, was the best

source for recruiting twins into our study. Another source

of twins existed in the lab of an investigator at UCSD

who performs research on twins. This investigator agreed

to mail out a flyer about our study to his previous

subjects. This was helpful in recruiting two sets of local

twins, in case twins outside the San Diego area failed to

report or were subsequently disqualified. Recruitment was

a time consuming and challenging effort. To save time we

asked twins to respond to some questions for screening

purposes; for example, age, history of smoking and

exercise, fitness, weight and height to calculate BMI, and

previous absence from home for 40 days. If twins passed

these initial screening questions, we then explained our

study in detail. If they were still interested, we obtained

all of their contact information (phone number, cell phone

number, mail address, email address, parent’s phone

number) and mailed them our consent form and a video of

previous subjects exercising in the LBNP chamber. A

week later we called to follow up if they were still

interested in our study. Also, we arranged a

teleconference with both twins to answer specific

questions about bed rest, testing and exercise. Most twins

were concerned about gaining weight, using the bed pan

and monetary payment. Many twins could not or did not

want to participate because of job or family commitments.

The older twins expressed interest, but their established



Table 1. Daily order of events

Day Procedure Time required

Familiarization day 1 Report to GCRC, check in 15 min

Orthostatic tolerance 90 min

Peak upright exercise capacity 60 min

Noninvasive Intracranial Pressure (ICP) 30 min

Familiarization day 2 Muscle strength 90 min

LBNP exercise for selected twin 90 min

Familiarization day 3 Plasma volumes and hematocrit 30 min

Regional arterial compliance 15 min

Echocardiography 60 min

Control day 1 Serum and 24 hr urine collection 30 min

Discomfort questionnaire 15 min


Sprint speed 10 min

Posture and balance analysis 20 min




DXA 60 min

Muscle strength 90 min

Regional arterial compliance 15 mm

Sleep quality 9 hr



Spinal compression and heart mass 60 min

Hamilton and Beck tests 30 min

Sleep quality 9 hr

Bed rest day 1 Begin bed rest


Bed rest (all days) Discomfort questionnaire 15 min

Bed rest Exercise for one twin 90 min

(all days except 7, 14, 21, 28, and 30)

Bed rest 4,11,18,25 Noninvasive ICP 30 min

Transcranial Doppler (TCD) 30 min

Bed rest 5,12,19,26 Serum and 24 hr urine collection 30 min

Bed rest 17/18, 26/27 Sleep quality 9 hr during sleep

Bed rest 14 Regional arterial compliance 15 min

Bed rest 24 Hamilton and Beck test 30 min

Bed rest 28 Spinal compression and heart mass 60 min

Bed rest 30 Plasma volume and hematocrit 30 min

Echocardiography 60 min

Regional arterial compliance 15 min

Recovery day 1 Serum and 24 hr urine collection 30 min



Sprint speed 10 min

Posture and balance analysis 20 min


Recovery day 2 Serum and 24 hr urine collection 15 min


DXA 60 min

Muscle strength 90 min

Recovery day 3 Discomfort questionnaire 15 min

Ambulation, discharge from GCRC 60 min



careers and family obligations usually prevented us from

recruiting them. However, younger twins, especially those

in college, had the time during the summer and were

usually without family and work commitments. We relied

heavily on the twins’ answers to our questions because we

could not support their travel to UCSD for selection

purposes. Only one set of twins could not participate

because of their extremely low fitness level, i.e., they

were unable to jog. Otherwise, all the other twins who

traveled to UCSD for the study were healthy and

reasonably fit candidates. Typically we flew twins to San

Diego one day before familiarization day 1. We had a list

of items we asked the twins to bring: black exercise shorts

for gait studies, comfortable running shoes and socks,

running shorts and t-shirts, toiletries, clothes for 2 weeks,

bathroom air freshener, videotapes and DVDs, music,

books, and a camera. All subjects were encouraged to

have a personal goal during their stay at UCSD. Affiliated

Genetics performed zygosity tests to verify that the twin

sets were monozygotic. The test kit was sent by mail and

the company provided results in a timely manner.

We had an orientation the afternoon all twins arrived in

order to review the consent form, all protocol risks and so

they could meet key research, medical and GCRC

personnel. Then twins were asked to sign both the UCSD

and NASA Johnson Space Center (and initially NASA

ARC) consent forms. After consenting to the study, the

twins were admitted to the UCSD GCRC and history and

physical exams were performed. It typically took one

week for the exercising subject to adjust and attain their

prescribed exercise protocol speeds. The twins liked to

watch TV and/or listen to music during the LBNP

exercise session. It was important to make sure that the

twins had eaten and used the bed pan and/or urinal prior

to exercise. Also, the twins brought their graduated water

container to each exercise session.

LESSONS LEARNED FROM CARE OF TWINS IN

THE UCSD GCRC

Conducting a clinical research study on the effects of

microgravity in the GCRC was interesting and

challenging from the Nurse Manager’s standpoint. It

required weeks of planning and preparation that consisted

of the following: 1) assessment of physical and staff

resources, 2) procedural preparation, including label and

tube set-up, 3) coordination of activities that required

accurate and frequent communication, 4) collection and

management of data, 5) management of subject care, and

6) management of staff, investigators’ and subjects’ issues

and concerns.

Physical Resources:

For the nurse manager, identifying a space to house large,

heavy equipment such as the LBNP exercise chamber and

treadmill apparatus required departmental and

administrative approvals from the Medical Center CEO

and from the Director of Patient Care Services. In

addition, locating a comfortable and functioning bed scale

was also a challenge. An accurate bed scale is essential to

measure daily weights of all twins so that diets can be

modified to maintain constant body weight. Usually, bed

scales were assigned to critical areas in the hospital such

as the ICUs. Careful negotiation with our Clinical

Engineering Department provided the four bed scales

needed upon admission of each two sets of twins. The bed

scales required fine-tuning and calibrations at repeated

intervals prior to use.

The GCRC is often not equipped with resources suitable

to conduct bed rest studies for an extended period of forty

days. One example is the shower room. It is located on

the 5th

floor of the hospital in the Burn Unit. The

availability of the shower room depended on the schedule

for the burn patients. The shower was not readily

accessible for our twins after the 40-minute daily exercise

Monday through Saturday. The twins were hot and

sweaty and this situation sometimes resulted in

inconvenience and annoyance. Twins were transported

within the hospital via gurney with HDT to maintain the

head-down posture for all subjects. Other resources

needed for the study and located outside the hospital were

the MRI Facility and the bone density (DXA) scanner.

The transport of patients to these outside facilities became

an issue and required the involvement of our Risk

Management Office due to liability issues. A waiver

allowed use of a UCSD van by the research personnel

versus a more expensive ambulance. A research-

designated MRI facility and DXA scanner would be

optimal to prevent scheduling conflicts and liability

issues.

Staffing Resources:

The staffing patterns were adjusted to meet the needs of

the study. Experimental volunteers on bed rest are

considered “total care” subjects; hence contract labor was

utilized to augment the staffing for the evening and night

shifts.

Procedural Preparation:

Preparation of computerized labels, tube set-up (185

tubes/per twin), charts, log sheets, pain questionnaires,

physician’s orders for the entire 40-day admission for two

to three sets of twins required two full-time nurses. These

two research nurses worked together and coordinated the

daily care and schedule of the GCRC staff. They updated

the entire staff including the dietitians and sleep

technicians on a daily basis. Communication among the

staff and research team was extremely critical in

preventing scheduling dilemmas. Communication with

the twins was equally critical to maintain compliance and

positive attitude with the study. Often the twins wanted a

set schedule and to know at least a day in advance when a

particular test would occur during the bed rest period.

Therefore, the subjects were provided summarized testing

schedules. For example, our twins preferred to watch TV

programs and if they were involved in a test, they

preferred to record the TV show on video.

Data Collection and Management:

Data collection and management required extreme



attention to detail due to the volume generated during

each procedure time. Correct tube labeling with the twin’s

identifier, date, time, and type of sample was checked

carefully and matched with the log sheet. The

computerized labeling of sample tubes was efficient and

clear, but became problematic when it was discovered

that the label used was not intended for long-term storage.

Dry ice delivery and supply was increased by two-fold to

maintain the quality and integrity of the samples.

Frequent auditing and monitoring of the data sheets

helped achieve accuracy, quality and completeness. Dry

ice was needed to maintain urine and serum samples at

−79° Celsius for sample storage within the hospital and

for shipment via overnight air mail. Additionally, labeling

the samples at times was an issue because the ink faded.

This required the GCRC to purchase a labeling system

with special ink for dry ice storage samples.

Management of Care for Our Twin Subjects:

Primary care was the model used for patient care. Ideally

two RNs were in charge of the total care for the twins.

However, with the GCRC being small and understaffed,

the care was divided among the staff on the day shift

during the 40-day admission period. The first day began

with the orientation of the twins to the unit’s routine

which included the following:

• Meal time

• Bedtime

• Phone system

• Entertainment

• Massage therapist

• Volunteer services (haircut, manicure, and pedicure)

• Chaplain services

• Open door policy to monitor subject well-being and

compliance to strict HDT

• Visiting hours

• Sleep studies

• Protocol for obtaining daily metabolic weight by the

night nurse

• Daily strict intake and output measurements

• Availability of the internet access

• Laundry services

The maintenance of strict HDT bed rest was emphasized

and all orientation sessions with the volunteers,

investigators and staff ended with a question and answer

period. With this orientation process, many of the twins

had difficulty remembering the open door policy. This

was a challenge with which the nurses were faced with

quite frequently. As one staff member stated, “I have an

issue with trust in caring for the twins.” There were a few

sets of twins who wanted their door closed at all times. A

one-way window or a camera in the room may be a good

addition in designing a bed rest study room in the future

to be able to monitor subject well-being and compliance

to HDT more effectively.

During the first week of the bed rest study, the biggest

dilemma for the female twins was adapting to the female

urinal and bedpan, while remaining in the required HDT

posture. Some twins had so much difficulty that they

would end up in tears out of frustration. Some would even

“hold it” for as long as they could to avoid using the

bedpan which sometimes resulted in being constipated.

This was probably the biggest complaint of the twins.

There is a need to design a practical “female urinal” and a

bed that can convert into a commode while maintaining

the HDT position. One possible solution may be a bed

with a hole in the middle such a “cholera cot.”

Because each of the twin pairs was in the same room for

almost 24 hours a day for 40 days, some twins developed

“cabin fever” during which they would constantly bicker

and sometimes throw objects at each other. In one

instance, one twin fell off the bed during a heated

argument with their sibling. The personality of the twins

played a major role in the degree of ease for their care.

Those who were young, gregarious, and college students

were easy to care for and had an easier time fulfilling the

requirements of the study. The staff spent more time in

the room with them compared to those who had a litany

of complaints whenever they were visited by the staff.

Unfortunately, because of being on bed rest, the twins

spent a majority of the time in their rooms, so trying to

keep them entertained was also difficult. On some days,

twins became so bored with the monotonous daily routine

that they appeared depressed and withdrawn. The staff

would try to encourage conversation, a new activity or

take them outside for some “fresh air” and sunlight to

boost their spirits. Thus, it is very important for bed rest

subjects to keep themselves busy with a goal or interest

throughout their entire period of confinement. On the

other hand, we found that twin pairs often supported each

other in completing their full period of bed rest.

Management of Staff, Research Team and Twins

Complaints:

Working with a diverse group of people is exciting,

interesting and requires tact. Issues and concerns need to

be dealt with in a fair manner and also need to be brought

to the table and investigated in an objective fashion. The

skills and knowledge of the Principal Investigator were

very important to complete the project successfully.

Issues and concerns were dealt with on a professional

level. The GCRC staff (nurses, dieticians and sleep

technicians) worked hard to perform a meaningful study

and the last female set of twins made the completion of

the bed rest study a great pleasure.

LESSONS LEARNED FROM NUTRITION OF

TWINS

Prior to initiation of the study, an 8-day menu cycle was

developed using Nutritionist IV analysis program. These

menus had a consistent macronutrient composition of

15% protein, 55% carbohydrate, and 30% fat. Sodium

was kept constant at 3.5 grams/day. Other nutrients that

were controlled included calcium (between 800 to 1000

mg/day) and fiber (1 gram per 100 calories or at least 20



grams for subjects whose menus were less than 2000

calories). Menus were randomly assigned throughout the

study.

Foods that were used to develop the menus were “typical

American foods” that are easily obtained through the

years and easy to store and prepare. Some of the foods

included dry cereal, low fat or non fat milk, yogurt,

American cheese, Swiss cheese, chicken breast, canned

tuna in water, whole wheat bread, white rice, pasta,

tomatoes, romaine lettuce, salad dressing, and tapioca

pudding. In order to control sodium, we used distilled

water to cook all the foods as well as for ice and drinking

water. Once the menus were calculated, salt was added to

meet the sodium requirement of the study. After the

menus were developed and tested, a food preference sheet

was developed and used for each participant. Once food

preferences were known, menus were individualized.

Calories were determined using the Harris-Benedict

equations plus an activity factor. Weight goals for each

subject were determined by using the average of the

initial two through four days of HDT bed rest. This

allowed time for the cephalad fluid shift and loss of body

weight during the first day of bed rest due to diuresis.

Calories were adjusted to maintain the constant body

weight within 1 kg. Importantly, we were able to maintain

body weight in exercise and control groups, thus avoiding

confounding effects of variable weight changes on the

post-bed rest test parameters. A detailed record of all

foods and fluids consumed was kept and all such data

were later provided to the investigators.

The following are key points/issues for the success of

future bed rest studies from a nutritionist’s point of view:

1) use foods that are easy to obtain all year long, 2)

develop menus with variety to avoid food boredom, 3)

explain with as much detail as possible the diet the

subjects will be following, 4) no matter how much you

explain, subjects will have issues with food, 5) use the

same nutrition analysis program for the entire study, 6)

keep in mind that food formulations change overtime, so

make sure to update values as needed, and 7) be flexible

and have fun with the food preparation. Twins were very

aware of the menu and that items repeated day to day. A

creative chef to develop meals that can be easily adjusted

in composition is recommended.

Non-select menus offered to the subjects became more

streamlined and labor-intensive menu items were omitted.

Subjects were contacted a month before the start of the

study to obtain a list of foods that they do not eat. Menus

were then created in advance that would work for all the

subjects. Nutrition staff communicated several times a

day with the subjects. They then communicated with the

nutrition manager (who was in charge of menu updates

and changes) regarding changes in food preferences so the

diets could be altered and adjusted accordingly.

As the menus became more streamlined, the food

ordering and preparation became more accurate. Storage

problems and over-ordering of food were prevented. At

the inception of the study we were weighing out raw

foods before cooking, and then reweighing the cooked

food before serving the subject. We found this to be an

unnecessary and time-consuming step. The diets

gradually incorporated more pre-packaged cold foods

(juices, granola bars) that helped streamline food

preparation time. As a result, volume of food presented to

the subjects gradually decreased. We found that the

volume of food presented to the subjects in the beginning

was too great. Foods were too bulky and overwhelmed the

subjects (salads, vegetables for example), especially with

the requirement that all subjects eat all food. We made

diet modifications to balance food volume (adding more

juices for example) and thus alleviated volume problems.

The subjects’ tight schedules because of testing and

exercise sessions sometimes resulted in dinner served

very late or breakfast very early. The problem was

compounded if they were slow eaters. Very early and very

late eating schedules were more labor-intensive for the

Nutrition and GCRC staff. The more we were able to

streamline the subjects’ schedules so they had

overlapping meal times, the more efficient our nutrition

team became with meal preparation and delivery.

Subjects’ satisfaction also increased.

Lastly, data entry was important to maintain on a

consistent basis in order to assess the subjects’ energy

balance. Towards the end of the study, we found it

beneficial to enter nutrition data (on Excel spreadsheets)

on a daily, versus weekly basis.

LESSONS LEARNED FROM MEDICAL CARE OF

TWINS

The identical twins used in this study were healthy

volunteers between 21 and 48 years old and provided

informed, written consent prior to arriving at the UCSD

GCRC. On the first day in the GCRC, a complete history

and physical exam were performed on each subject to

ensure that all inclusion/exclusion criteria were met. All

male subjects were free from medications. If female

subjects were taking prescribed oral contraceptives, these

medications were continued for the duration of the study

as the risk of deep vein thrombosis during bed rest was

considered low in this normal population. A urine

pregnancy test was administered on each female subject

and all results were negative. Two of our female subjects

were using inhalers for mild asthma and were allowed to

use this medication as needed and one female subject had

a history of gastroesophageal reflux disease and was

placed on famotidine, histamine-2 receptor antagonist.

These subjects developed no problems related to their

disease or medications. The physical examinations

revealed no significant findings and all subjects were

allowed to participate in the study.

During their 3-day familiarization period, the subjects

underwent orthostatic tolerance and exercise capacity

tests. These tests were then administered again two days

later to obtain baseline, pre-bed rest data and immediately

after 30 days of bed rest. All subjects performed well



during orthostatic tolerance testing and no subject

experienced syncope or any other medical problem. All

subjects gave excellent efforts during exercise capacity

testing and no medical problems were encountered during

this test either. One set of female twins, however, had

significantly limited exercise tolerance and, after

determining that neither of them could complete the

LBNP exercise familiarization session, they were

disqualified from the study prior to the start of bed rest.

Despite their lack of admission of smoking during initial

screening, both of these twin subjects were found to be

smokers with relatively sedentary lifestyles during the

baseline testing period. In future bed rest studies, it is

important to screen out subjects who have this type of

lifestyle. However, in the past we trusted that all subjects

were honest in reporting their qualifying habits and

activity levels prior to arrival at the UCSD GCRC.

Shortly after being placed into HDT bed rest, all subjects

reported mild back pain. This pain typically lasted a few

days and was never associated with any neurologic

complaint or deficit. Several exercising subjects

developed mild discomfort in their lower extremities and

one female subject developed tendonitis of her knee. Her

physical examination and an MRI of the knee failed to

show any intra-articular derangement. She was able to

work through her discomfort and complete the study

without any problems. No exercising subject developed a

specific problem, such as hernia, from the lower body

negative pressure exercise.

During the 30 days of HDT bed rest, there were no

unexpected events in any of the male subjects and all, but

one, of the female subjects. When some subjects

developed minor problems related to HDT bed rest, they

sometimes tried to self-diagnose and exaggerate the

problem. This occurrence was frustrating to the attending

physician and nursing staff, especially if similar problems

occurred frequently for a given subject. One female

subject developed constipation and abdominal pain and,

despite a normal medical workup and physical

examination, she and her twin sister disqualified

themselves from the study several days into the bed rest

phase. An overriding issue was their inability to comply

with the strict HDT regulation during use of the bed pan.

These twin sisters were very close to the 50 year age limit

for our protocol and were relatively less physically fit

than the other subjects. It is likely that older subjects are

less able to adapt to the rigors of HDT bed rest compared

to younger volunteers. This may be important to consider

when recruiting subjects for other bed rest studies. All

other subjects completed the entire 30 days of bed rest

without difficulty. In fact, a common finding was that

each twin helped support their sibling to complete the

entire study.

In summary, all male sets of twin and all but one female

set tolerated the study well and no medical complications

occurred. Future bed rest studies should focus on the

recruitment of healthy, young, non-sedentary subjects.

Mild controlled medical conditions such as asthma or acid

reflux are not contraindications to participating in bed rest

studies successfully.

PERSPECTIVES FROM ONE SET OF TWIN

VOLUNTEERS

Participating in the NASA Twin Study was a great

experience for both of us (Andrea Hawkins and Kristina

Hawkins). Like anything in life, there were challenges

that we inevitably encountered. We entered this study

expecting to encounter many challenges, but it was

surprising that we had more positive experiences than

negative experiences. We used each day as a new way of

making the next day more enjoyable. It was an

opportunity that many people aren’t given and we’re both

glad we seized it.

Using the “female urinal” was by far the most challenging

aspect of the study. It was such a foreign object to us and

we both went into bed rest with the mind-set of “we’ll

worry about it when the time comes.” This was a bad

idea. It just caused a lot of anxiety and frustration. A

week later, we became more at ease with the whole

situation. We highly recommend getting comfortable with

the urinal prior to bed rest. After my twin sister told me

that the handle had to be facing upward, I tried it again

and it worked fine. That is why we recommend that other

female subjects try to get comfortable with the urinal

prior to bed rest. Once we mastered the urinal, drinking

water became more enjoyable and in the long run was

probably better to maintain fluid balance and digestive

processes.

The bedpan was also a very unpleasant issue for us.

Similar to the urinal, it just took time to adjust to a new

way of doing business. The entire process was never

anticipated. It was awkward in the beginning because we

did not quite understand how we could do it without

making a mess. Once we used it the first time, it was fine.

We just made it part of our routine to use it immediately

after being weighed in the morning. The nurses did their

best to make us feel comfortable. We felt guilty that they

had to come to collect our “stuff,” but they were very

good about it. It was more of an issue for us than it was

them. After a week or so, it was no big deal. It was

actually better having my sister in the room with me

because she would make me laugh every time I had to use

the urinal or bed pan. We think it is unlikely that we

would be able to take care of these normal body functions

in a room of other subjects without significantly more

privacy.

As far as physical discomfort, the most significant pain

we encountered was an intense headache. Day one of bed

rest was the most severe. Within hours of bed rest the

pressure built up in our heads from the HDT and caused

an intense headache. By the next day we had adjusted to

the change. We discovered that every time we went

outside for a short period of fresh air and sun, we’d

always develop a headache an hour after re-entering our

room. We were given 400 mg of Motrin pain reliever for



our headaches, which really did not take the pain away

completely, so we found that putting an ice pack over our

eyes and turning the lights off helped ease the pain.

Minor challenges we encountered on a daily basis were

morning weigh-ins and late showers. We were awakened

every morning at 6am and weighed. The routine entailed

our first morning urination, putting on a hospital gown,

transferring to a gurney, and transferred back to the bed

for weighing, which was not a challenge, but more of a

minor inconvenience. Another adjustment was the

evening shower. We were always accustomed to

showering first thing in the morning, so it took some

adjusting to showering at night. Every morning we would

“bathe” with a tub of water and washcloths. It was not the

same, but it helped. It was a slight challenge to shower

horizontally because the shower bed was also tilted down.

The slippery surface and soapsuds caused us to slide

down. Laying a towel beneath us prevented the sliding

problem.

Challenges for control, non-exercise twin

We were fortunate to have the support of each other

throughout this journey. We were always able to keep

each other’s spirits up by making each other laugh. There

were brief moments when we’d had time to ourselves.

These were times when one of us was showering and the

exercise twin was running. Those moments were often

used to write in journals. However, after a while, boredom

set in and in a situation like this, boredom is the enemy.

We would find things to do such as e-mailing friends on

the computer or socializing with the nurses. The nurses

played a crucial role in making this a great experience for

us. They kept us entertained throughout the days. We

tried not to watch TV throughout the day because it made

us feel like we were “sick patients” in bed. We found it

more enjoyable to watch movies at night.

Challenges for exercise twin

The first exercise on the treadmill was the most difficult

of all. Running on a vertical LBNP treadmill in supine

posture is different from any other form of running. The

first problem I encountered was developing a rash from

wearing shorts during the run. Because of the rubbing

from the shorts, I developed a painful rash during a few

more exercises. After the first week, the exercise started

to become more familiar. Having a visual print out of my

exercise protocol allowed me a greater chance of meeting

my prescribed exercise goals and knowing in advance

what was expected prior to my run made me more

comfortable. I discovered during the first run how

important it was to use the urinal before each run. Even

though I didn’t have the urge to urinate, I made sure I

always tried because once I was inside the chamber the

negative pressure seemed to expand my bladder. A V02

test was performed once a week during the study. This

posed a challenge because I ran with a mouthpiece in

place that restricted heavy breathing. To help eliminate

outside stressors, it was helpful to listen to my Walkman

and run with my eyes closed. This gave me the chance to

listen to my favorite songs and daydream about the

outside world.

Useful Tips

A significant amount of time is needed to become familiar

with everything and become comfortable with the new

environment. We have compiled some beneficial

instructions that could help future subjects have a more

enjoyable experience. The most valuable suggestion we

can offer is to familiarize oneself with the urinal prior to

bed rest. The first week is somewhat overwhelming so if

you can eliminate the stress of getting comfortable using

the urinal ahead of time, it will make the transition more

comforting. It would be useful for subjects who are

dependant on caffeine to eliminate it at least a week prior

to bed rest. This is something that might prevent

headaches during the initial phase of bed rest. Also,

waking up at 6am every morning is an adjustment. We

suggest that the subjects go to sleep early and avoid

napping during the day.

Finding things to do during the day may pose a challenge

for some subjects. We found it very useful to incorporate

activities of daily living into our bed rest routine. This

included: grooming, eating, correspondence, and reading.

Showering was always an anticipated activity during the

day. To avoid sliding during the shower we recommend

lying on top of a towel. Also, we suggest keeping a towel

near bedside to avoid getting the pillow wet after a

shower.

Being confined to the same room for a long period of time

can lower your spirits. We found it uplifting to have our

room decorated with familiar items from home such as

pictures, flowers, blankets, and other memorable items.

No matter how unmotivated we were feeling, we always

kept our room bright by having the curtains opened. It

always cheered us up and helped to maintain a positive

attitude.

QUANTITATIVE ADVANTAGES OF USING

IDENTICAL TWINS

In the present review, we compared paired analysis

against an unpaired analysis to determine if we needed

more subjects to detect significant differences in select

physiologic tests. As previously stated, our experimental

design and subsequent analyses were intended to use a

paired model with identical twins.

Paired Model

Eight male and seven female identical twin sets were

recruited. One twin was randomly assigned to the control

group (CON) and his or her twin was assigned to the

exercise group (EX) via a coin toss. The CON and EX

groups were compared before bed rest using a paired t-

test to determine if the measured parameters were

significantly different. Paired t-test revealed that the



groups were not significantly different. Based on these

results and the assumption that the identical twins have

identical genetics, paired t-tests were preformed

comparing the orthostatic tolerance test time, treadmill

exercise capacity test time, maximal oxygen consumption,

and urine markers in the CON and EX group. After bed

rest, the paired t-test yielded significant differences

between the CON and EX groups (Table 2).

Unpaired Model

Sixteen males and fourteen females were recruited. The

subjects were randomly assigned to the CON and EX

group. The CON and EX groups were compared using an

unpaired t-test to determine if the measured parameters

were significantly different before bed rest. Unpaired t-

test revealed that the groups were not significantly

different before bed rest. The following parameters,

orthostatic tolerance test time, treadmill exercise capacity

test time, maximal oxygen consumption, muscle back

strength, and urine markers were compared using an

unpaired t-test after bed rest between the CON and EX

group. The unpaired t-test p-values for orthostatic

tolerance test time, maximal oxygen consumption, muscle

back strength, and urine markers were not significantly

different between the CON and EX groups (Table 2).

Table 2: P Values Obtained Using Paired and Unpaired Models

Paired p-value Unpaired p-value

VO2pk 0.001 0.2

VORTTT <0.0005 0.0005

TUVol. <0.05 >0.25

Ca 0.01 0.15

Oxalate 0.055 >.25

OT Time 0.0025 0.1

Back Strength 0.005 0.2

Key: Orthostatic tolerance (OT); Time Treadmill exercise

capacity test time (VOKTT); maximal oxygen

consumption (VO2pk); urine markers (TUVol., Ca,

Oxalate)

When using an unpaired model, p-values increase,

suggesting that we would need to study more subjects in

order to detect a significant difference with bed rest. The

paired model using identical twins as used in a crossover

study decreases the group variation. Some of the loss in

statistical power using an unpaired t-test may be due to

pooling male and female data. For example, extensive

previous research has documented that orthostatic

tolerance time, exercise capacity and muscle strength are

less in females than males. Therefore, when pooling male

and female data, a larger range and variance occurs,

reducing statistical power when using an unpaired t-test.

SUMMARY

Our experimental design and use of identical twins may

represent an innovative method for evaluating the

physiologic efficacy of a treatment when comparing

experimental and control groups. As reported elsewhere

(Cao et al., 2005; Hargens et al., 2003; Macias et al.,

2005; Monga et al., 2006b; Smith et al., 2003), our

treadmill exercise protocol within LBNP maintained

plasma volume, orthostatic responses, upright exercise

capacity, muscle strength and endurance during bed rest.

Future bed rest studies should focus on the recruitment of

healthy, non-sedentary subjects and may want to follow

our recommendations in terms of screening, testing

sequence, nutrition, medical care and subject support.

Identical twins offer unique insights into countermeasure

development and heritability of physiologic traits (Monga

et al., 2006 a).

ACKNOWLEDGMENTS

The authors gratefully acknowledge the enthusiastic

participation of all of our outstanding twins, excellent

support from staff at the UCSD GCRC, MRI and DXA

facilities. This study was supported by NASA grants 199-

26-12-34 and NAG 9-1425 as well as by an NIH grant to

the UCSD GCRC M01RR00827.

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TRANSLATIONAL MEDICINE: FROM GROUND-BASED STUDIES OF TRAUMATIC INJURIES TO

ASTRONAUT HEALTH AND EARTH BENEFITS.

Charles E. Wade

US Army Institute of Surgical Research, Fort Sam Houston TX 78234

ABSTRACT

Traumatic injuries are an everyday occurrence, from minor

scratches to the massive injuries of auto accidents. Based on this

reality NASA has prepared for a range of contingencies to care

for injured astronauts. These preparations have been based upon

the clinical incidence of traumatic injuries in environmental

analogs such as Arctic habitats. However, information is needed

as to the effect of acclimation to the space environment on

responses to injury, subsequent treatment and rehabilitation.

Deconditioning of a subject leads to significant alterations in

responses to hemorrhage, susceptibility to infection, and the

healing process. The treatment of the injured astronaut may have

to be altered to compensate for the effects of acclimation in light

of these findings. Furthermore, upon return to Earth's gravity

therapeutic interventions, such as administration of anesthetics,

may have to be adjusted. The response of patients to the

deconditioning of acute bedrest associated with the care of

traumatic injuries provides insight into care of injured

astronauts. In order to assure the mission success of long

duration space flights the care of even minor injuries should be

extensively evaluated.

Traumatic injuries are often the result of a single acute

event. However, the consequences of traumatic events can

be far reaching, extending from the patient to families,

communities, nations, and the world as a whole (Kauvar

& Wade 2005). Spaceflight programs have not been

immune to traumatic injuries. While most of these injuries

have been lethal, space agencies have focused extensive

resources on consideration of the traumatic injuries that

may occur during the course of space flights. As the

duration and planned scope of space flight missions is

expanded the probability of traumatic injuries is

increased. Of concern is the health and well being of the

injured astronaut as well as the impact on mission

success.

Epidemiology of Emergencies

Traumatic injuries take many forms, from the very simple

scratch to the massive crush injury with broken bones and

hemorrhage. The space agencies have focused on the

injuries serious enough to be classified as emergencies,

where the life of an individual or the success of a mission

is threatened (Safe Passage: Astronaut Care for

Exploration Missions 2005; Billica et al. 1996;

Kirkpatrick et al. 2005, 1997; McCuaig 1994; McCuaig

& Houtchens 1992; Summers et al. 2005). In the 44 years

of manned spaceflight 5 major accidents have resulted in

21 deaths (Safe Passage: Astronaut Care for Exploration

Missions 2005). Other accidents have resulted in

traumatic injuries, but most of these have occurred on

Earth where traditional emergency advanced life support

measures were applied (Summers et al. 2005). During

flight in the microgravity environment few medical

emergencies have been documented, with only two

reported incidents of traumatic injuries (Safe

Passage:Astronaut Care for Exploration Missions 2005;

Billica et al. 1996; Summers et al. 2005).

In the general population, the incidence of medical

emergencies is 0.06 events per person year (Safe Passage:

Astronaut Care for Exploration Missions 2005; Billica et

al. 1996; Summers et al. 2005). Using this model, a ten

member crew on a 2.4 year mission to Mars would have

at least one medical emergency (10 people x 2.4 years x

0.06 incidence per person-year = 1.4 emergent medical

events). It could be expected that the incidence in the

extreme environment of space would be higher than the

rate observed in the general population. However, studies

conducted in different injury settings have helped to

formulate the direction and policies of the NASA

emergency medical program. These environments include

the US Navy experience in nuclear submarines and the

Antarctic McMurdo Station (Thomas et al. 2003; Lugg

2000). Estimates of the occurrence of medical

emergencies during spaceflight are based on the data from

these environments. At McMurdo Station the incidence of

medical emergencies requiring medical evacuation is

reported as 0.036 events per person-year (Lugg 2000).

Review of the Russian program found four medical

evacuations over 72 person-years in space with an

incidence of 0.056, very similar to that of the general

population (Summers et al. 2005). Thus both the data

from spaceflight and simulation environments yield rates

of 0.04 to 0.05 per person-year for a medical emergency

requiring evacuation. In planning for long-duration

spaceflight the probability of an emergent event must be

considered and appropriate measures to deal with the

situation must be in place.

There have been 17 medical emergencies during

spaceflight in the NASA program, two of which were

traumatic injuries (Safe Passage:Astronaut Care for

Exploration Missions 2005; Summers et al. 2005). In the

NASA program the incidence of emergencies related to

trauma (12%) falls far below that observed in the

McMurdo Station (48%) (Lugg 2000; Safe Passage:

Astronaut Care for Exploration Missions 2005).

Irrespective of the present rate of injury, traumatic

injuries are of great concern to the NASA program as they

____________________

* Correspondence to: Dr. Charles Wade

US Army Institute of Surgical Research

3400 Rawley E. Chamber Dr.

Fort Sam Houston, TX 78234


Phone: 210-916-3698; Fax: 210-916-2942

C.E.Wade — Translational Medicine


are mentioned in 44% of the 45 risk areas in the

Bioastronautics Critical Path Road Map

(http://bioastroroadmap.nasa.gov/index.jsp). Further, five

questions under the heading “Major Illness and Trauma”

present the following:

18d) What resources are required for telemedical

consultation, diagnosis, and management of major

trauma?

18f) What are the resources and procedures needed to

perform basic and advanced management of trauma?

18I) What procedures and protocols are necessary for

rehabilitation after an acute medical illness or trauma?

18AH) What resources and procedures are needed for the

surgical management of major illness, injury, and trauma?

20E) What are the resources and procedures required for

the treatment of minor trauma, emphasizing autonomous

decision-making, based on known space flight illnesses,

injuries, and expedition analogs? How might they be

adapted to reduced-G operations?

NASA has focused its efforts on applying the standard

medical treatments used on Earth to spaceflight (Safe


Kirkpatrick et al. 2005, 1997; Summers et al. 2005). The

agency has contributed significantly to the medical field

by making equipment/devices simpler, smaller, and

autonomous. Furthermore, efforts have been made to take

into account the physical environment (microgravity,

closed-loop environmental systems, etc.) when caring for

the spaceflight patient. For example, alternatives to the

use of volatile anesthetics and the means of restraint of

the surgical patient have been evaluated (Campbell 2002,

1996, 2002, 2004, 2001). However, the physiological

changes of the patient due to acclimation to the

microgravity environment have not been adequately

considered. In formulating and reviewing NASA’s

policies regarding the care of the astronaut with an

emergency due to traumatic injuries, the altered

physiological status of the patient is recognized and the

need for additional information noted. Though the

problem is clearly defined it has not been adequately

addressed.

The study of the care of traumatic injuries is conducted in

animal models that provide evidence for the formulation

of guidelines for treatment of human patients. The

efficacy of the guidelines is then evaluated over the

course of patient care. An example of this process is the

study that led to the classification of astronauts as at risk

when undergoing anesthesia. On the Bion 11 mission

anesthetic complications occurred in two primates who

had been in spaceflight for 14 days (Ilyin et al. 2000).

This project was a multinational effort to study the effects

of spaceflight on bone, muscle and the vestibular system.

Anesthetic was administered to the animals by a

competent team of veterinarians on the first day post-

flight for minor surgery, muscle biopsies. One animal

died during the procedure and the other experienced

serious complications. Previously, surgeries had not been

performed until the seventh day of recovery following

Bion flights, and no complications were observed.

Furthermore, the procedure done multiple times on

control animals produced no adverse events. This

experience pointed out that the knowledge of risk

imposed by anesthesia following acclimation to the

spaceflight environment is limited (Safe


Norfleet 2000). This incident led to the reclassification of

astronauts by the American Society of Anesthesiologists

(ASA) 4: A patient with severe systemic disease that is a

constant threat to life. The change in classification means

increased vigilance when administering anesthetic to

patients and during recovery. Another example of the

value of animal studies in space was the series of surgical

procedures performed on rats during Neurolab mission

which confirmed the expectation that surgery can be

conducted in microgravity (Safe Passage: Astronaut Care

for Exploration Missions 2005; Campbell et al. 2005;

Kirkpatrick et al. 2005). These examples of the findings

derived from the study of animals subsequently impacted

patient care. While these are anecdotal observations there

is increased interest in establishing requirements for the

acute care of astronauts by performing classically

designed studies, as noted in a recent National Academy

of Science (Safe Passage: Astronaut Care for Exploration

Missions 2005) report: “Although it may not be realistic

to replicate these myriad physiological studies during the

postoperative period, carefully designed experiments with

simple outcome measures (supplemented by highly

selected physiological, histological, or metabolic studies)

performed in a microgravity environment such as the

International Space Station (ISS) after surgery in animals

might yield significant information of value.”

Presented below is an example of how animal

experimentation provides information to predict the

possible impact of the microgravity environment on the

care of astronauts with traumatic injuries. This example,

fluid resuscitation in response to hemorrhage, was

selected as it is part of the initial response to injury and

was called out in the National Academy Report (Safe

Passage: Astronaut Care for Exploration Missions 2005).

Fluid Resuscitation in Response to Hemorrhage On Earth the care of the patient with severe traumatic

injuries is compartmentalized into a series of care

responses. The first responder, in most developed

countries is a paramedic, who renders first aid assuring

that the airway is open, there is adequate ventilation, and

bleeding is controlled. Once this is accomplished,

resuscitation is initiated, often entailing the administration

of fluids. It is expected that in the course of a continued

human presence in space a number of situations will

require the use of resuscitation solutions. These include

traumatic injuries, blood loss during surgery and a

number of circumstances resulting in dehydration. At

present the requirement for resuscitation fluids has not

been documented in the space programs. However, one

could assume that the need would be similar to those

observed in other extreme environments such as

submarines or Antarctica.



The primary use of resuscitation fluids in spaceflight

would be associated with emergency and surgical

procedures (Kirkpatrick et al. 2005, 1997). Emergency

surgery might have to be performed in a variety of

scenarios. While resuscitation fluids are mostly used as

volume support in the course of a conventional surgery,

the rate of blood loss could be greater during spaceflight

as blood clotting may be compromised, and a trained

surgeon may not perform the procedure. Thus, a greater

amount of blood loss would be expected.

Attempts to evaluate the performance of surgery in

microgravity generally concluded that operative

procedures may be conducted (Safe Passage: Astronaut

Care for Exploration Missions 2005; Campbell et al.

2005; Kirkpatrick et al. 2005). However, the limitations

of the procedures were not defined. Fluid would also be

required for the care of an astronaut requiring surgery

immediately upon return to Earth for an emergency

procedure. These two surgical environments may

necessitate different types of fluids or different procedural

approaches to fluid administration than those normally

used.

Traumatic injuries may also occur in space. For example

the ISS is a construction zone with astronauts often

moving large objects during extravehicular activity (EVA

or “space walk”). These activities are believed to be the

riskiest for traumatic injuries. Though the utmost safety

precautions are in place, the probability of injury is still

high. These injuries could be both blunt and penetrating

and could result in bleeding from an open wound or into a

body cavity. On Earth hemorrhage is the leading cause of

death due to traumatic injuries and accounts for 80% of

intraoperative trauma deaths (Champion 2004; Sauaia et

al. 1995). Fluids for the patient with traumatic injuries

would stabilize the individual prior to definitive care,

such as a surgical procedure, which would in turn

necessitate further fluids.

Resuscitation solutions would also be used for the

treatment of hypovolemia induced by dehydration.

Astronauts spend extended periods of time doing work in

space suits and in exercise with significant dehydration. It

is conceivable that extreme dehydration could require the

administration of fluids. Another likely cause of fluid loss

would be food poisoning or gastroenteritis resulting in

excessive diarrhea and vomiting, though oral rehydration

has been demonstrated as the ideal means of replacement

for gastrointestinal losses.

The goal of fluid administration for these conditions is

hemostasis (correcting physiological parameters to

normal levels). On Earth, accomplishing this goal is often

difficult, time consuming and requires extensive

resources. In spaceflight hemostasis for a victim of

hypovolemia by fluid resuscitation may be insufficient or

impossible to attain due to limited resources and

expertise. A comprehensive review of resuscitation fluids

for spaceflight by Kirkpatrick et al. (Kirkpatrick et al.

2005) reports that, “Present medical care in space has

been developed on the basis of proven conventional

terrestrial strategies of proven health care.” There has

been no attempt to systematically evaluate resuscitation

procedures or solutions to meet the unique requirements

of the altered physiology of an astronaut acclimated to

spaceflight.

Presently, normal saline (NS; 0.9% NaCl) is the primary

solution carried aboard the Shuttle and the ISS. The total

volume available is 4 L in the Advanced Life Support

Pack (ALSP) and 7.5 L in the Health Maintenance System

Ancillary Support Pack. The ALSP carries an additional

0.5 L of dextrose (D5W) (information provided by the

NASA Crew Office). Therefore, the total available fluid is

12 L. For the care of a major traumatic injury this would

equate to a blood volume expansion capability of only 4 L

(American College of Surgeons, C. O. T. Advanced

Trauma Life Support Student Manual (ATLS) 1997;

Fluid Resucitation: State of the Science for Treating

Combat Casualties and Civilian Injuries. 1999). In a

recent study of penetrating trauma we found the fluid

requirements to be 12.5 L over the first 24 hours (Wade et

al. 2003). Therefore, the volume and type of fluid

presently stored on space vehicles appears insufficient.

In the administration of normal saline to critically injured

patients it is recommended that the patient receive three

times the volume of blood loss (American College of

Surgeons, C. O. T. Advanced Trauma Life Support

Student Manual (ATLS) 1997; Fluid Resucitation: State

of the Science for Treating Combat Casualties and

Civilian Injuries.). However, the Advanced Trauma Life

Support recommends the limitation of fluid administration

to maintain systolic pressure to reduce uncontrolled

bleeding and the use of normal saline has been

questioned, as large amounts of fluid collect in the

extravascular space (American College of Surgeons, C. O.

T. Advanced Trauma Life Support Student Manual

(ATLS) 1997). This “third spacing” has been implicated

in subsequent morbidity, specifically, an increased

incidence of multi-organ dysfunction and acute

respiratory distress. Another risk factor in the use of large

volumes of normal saline is the hemodilution of red cell

mass that decreases oxygen carrying capacity and dilution

of clotting factors that would result in secondary re-

bleeding.

At present the various space programs recognize the need

for resuscitation but specific requirements are not clearly

defined. What is not understood is the impact of the space

environment on the response of the patient to

hypovolemia and the ability to adequately resuscitate the

individual. The spaceflight environment presents a

number of other challenges in the treatment of the patient

with hypovolemia. These include alterations in the

cardiovascular, endocrine, immune, and hemopoietic

systems as well as metabolic changes coupled with the

closed environment of the spacecraft. Below we present

possible modulators to the response of hypovolemia and

subsequent resuscitation. In addition we discuss the use of

a ground-based animal model in light of these changes.



Based on the plethora of changes in spaceflight any

studies on the ground should employ a model emulating

as many of these alterations as possible. In human

subjects this has been accomplished by extended periods

of bed rest. While this model is appropriate for studying

countermeasures and provides some information as to

acute fluid shifts, the use of humans to evaluate

hemorrhagic hypovolemia due to injury is not warranted.

An animal model is the method of choice, especially if the

influence of long-term acclimation to the spaceflight

environment is to be evaluated.

The animal model we have focused on is the hind limb

suspended (HLS) rat (Morey-Holton & Globus 2002).

This model reproduces many of the characteristics

observed in humans and rodents in response to

spaceflight. In addition, if efficacy of resuscitation

solutions is to be evaluated in the microgravity

environment of spaceflight the rodent is the model

specimen of choice. Rodents have an extensive flight

history, and have been manipulated during spaceflight and

have undergone surgical procedures. Thus, rats provide

the basis for subsequent studies evaluating fluid

resuscitation procedures and techniques in actual

spaceflight.

Response to Spaceflight: human vs. hind limb

suspended (HLS) rat model

Spaceflight results in pronounced changes in the

physiology of astronauts and rodents. We will focus on

the changes observed in the acclimated individual and do

not discuss the acute responses before acclimation upon

insertion into orbit. These changes have been extensively

reviewed. We will use the reviews as a basis for the

discussion of the impact of these changes on the ability to

compensate for blood loss due to hemorrhage and the

subsequent ability to resuscitate the patient. We will then

address the response of the animal model and its viability

as a surrogate to study the responses in astronauts.

Cardiovascular changes Hemodynamics: The hemodynamic status of the astronaut

once acclimated to space flight is relatively normal

(Convertino 1996; Hargens & Watenpaugh 1996; Sawin

1998; Watenpaugh 2001). Blood pressure and heart rate

are within normal limits. Some reports note decreases in

resting heart rate, as well as a trend toward reduction in

diastolic pressure. These changes have been highly

dependent upon the flight mission and are within normal

clinical variability. Rodents exposed to HLS have an

initial period of acclimation similar to that of astronauts in

spaceflight (Overton et al. 1989; Ray et al. 2001).

Following this period mean arterial pressure (MAP), heart

rate (HR), systolic arterial blood pressure (SBP) and

diastolic blood pressure (DBP) seldom differ from those

of control animals.

Cardiac output following acclimation to spaceflight is

normal in astronauts (Hargens & Watenpaugh 1996). If

there is a reduced heart rate there is an accompanying

increase in stroke volume to sustain cardiac output. In the

rat HLS model there is no difference in cardiac output at

rest compared to control animals (Overton et al. 1989;

Woodman et al. 1995). As with the astronauts, reduced

responsiveness in cardiac output paralleled increasing

exercise loads. Failure to increase cardiac output in the

presence of increased metabolic demands as noted during

exercise would not bode well for correction of the loss of

oxygen-carrying capacity, and thus delivery, during

hemorrhage or the subsequent hemodilution and volume

expansion of resuscitation.

Blood Volume: Cardiovascular changes include a

reduction in blood volume on the order of 15% in

astronauts (Convertino 1996; Hargens & Watenpaugh

1996; Leach et al. 1996; Strollo 1999; Watenpaugh 2001).

The decrease in blood volume occurs by reductions in

both plasma volume and red cell mass, such that

hematocrit is not changed. This decrease in blood volume

could be detrimental upon return to Earth in the

hypovolemic patient. With re-exposure to Earth’s gravity

a shift in fluids has led to orthostatic hypotension

necessitating countermeasures. It would be logical that the

reduced blood volume would adversely impact a patient

brought back to Earth exacerbating the hypovolemic state.

In flight the reduced volume appears to be defended (see

Fluid Homeostasis below). The blood volume of HLS

animals appears to be reduced, but not to the same

magnitude as observed in astronauts. The reduction in

HLS animals is on the order of 6% and appears to be

dependent upon the duration of the HLS. The majority of

the literature reports the change in blood volume during

HLS not to be significant (Bouzeghrane et al. 1996; Chew

& Segal 1997; Tipton et al. 1998). A reduction in blood

volume prior to hemorrhage drastically reduces survival

and the success of resuscitation (Ho et al. 1996;Wade et

al. 1992).

Transcapillary fluid flux: Spaceflight appears to

redistribute fluid between body compartments in

astronauts (Convertino 1996; Hargens & Watenpaugh

1996; Leach et al. 1996; Watenpaugh 2001). There is the

decrease in the extracellur fluid volume comprised of a

contraction of blood volume previously mentioned and a

reduction in interstitial fluid volume. Simultaneously,

intracellular fluid volume increases on the order of 10%.

A major compensatory response to hemorrhage is the flux

of fluid from the interstitial space into the vascular

compartment (Hannon et al. 1990; Wade et al. 1992). In

time there is movement from the intracellular space. This

response is referred to as “auto-transfusion”. The lack of

adequate movement from the intracellular compartment

during hemorrhage would reduce survival. In the HLS

model this has yet to be addressed.

Fluid homeostasis

The reduction in blood volume of astronauts during

spaceflight suggests a new set-point for the maintenance

of fluid homeostasis. The regulation of fluid balance in

astronauts is altered with longer retention of a fluid load



as a result of a reduced rate of urine excretion due to

alteration in the response of regulatory hormones

(Convertino 1996; Watenpaugh 2001). In the HLS rat

model though blood volume is slightly reduced, fluid

intake and urinary excretion are normal, suggesting a

shifted fluid homeostasis similar to that observed in

astronauts. This finding has led to the supposition that

there is attenuation of the responses to changes in blood

volume (Bouzeghrane et al. 1996; Deavers et al. 1980;

Steffen et al. 1984; Tucker & Mendonca 1995). Upon

return to Earth both astronauts and rats have a pronounced

diuresis (Wade & Morey-Holton 1998; Wade et al. 2000).

This increase in urine output is not immediately

compensated for by an increase in fluid intake and results

in hypovolemia. This response to landing, coupled with

hemorrhagic hypovolemia, would put the patient at risk

for a poor outcome. Thus, in the case of hypovolemia due

to hemorrhage the compensatory responses may be

attenuated in flight and after landing.

Endocrine System Pressor hormones: In response to hemorrhage there is a

progressive recruitment of pressor hormones. These

hormones, norepinephrine, epinephrine, angiotensin II

and vasopressin, increase peripheral resistance, leading to

maintenance of blood pressure and blood flow to essential

organs. In addition, the sympathetic hormones play a

major role in substrate mobilization. Reports as to

alteration of the sympathetic tone of astronauts are

variable, appearing to be highly mission dependent

(Leach et al. 1996, 1983; Macho et al. 1996; Stein &

Wade 2001; Tipton et al. 1996). Changes in sympathetic

tone and responsiveness are essential compensatory

responses to hemorrhage. With HLS animals, as with

astronauts, the reported plasma norepinephrine levels are

variable(Hasser & Moffitt 2001). Furthermore, changes in

sympathetic output in responses to manipulation are not

definitive, but appear to be reduced. The renin-

angiotensin system of astronauts is reduced (Leach et al.

1996, 1983). In addition, the response of this system as

determined by response to fluid load was attenuated. In

the HLS rodent plasma renin activity (PRA) decreased

after 7 days of suspension (Bouzeghrane et al. 1996).

However, no difference in PRA from control was noted

after 14 days of acclimation to HLS.

Astronauts experience reduced vasopressin (Leach et al.

1996,. 1983). In response to HLS the response of

vasopressin in animals is variable. An acute increase in

the rate of urinary excretion of vasopressin is found in

response to HLS, however, 14 days of suspension

produced no difference in plasma levels (Bouzeghrane et

al. 1996). Alterations in astronauts and HLS rats of most

of the pressor hormones occur during the period of

acclimation with control values attained after a period of

time. However, the alteration in responsiveness of the

hormones to volume manipulations suggests

compensatory changes due to hemorrhagic hypotension

would be altered, contributing to an inadequate

compensatory response.

Reproductive hormones: Spaceflight consistently reduces

testosterone levels in astronauts and rats (Ortiz et al.

1999; Strollo 1999; Tou et al. 2002). The cause of this

change is not well defined. There is no information as to

the change of reproductive hormones in female astronauts

(Ortiz et al. 1999; Strollo 1999; Tou et al. 2002). The

level of reproductive hormones has been demonstrated to

account for the pronounced gender differences in the

response to hemorrhage (Jarrar et al. 2000; Remmers et

al. 1998a; 1997, 1998b). A reduction in testosterone in

male rats is beneficial in the presence of hemorrhage. The

addition of estrogen contributes to an increase in survival.

With the HLS model male rats have a significant

reduction in plasma testosterone levels (Ortiz et al. 1999;

Tou et al. 2002). In female rats HLS reduces estrogen and

disrupts the reproductive cycle (Tou et al. 2005, 2004).

Therefore, changes in reproductive hormones during

spaceflight may be of benefit to male astronauts whose

injuries result in hemorrhage, and a decrement in female

astronauts.

Insulin/Glucagon: Investigations during spaceflight, in

astronauts and rats, have demonstrated changes in glucose

and insulin metabolism suggestive of a diabetic-like state

(Macho et al. 1996; Stein et al. 2000; Tobin et al. 2002).

There is an increase in circulating glucose and a reduction

in insulin concentrations for the associated plasma

glucose level (Macho et al. 1996). Furthermore, there

appears to be a decrease in insulin sensitivity. Plasma

glucagon levels are not changed. Hemorrhage decreases

sensitivity to insulin in liver and muscle tissues (Carter

1998; Custalow et al. 2001). This leads to a catabolic

state. Muscle atrophy and reduced insulin sensitivity as a

result of spaceflight would theoretically adversely affect

the normal compensatory response to hemorrhage. HLS

rats exhibit enhanced sensitivity to insulin-induced

glucose uptake when compared with normal control rats,

and resistance to the actions of insulin when contrasted

with rats similarly matched for the reduction in body mass

gain (Mondon et al. 1992). Insulin binding is also

reduced. A number of studies have produced conflicting

results in this area (Koebel et al. 1993; Stump et al. 1992,

1993), but the ability to mobilize and transport glucose in

response to hemorrhage is important to survival (Barton

& Passingham 1982; Carter 1998; Chang et al. 2000; Ma

et al. 2003). The diabetogenic state of space flight would

adversely affect this compensatory response and alter

outcome from hemorrhage.

Metabolic Changes

Negative energy balance: Astronauts experience a

negative energy balance that is highly mission dependent

and related to work load requirements (Stein 2000; Wade

et al. 2002). Over the course of a Shuttle mission body

mass is reduced (Sawin 1998; Stein 2000). A negative

energy balance adversely impacts survival following

hemorrhage (Nettelbladt et al. 1996). Bacterial

translocation from the gut may also increase after a

normally non-lethal hemorrhage if the patient is food

deprived (Bark et al. 1995). A consistent finding in the

HLS rat is a decrease in body mass on the order of 10%



(Morey-Holton & Globus 2002; Thomason & Booth

1990). Attempts to determine energy balance have been

limited. We reported that food intake adjusted for body

mass was similar for HLS and control rats (Wang &

Wade 2000). Further, Blanc et al. (Blanc 2001, 2000)

reported no change in energy expenditure during 7 days

of suspension, and a net reduction in energy balance as

intake was reduced. We have reported no change in the

body mass of space flown rats (Wade et al. 2002). In this

case the HLS model may be more representative of the

astronaut than an animal on board the space craft.

Substrate metabolism: There are numerous reports of

changes in substrate metabolism of astronauts and rats

flown in space (Fitts et al. 2001; Stein 2000; Thomason,

1990). Many of the changes are associated with muscle

disuse (Fitts et al. 2001; Picquet et al. 2000; Thomason &

Booth 1990). There appears to be a preferential shift in

large normally weight-bearing muscle from fat

metabolism to glucose. In rats flown in space or HLS the

changes in fiber type are similar to those observed in

astronauts. This change in fiber type is associated with a

reduction in the ability of the muscle to oxidize long-

chain fatty acids, and an increased reliance on

carbohydrate metabolism (Stein et al. 2000; Thomason &

Booth 1990). In response to exercise these changes have

led to a reduction in exercise performance due to limited

metabolic reserves. Reliance on carbohydrates as a

metabolic substrate is high in the presence of major

hemorrhage (Boija et al. 1988; Custalow et al. 2001).

Thus, increased reliance on this metabolic pathway for

normal metabolism may limit the ability of this energy

pool to serve as a reserve during hemorrhage. The end

product of the use of carbohydrates in the presence of

limited oxygen is lactate. Lactate increase is indicative of

the magnitude of the oxygen debt incurred and is related

to subsequent outcome (Wade et al. 1989). In the HLS

model Fitts and colleagues (Fitts et al. 2001) have

performed studies showing there is an increase in muscle

glycogen, but in response to stimulation this reserve is

rapidly used. Further, at rest there was an increase in

tissue lactate that rapidly increased during stimulation. As

blood flow is shunted away from muscle during

hemorrhage the lack of shift in the metabolic substrate

would lead to rapid depletion in energy reserves and a

greater production of lactate and acidosis. These shifts in

metabolism would have a negative impact on survival

from hemorrhage and may preclude some commonly used

resuscitation solutions.

Changes in liver metabolism during spaceflight will also

influence responses to hemorrhage (Merrill et al. 1992;

Stein et al. 1994). Flight alters a variety of metabolic

pathways in liver tissue, reducing P-450 pathway

enzymes and cholesterol biosynthesis. There appears to be

a shift favoring gluconeogenesis as glycogen stores are

increased. The liver is one of the first organs to show

signs of injury during hemorrhage (Boija et al. 1988). The

pathway changes during spaceflight may impact the

ability of the liver to provide adequate substrate during

hemorrhage, leading to a reduction in ATP stores. A

reduction in liver ATP levels is associated with an

increase in apoptosis after hemorrhagic hypotension

(Mongan et al. 2002; Paxian et al. 2003). Alteration of

substrate metabolism during spaceflight may adversely

impact outcome following hemorrhagic hypotension due

to traumatic injuries. The metabolic imbalance may

persist during resuscitation as normal compensatory

pathways diminish. The capability to stabilize the patient

for an extended period of time following resuscitation is

in jeopardy as well.

Immune system:

The immune system of astronauts and rats is dramatically

altered during space flight (Sonnenfeld 2005b, 2005a,

2005c). The HLS model emulates many of the changes in

the immune system reported during spaceflight. HLS has

been used to assess possible countermeasures to immune

dysfunction associated with space flight. The response of

the immune system to hemorrhage plays a major role in

outcome of the patient and subsequent morbidity. The

dysfunction of this system could lead to a variety of

differences in the response to hemorrhage and

resuscitation.

Environmental:

The spaceflight environment is often thought of as only

involving responses to microgravity. However, there are

numerous other environmental factors that could

adversely affect the ability of a patient to survive

hemorrhage. A major environmental factor may be the

transition from the microgravity to the normal gravity of

Earth and the experience of hypergravity during landing.

An example of the influence of the return to Earth’s

gravity is the high incidence of astronauts experiencing

orthostatic intolerance (Hargens & Watenpaugh 1996;

Sawin 1998). Brizzee and Walker (Brizzee & Walker

1990) examined the response of reloading on blood

pressure, heart rate and cardiac output following hind

limb suspension for 7 days. They noted no difference in

any of the measured hemodynamic parameters. With 10

minutes reloading, mean arterial pressure did not change,

with trends towards an increase in heart rate and total

peripheral resistance and a fall in cardiac output. Others

have noted a significant reduction in blood pressure with

reloading (Bayorh et al. 2002). These responses are

similar to those observed in astronauts and do not bode

well for an individual injured in space who is returned to

Earth in a hypovolemic state.

Response of the HLS Model to Hypotension:

Studies of the HLS model response to hypotension are

limited and only one examined the response to

hemorrhage. Studies of hypotension have been directed at

the baroreflex to assess the impact of return to the gravity

of Earth on orthostatic intolerance, not the study of

responses to hemorrhagic hypovolemia. In studies of the

baroreflex the animals were administered a vasodilator at

increasing doses. The heart rate response to a given fall in

blood pressure provides an index of the responsiveness of

the system. This compensatory response is mediated by

the sympathetic nervous system. The response of heart



rate to administration of the vasodilator was not affected

by HLS (Brizzee & Walker 1990; Hasser & Moffitt 2001;

Moffitt et al. 1998). However, the response of

sympathetic nerve activity decreased (Foley et al. 2005;

Hasser & Moffitt 2001; Moffitt et al. 1998; Mueller et al.

2005; Mueller & Hasser 2003). The authors concluded

that, as previously reported in astronauts (Hargens &

Watenpaugh 1996; Sawin 1998), HLS alters the arterial

baroreflex, possibly contributing to orthostatic intolerance

(Foley et al. 2005; Hasser & Moffitt 2001; Moffitt et al.

1998; Mueller et al. 2005; Mueller & Hasser 2003). The

inability to increase sympathetic tone in response to a

decrease in arterial pressure would also suggest a negative

impact of HLS on the response to hemorrhage, adversely

affecting outcome. The same authors have conducted

preliminary experiments of the response to hemorrhage

(Hasser & Moffitt 2001). The rats were exposed to HLS

for 12 days. Mean arterial pressure, heart rate and

sympathetic nerve activity were recorded. In response to

HLS mean arterial pressure did not change, but heart rate

increased. Rats were hemorrhaged at 3 ml/kg/min until

attaining a mean arterial pressure of less than 40 mmHg.

The amount of blood removed to reach the desired blood

pressure was similar (control 20.2 ± 2.1 ml/kg and HLS

19.0 ± 1.8 ml/kg). However, the increase in sympathetic

nerve activity in response to a reduction in pressure was

attenuated in HLS animals. This preliminary work on

hemorrhage, coupled with a body of literature that

addresses the modification of the cardiovascular

responsiveness of the HLS model, supports use of this

method. Further, as the HLS model produces many of the

metabolic, cardiovascular, immunologic and endocrine

changes observed in astronauts during spaceflight we

propose it as an appropriate means for the initial

evaluation of resuscitation procedures to be used to treat

hemorrhagic hypovolemia in astronauts.

Hemorrhage Models: As noted previously, the traumatic injuries that may

induce loss of blood volume necessitating fluid

replacement in an astronaut are quite diverse. Fortunately,

a number of experimental hemorrhage models can mimic

a range of clinical scenarios, such as fixed pressure, fixed

volume, uncontrolled and crush hemorrhage models. The

first model, fixed pressure, is one of the most common.

This method developed by Wiggers and colleagues bleeds

the animal to a fixed pressure and sustains that pressure

by removal of additional blood or replacement of the shed

blood volume (Wiggers 1950). This model contributed to

the hypothesis of irreversible hemorrhage in that at some

point, no matter what resuscitation interventions are used,

survival is not possible (Shah et al. 1998). This model

provides insights into cardiovascular compensation and

other endogenous factors mediating restitution. The

second is the fixed volume hemorrhage model with the set

removal of volume of blood per kilogram of body mass

(Hannon et al. 1990; Shah et al. 1998). This approach

allows differences between initial conditions to be

assessed. It also allows the condition of the animal to be

evaluated in terms of the net response to hypervolemia.

We have used this approach with a slight modification,

extending the period of bleeding at a slower rate to mimic

the drop in the drive pressure observed clinically. The

third model is of uncontrolled hemorrhage to simulate a

major injury in which the control of bleeding is not easily

attained (Bickell et al. 1991, 1989, 1994; Krausz et al.

2000). This simulates major traumatic injuries to the

torso. The model has been used extensively to assess

initial resuscitation in the pre-hospital setting. The fourth

model includes major tissue damage in the presence of a

fixed volume of bleeding (Jarrar et al. 2000; Remmers et

al. 1998al., 1997, 1998b). This model allows the study of

responses to tissue injury which modifies compensatory

adjustment. In evaluating approaches to resuscitation the

use of a number of the animal models of hemorrhage is

highly recommended (Fluid Resucitation:State of the

Science for Treating Combat Casualties and Civilian

Injuries. 1999). Although there are other approaches to

the study of hemorrhage however, the above methods are

widely used to assess the efficacy of resuscitation

procedures. We have deliberately not addressed burn or

septic shock. However, these forms of hypovolemic shock

would also be modified by acclimation to the spaceflight

environment and should be evaluated.

Resuscitation Parameters:

The approach to resuscitation following traumatic injuries

and the appropriate endpoints are controversial. Recent

studies using physiological endpoints have been effective

in increasing survival and reducing morbidity. However,

they require extensive physiological monitoring and

staffing. Thus, they have not been used outside the

hospital. This has led to the approach of stabilizing the

patient until definitive care can be administered, which

would be the optimal situation in the spaceflight

environment. These methods include delayed fluid

administration, hypotensive resuscitation, modifications

of the fluids administered and mechanisms for lowering

metabolism (Fluid Resucitation:State of the Science for

Treating Combat Casualties and Civilian Injuries. 1999;

Bickell et al. 1994; Dutton et al. 2002; Knudson et al.

2003; Kramer 2003).

Another area of controversy is the endpoint for evaluation

of resuscitation efficacy (Fluid Resucitation: State of the

Science for Treating Combat Casualties and Civilian

Injuries. 1999; Revell et al. 2003; Rhee et al. 1998;

Shoemaker et al. 1996; Wade & Holcomb 2005; Zhao et

al. 2002). The ultimate endpoint is survival. However,

these studies are expensive and involve large numbers of

subjects and thus are not appropriate for the initial

assessment of an intervention. Later in the development

of a process they are necessary. Many have felt that

restoration of blood volume and cardiovascular function

are adequate indices of resuscitation. This is not always

true. For example, the use of crystalloids may replace

blood volume and improve cardiac output in the short

term. However, due to hemodilution, oxygen delivery is

reduced, leading to death. Therefore, long-term survival

must be assessed. This is especially true in the spaceflight

environment where the extraction of the patient will be

days, if not weeks, and the acclimation process may



necessitate alteration of the standard of care approaches to

resuscitation.

SUMMARY

The question is not if a traumatic injury requiring

resuscitation during spaceflight will occur, but what

systems and clinical guidelines will be in place to handle

it when it does occur, without loss of life or compromise

of the mission. The need for the care of traumatic injuries

has been recognized by the space agencies. Extensive

efforts and resources have been expended to meet these

contingencies by applying successful Earth-based clinical

practices. However, acclimation to the spaceflight

environment results in alterations in the cardiovascular,

endocrine, immune, and hemopoietic systems as well as

metabolic changes. These changes will drastically alter

the response of the patient to injury. These alterations

have to be considered in clinical guidelines to care for the

injured space traveler, and must be validated on the

ground and in flight in clinically relevant animal models.

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Symposium III: Planetary Biology and

Terraforming

Paul Todd and David Thomas, Editors


PLANETARY BIOLOGY AND TERRAFORMING

Paul Todd

SHOT, Inc., 7200 Highway 150, Greenville, IN 47124 ABSTRACT

Planetary biology can be considered in terms of four

components: (1) planetary protection, (2) the search for life, (3)

human life support and (4) ecopoiesis and terraforming.

Initially, contamination of a planet with Earth life is to be

minimized in order to facilitate a search for planetary life.

Meanwhile, humans, if present, must be sustained.

Subsequently, large-scale modifications of a planetary

environment can be considered. “Ecopoiesis” is a term

introduced by McKay and Haynes to describe the initiation of a

living, self-sustaining ecosystem in a planetary (Mars)

environment. “Ecosynthesis” refers to the development of an

ecosystem that includes succession (ecosystem maturation by

the replacement of organisms). “Terraforming” refers to

creating an Earth-like world and includes planetary engineering.

A considerable amount of debate, discussion and publication has

been devoted to these subjects, but, at least in the case of

ecopoiesis, there has been very little, if any, experimental

research. The speakers in this session address, in order, issues

of planetary environments and habitability with reference to

Earth analogues, the role of extremophiles as pioneer organisms

in ecopoiesis, and the concept of succession as it applies to

terrestrial ecology and ecosynthesis.

KEYWORDS planetary biology, ecopoiesis, ecosynthesis, terraforming,

Mars, extremophiles

INTRODUCTION

Mars is considered the ultimate target for terraforming.

There are at least four levels of inquiry concerning biology

and Mars:

(1) Planetary protection, contamination and quarantine

issues (NRC, 1992; Rummel, 2001),

(2) The search for life on Mars (Banin and Mancinelli,

1995; Ivanov & Lein, 1995; Koike et al., 1995),

(3) Human expeditions to Mars and biological life support

(Meyer & McKay, 1984, 1989) and

(4) The terraforming of Mars starting with ecopoiesis

(McKay, 1982; Fogg, 1995a, b; Haynes, 1990; McKay and

Haynes, 1990; Haynes and McKay, 1992; McKay et al.,

1991; Hiscox, 1995) and continuing with ecosynthesis

(Graham, 2005),

The discussions that follow are concerned with the fourth

subject, terraforming.

A variety of claims have been made concerning the future

of ecopoiesis (starting an ecosystem) and terraforming

(creating an earth-like environment) – highly

controversial subjects. Planetary protection is a

significant component of the ecopoiesis debate. One of

the significant outcomes of performing research in this

area is expected to be the informing of a scientific

community consensus concerning these subjects.

Today’s thinkers hold varying opinions concerning

ecopoiesis, the process of evolving a physical and

biological environment that can lead to “terraforming” –

the deliberate introduction of terrestrial-type ecosystems

on remote celestial bodies such as planets, moons and

asteroids (Haynes, 1990). On one extreme, principles of

planetary protection prohibit the introduction of any

living organisms onto Earth’s neighbors in the solar

system while, on the other extreme advocates of

terraforming propose the early modification of Mars (for

example) to initiate processes that will make the planet a

more hospitable place for humans should they decide to

go there (Zubrin and Wagner, 1996). Either way,

knowledge is lacking, and experimental testing is non-

existent, related to the basic understanding of the

ecopoiesis process. Since the implementation of any

concepts related to ecopoiesis would occur several

decades in the future, the experimental study of

ecopoiesis now would seem an ideal objective. While

ethical considerations are outside the scope of this

presentation, such a research program could inform this

debate.

It is of great value to cause the visions of the prophets of

the field to be extended an entire century forward.

Meanwhile, a series of benign, but difficult experiments

(laboratory, field and theory) can and should be

performed. The undertaking of ecopoiesis/terraforming

research can be considered a direct response to the challenge

originally put forth by Christopher McKay and the late

Robert Haynes: “ . . if it is decided to implement such a

program of planetary engineering, a slow and conservative

approach is essential. Sufficient time must be allowed for a

wide range of studies of Mars as it exists at present, and for

careful planning, modeling and ‘pilot-plant’ trials (where

possible) of all successive steps in the enterprise.” (Haynes

& McKay, 1995). This challenge calls for research that

includes biological experiments and theoretical modeling

directed at the implementation of the enabling notions in

terraforming offered by Fogg (1995), McKay et al.(1991),

and Boston and Thompson (1991) among others. To date,

only individual components of ecopoietic systems have

been investigated – a few species of microorganisms and

certain primitive and vascular plants.

____________________

* Correspondence to: Paul Todd

SHOT, Inc.

7200 Highway 150

Greenville, IN 47124


Phone: 812-923-9591x242; Fax: 812-923-9598

P. Todd — Planetary Biology and Terraforming


THE MARTIAN ENVIRONMENT AND

PLANETARY ENGINEERING

A set of ecopoiesis research principles is offered, and they

are listed in the order in which they would logically be

implemented in an integrated ecopoiesis research program

(Boston et al., 2004):

1. Assume the ultimate problem to be addressed is

the terraforming of the surface of Mars.

Mars lies within the habitable zone of the solar system

(Fogg, 1998, McKay et al., 1991) and is second only to

Earth in biocompatibility. If the laws of physics and

chemistry are universal laws, and if the sequence of

events that led to the origin of life on Earth occurs

elsewhere, Mars may thus be considered a prime

candidate for both the existence of life and human

settlement (Meyer and McKay, 1995, 1984, 1989; Zubrin

and Wagner, 1996). The chemical-elemental inventory

of Mars' surface (both absolute and relative

concentrations, Table I) is quite similar to the Earth's crust

with the exception of a potentially low nitrogen content

(Banin, 1989; Banin et al., 1992; Banin and Mancinelli,

1995).

The primary physical properties of the Martian

environment that are a challenge for the survival and

Table I. Physical properties of Mars______________________________________

Pressure 6 – 15 millibars (0.6 – 1.5 kPa)

Global temperature range -133oC -- +23

oC

Mean Solar irradiance 589 W m-2

(~50% of Earth's value)

Solar UV flux 6500 ergs cm-2

(190 – 300 nm)

(<190 nm absorbed by CO2)

Water Vapor 0.03% of atmosphere mass (saturated at 8.1 mbar)

Gravitational constant 0.387g

Escape velocity 5km s-1

Day length 24h 37min 22s

Year length 687 Earth days

Equatorial diameter 6788 km (~1/2 Earth)

Mass 6.4 X 1023

kg (1/10 Earth)

Water ice North polar ice cap

Atmospheric composition

CO2 95% (<0.05% on Earth)

N2 2.7% (78 % on Earth)

O2 0.13% (21% on Earth)

CO 0.07%

Argon 1.6%

Neon ppm

Krypton ppm

Xenon ppm

Lithosphere elemental composition excluding oxygen (most elements are in oxides)

SULFUR 3.1%

MAGNESIUM 5%

CALCIUM 4%

IRON 12.7%

POTASSIUM 0.08%

CHLORINE 0.7%

SILICON 21%

ALUMINUM 3%

SODIUM 2.3%

PHOSPHOROUS 0.3%

Mn, Co, Cu, I, Zn, B, V, Mo, Trace

F, Cr, Se, Tl, Br

CARBON, NITROGEN Low levels (Abundant on Earth)



growth of terrestrial organisms include; low atmospheric

pressure (~ 7 millibar), low temperature ranges (-100°C to

- 5°C, with a rare high of 26°C), no observed liquid water

to date (Soderblom, 1992), unfiltered ultra violet (UV)

radiation, no significant ozone layer, no organic material,

and high concentrations of surface oxidants (Kieffer et al.,

1973; Rothschild, 1990; Banin & Mancinelli, 1995). The

atmospheric pressure on Mars, mostly due to CO2, varies

from approximately 7.4 to 10 millibar (mbar). Extremely

low pressure can damage organisms and affect efficient

DNA repair (Ito, 1991; Koike et al., 1991).

2. Starting conditions to be studied on Earth should

resemble those expected to exist at the best possible

location on the surface of Mars (a temperate location at

low altitude or near potential water sources at the north

polar cap).

The physical and chemical properties of the planet are

summarized in Table I.

On Mars there are difficult trade-offs between water

availability and temperature. In general, water is

available only at or near polar caps because the

temperature is sufficiently low to prevent sublimation at

such low pressure. Liquid water may exist transiently in

the Martian lowlands at mid-day in the summer months

on occasions when the pressure (15- 20 mbar) and

temperature (2-20oC) are both above the triple point of

water – that unique combination of pressure and

temperature at which all three phases co-exist, 0.006oC

and 6.1 mbar (Kuznetz and Gan, 2002).

3. The regolith must be heated to a temperature

compatible with the most robust form of terrestrial life at

low temperature.

Shortly after the Viking missions, Averner and MacElroy

(1976) and Kuhn, Rogers and MacElroy (1979) launched

a program to study the habitability and planetary

ecosynthesis of Mars by designing an energy-balance

model. The initial studies evaluated the effects of a

Martian atmosphere on photosynthetic microorganisms by

approximating equatorial equinox parameters for solar

and thermal radiation, convective and conductive energy

transport and evaporative cooling. These early

investigations took into consideration the effect of diurnal

variation on the organisms’ temperature, transpiration and

photosynthetic rates, all of which are equally applicable to

current ecosystem models. Initial heating of the regolith

is to be achieved by the addition of black material (soot,

soil or microorganisms) to the near-polar ice to initiate

melting of water and CO2 to increase atmospheric

pressure and solar heat retention, and heating of the

atmosphere is achieved by the addition of fluorocarbon

gas into the planetary atmosphere, a very effective

greenhouse gas. Laboratory investigations of heating

strategies will require a combination of experiment and

modeling, since the laboratory scale differs significantly

from the planetary scale.

4. The first organisms must derive energy from the

mineral content of the regolith and/or sunlight, and their

metabolism must produce a net increase in greenhouse

gas(es).

Since the advent of MacElroy's program in 1975, several

living systems on Earth have been identified as potential

pioneer organisms for the Martian environment. These

biota may be classified as "extremophiles" and include

classes of anhydrobiotes (Dose et al., 1995), cryophiles,

thermophiles and halophiles, to name a few. Examples

include cryophiles found in the ice-covered lakes in the

Bunger Hills Oasis of Antarctica (Andersen et al., 1995;

McKay, 1991, 1993) and in Arctic permafrost in the

Kolyma lowland of Siberia, Russia (Ostroumov, 1995;

Soina et al., 1995). Thermo- and halophiles including

endolithic bacteria found in extreme arid deserts, high

Alpine rocks, and hot springs (Boston et al., 1992;

Friedmann, 1982, 1986; Friedmann & Weed, 1987),

evaporite biota found in the hypersaline habitats

(Rothschild, 1990; Rothschild et al., 1994) and cryptic

microbial mats in the Guerrero Negro, Baja California

Sur, Mexico.

An effective ecopoietic strategy requires knowing the limits

of habitability for Earth organisms as we understand them,

especially under conditions of limited water and oxygen

(Banin and Mancinelli, 1995; Carr, 1987; Fogg, 1995;

Hoffert et. al., 1981). To understand these limits, the

broadest possible range of genetically encoded capabilities

must first be considered, then, in a narrower way, conditions

that are and are not destructive to Earth's most extremophilic

organisms are to be identified (Hiscox, 1995; Hiscox and

Thomas, 1995). Using Mars regolith simulant (Allton et.

al., 1985) and planetary environment simulation chambers

(Sagan and Pollack, 1974), the vigor of such organisms can

be tested (Koike et. al., 1991; Kuhn et. al., 1979;

Hawrylewicz et. al., 1962; Thomas et al., 2006).

5. The first organisms must be capable of

withstanding, or be protected from, the ultraviolet and

ionizing radiation present on the Martian surface and the

high CO2 of the atmosphere.

The Martian atmosphere, devoid of an ozone layer, allows

solar ultraviolet (UV) radiation to penetrate to the Martian

surface in wavelengths ranging from 190 to 300 nm. In

living cells UV radiation is absorbed by nucleic acids to

activate the chemical formation of various adjuncts that

inhibit replication and transcription of DNA and can be

lethal (Lindberg & Horneck, 1991). The high

concentration of atmospheric carbon dioxide (~95.3%)

would, in most terrestrial organisms cause a low

intracellular pH, resulting in damage to cellular

components, proteins and metabolism.

6. Organisms early in succession should produce

significant amounts of O2.

At some point in the early stages of succession

(ecosystem maturation by the replacement of organisms),

O2 will be essential to drive the dark reactions of

photosynthetic organisms, which are O2 concentration

dependent and to support the metabolism of heterotrophic

aerobes (Graham, 2005).



7. Higher plants late in succession will need to be

studied under conditions that pioneer organisms

preceding them can create.

Cold-weather species that do not require pollination or

can pollinate without insect assistance, that can function

in low O2 and high CO2 and extreme drought and that are

useful to humans are among those to be considered and

investigated (Graham, 2005).

THE PRESENTATIONS IN THIS SERIES

The subsequent presentations in this series address, in a

similar sequence, the importance of and evidence for

liquid water on Mars, the habitability of extraterrestrial

and extreme terrestrial venues for Earth life, the capability

of extremophiles to serve as pioneer organisms in

ecopoiesis and the succession of organisms in

ecosynthesis required to fulfill the ultimate goals of

terraforming.

ACKNOWLEDGMENTS

Assistance with literature review efforts by Penelope

Boston and Kelli McMillen are gratefully acknowledged.

Material for this presentation was developed under the

support of subcontract agreement 07605-003-026 with

NASA's Institute for Advanced Concepts (NIAC), a

program of the Universities Space Research Association

(USRA) funded by NASA contract NAS5-03110 and also

with the support of SHOT, Inc.

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LAST PLACE TO BOIL AWAY, FIRST PLACE TO LOOK: THE HUNT FOR WATER

AND LIFE ON MARS

Lawrence H. Kuznetz

Space Spinoffs, Inc., Universities Space Research Associates (USRA) NASA Johnson Space Center, Houston,

Texas 77058

ABSTRACT

The primary goals of the robotic exploration of Mars are to

characterize the geology and climate of the planet; determine

whether or not life ever arose and establish the planet’s

suitability for human exploration. Inseparable in this quest is the

hunt for water and ancient seas. To date, the landing sites

associated with the quest have been driven in equal measures by

geology and conservatism (ie, a safe place in which to land).

This study uses principles of physics and thermodynamics to

target alternative landing sites that might maximize the

probability of finding water, past and present and extant life as

well. The phase diagram of water together with predictions of

temperature and pressure at specific locations is used with

elevation data from MOLA (Mars Orbiter Laser Altimeter) to

predict locations where water in liquid form last existed on the

surface and may, with accompanying extremophiles, exist today.

Calculations at Candor Chasma and Hellas are presented as

examples of how the principles of physics may be applied to

choose landing sites of the future that would optimize the goals

of robotic exploration.

KEYWORDS Mars, liquid water, landing sites, water phase diagram,

atmospheric pressure, Candor Chasma, Hellas, Mars probes

ABBREVIATIONS

MGS: Mars Global Surveyor, MOLA: Mars Orbiting

Laser Altimeter, RS: Radio Science Instrument on MGS,

VL1: Viking Lander 1, VL2 Viking Lander 2

INTRODUCTION

Follow the water: long the mantra of NASA’s robotic

program to explore Mars. Follow the water to discover

life, extinct or extant, and the other things. To date,

geology, and the safety net of relatively flat landing sites

have played the major role in looking for water, ancient

or otherwise. But thermodynamics has a role to play, too.

Hence the title, Last Place to Boil Away, First Place to

Look. Consider the following: If you were a liquid water

droplet millions or billions of years ago, and realized the

atmosphere above you was disappearing, and oceans were

boiling away, where would you rather be, in a high place

or low place, in a warm place or cold place? Physics

dictates low and warm, since that’s what it takes to stay

liquid. The lower the altitude, the higher the pressure,

and only the combination of higher pressures and warmer

temperatures will keep you and any life forms that you

support from freezing, boiling or sublimating away.

Granted, such places are rare on Mars today but they were

plentiful in the past. And they may exist today. Finding

them is the subject of this presentation.

TARGETS OF OPPORTUNITY, PAST AND

PRESENT

Logically, if you were an alien from Earth landing on

Mars, such as Viking, Pathfinder, Spirit or Opportunity,

and your task was to “follow the water,” you would likely

take the low, warm road to find it because that’s where

liquid water and any life forms within would have

survived the longest. This road has not been taken for

two reasons. First, low warm places are not as easy land

in; and second, we have assumed there is no liquid water

left on the surface today anyway and decided to look for

signs of past water only. Past or present, physics can help.

It leads us, in fact, to an entirely different set of landing

sites from the ones we have visited thus far. To

understand why, we must start with the water phase

diagram.

THE PHASE DIAGRAM OF WATER AND

CONDITIONS ON MARS

1. There is no liquid water on the surface of Mars

today; the pressure is so low that ice sublimates to water

vapor without ever passing through a liquid phase. This

statement, popularized by Norman Horowitz, the Cal

Tech Nobel laureate, reflected the thinking in the years

immediately prior and subsequent to the Viking missions.

The recent article in Nature, Evidence from HRSC Mars

Express for a Frozen Sea (Murray, et. al., 2005}, is a case

in point. The authors surmised a frozen sea at southern

Elyssium (+5o south, 150

o east) close to the Martian

equator. While adding to the growing body of knowledge

about recent geologic activities that lend credence to

active hydrothermal processes (with tantalizing hints of

microbial life), it repeats an oft-stated misconception that

detracts from its central theme. Specifically, the

statement, "Ice is unstable at the surface of Mars due to

sublimation in the 6-mbar atmosphere," is misleading.

While a frozen sea certainly could exist at temperatures

below freezing, ice is definitely not unstable. A close look

at the water phase diagram of Figure 1 reveals why. The

triangular region marked off shows the window of

opportunity in which water can exist as a liquid under

Martian datum level (mean surface altitude) conditions

today. At a 10mbar surface pressure, the liquid state exists

between 0 and 7oC. This window will be narrower at

lower pressures and wider at higher pressures but as long

as the pressure remains between 6.1 and 10 mbar, it exists

____________________

* Correspondence to: Lawrence H. Kuznetz

Space Spinoffs, Inc.

c/o NASA Johnson Space Center

Houston, TX 77058


Phone: 510-205-8056

L. Kuznetz — The Hunt for Water and Life on Mars


Figure 1. A selected portion of the water phase diagram (Weast, 1973-74). The phase diagram illustrates when pure water exists as a

solid, liquid, or vapor as a function of pressure and temperature. Triangle at the left end of the liquid region indicates conditions for liquid

water at Mars datum level.

nonetheless. Surface pressure data from the Viking and

Pathfinder landers have always been in this window, with

a 6.7 to 10 mbar pressure range. The above authors’ claim

of instability and sublimation is feasible only under two

conditions:

1. At higher elevations (hundreds or thousands of

kilometers above datum level) where the total pressure

will always be below the triple point of 6.1 mbar or

2. If the partial pressure of water vapor cannot build to

the total pressure within the boundary layer above the ice

source.

Neither of these conditions likely exists at Elyssium, the

site of the above authors’ proposed frozen lake. Firstly,

Elyssium is located approximately 2 km below datum

level, where total pressure should always be above the

triple point and secondly, unless strong winds exist to

disrupt the boundary layer, the partial water vapor

pressure should always build to levels approaching total

pressure. In other words, the surface of this frozen lake

should pass through a liquid phase as temperatures climb

above freezing. If this happens, the fundamental question

then becomes the fate of this liquid phase. Will it boil,

evaporate, or pool after it melts, and for how long?

This question is crucial because it speaks to nothing less

than the stability of liquid water and its link to life. If

liquid water can still form and pool on Mars then the

presence of such standing water increases the possibility

of extant life. If it is the evaporation (boiling) rate, not the

sublimation rate that determines the fate of the standing

water, one must ask the next question: Assuming frost,

snow or ice is available at or near the surface (an entirely

different question not addressed here), then under what

conditions will it form into a liquid and remain that way

for more than a few seconds?

EVAPORATION RATE AND THE LIKELIHOOD

OF STANDING WATER ON MARS TODAY

Studies by Kuznetz and Gan (2000, 2002); Quinn and

McKay (2000); Hecht (2002) and most recently by Moore

and Sears (2005) exposed ice to simulated Mars datum

level conditions and demonstrated conclusively that it will

pass through a liquid phase as long as the temperature

rises above freezing and remains below the boiling point.

Typical results are shown in Figure 2, which shows water

drops falling from an ice cube above 0oC in a Mars

vacuum jar (desiccator). Kuznetz and Gan (2002) also

photographed the melting sequence of frost on a

simulated Martian rock as it first forms a ring, then

coalesces to stable water droplets after it melts. In these

studies, water was not only observed to form after the ice

or frost melted, but once formed, lingered for substantial

time periods due to slow evaporation rates. How slow is

this evaporation rate? Kuznetz and Gan (2002) found

evaporation rates averaging 0.023 g/cm2-h in a Mars

simulation vacuum jar controlled to temperatures between

-2 to +20oC, and pressures of 6.3-14 mbar with and

without advection. Quinn and McKay (2000), Ingersol

(1970) and Hecht (2002) found similar scales of

evaporation, and Moore and Sears (2005) reported rates

around 1 mm/hr. These rates are slow enough to permit

standing water to remain liquid as long as it does on

Earth, assuming ice is present and a heat source above the

freezing point is there to melt it. Climate modeling studies

by Haberle (2000) supported these lab findings, predicting

in excess of 135 days per year (668 Earth days) during



which conditions along Mars’ equatorial region could

support liquid water (innermost contour areas of Figure 3,

surrounded by lines labeled “135”). These are not

"unstable," conditions as the Nature authors maintain, but

"metastable," as Hecht (2002) pointed out. (A glass of

water on a table top on Earth is similarly metastable).

Figure 2. Liquid water droplet formation in a vacuum

desiccator jar under simulated Martian conditions. The vacuum

desiccator is evacuated to 10 mbar and cooled with solid CO2,

which also provides Mars atmosphere stimulant. (Kuznetz and

Gan, 2002).

Examining the liquid-phase area in the phase diagram of

Figure 1 provides further insight. If standing water drops

can linger between 0 and 7oC at 10 mbar, they should

linger longer at the higher pressures that occur at lower

elevations on Mars. The window of opportunity at 15

mbar, for example, is 7 times greater than at 6.3 mbar (0

to 14.9oC, vs 0 to 2

oC on the phase diagram before

boiling occurs) and 12 times greater at 25 mbar. By

inference, the odds of standing water in the northern

lowlands should be substantially greater than in the

southern highlands, an intuitively obvious finding since

water flows toward the lowest level. This is predicated by

the temperature cooperating, i.e., staying between the

freezing and boiling points for extended periods of time at

the lower elevations.

THE INFLUENCE OF ELEVATION

Returning to our central thesis, if the window of

opportunity for liquid water increases at higher pressures,

where then are the locations associated with these

pressures? These would be the places where water would

have boiled away last, leaving its most recent signature,

as well as fossil traces if they exist. These would also be

the places where standing water and extant life, if it exists

at all, may reside. Insight to such locations is provided by

a combination of data from Viking Lander 1 ; MGS’

MOLA- Mars Global Surveyor’s Mars Orbital Laser

Altimeter, which shows the depth of Candor Chasm in

Figure 4; and by the MGS’ Radio Science instrument,

which shows the depth-pressure profile over Hellas in

Figure 5.

Figure 3. Predicted durations (given in Mars sols per year on

each contour line) suitable for liquid water brine on Mars. From

the Mars Global Climate model (Haberle, 2002).

Three approaches can be used to employ these data sets in

the search for optimum locations. In the first, Viking

Lander 1 (VL1) pressure and elevation data at Chryse

Planitia are referenced against elevations at other

locations derived from the Mars Orbital Laser Altimeter

(MOLA). The difference can be used to extrapolate the

pressure at these other elevations. In the second approach,

pressures measured by the MGS radio occultation

instrument at these same surface locations can be

compared to the pressures deduced by Approach 1. In the

third, the logarithmic relationship between altitude and

pressure, as shown in Figure 5, can be extrapolated to

lower depths below datum level and compared to the

other approaches. Examples of how this might be done for

Candor Chasma and Hellas follow.

CANDOR CHASM, -5 KM

Figure 4. The topography of Mars from MOLA, with a closeup

of Candor Chasm, showing elevation on a graded scale.

(http://ltpwww.gsfc.nasa.gov/tharsis/Mars_topography_from_M

OLA/M_-80_-70_-10_0.html)

In Approach 1, the elevation of Viking Lander 1 (VL1) is

known from MOLA to be -3683 meters, while the peak

pressure recorded by VL1 was 9.0 mbar (near Ls=270,



northern winter solstice). Extrapolations of pressure at

lower elevations were made assuming an atmospheric

scale height of 13 km (corresponding to a temperature of

250o K). The pressure calculated this way at Candor

Chasma, located at –5000 meters (about 1.3 km below

VL1), is roughly 10 mbar (~9 x exp(1.3/13) = 9 x 1.11).

The pressure at the low point in Hellas, on the other hand,

between –7500 and –8000 meters, could be as high as 14

mbar (using a low point for Hellas of 4.5 km below VL1

yields ~9 x exp(4.5/13) = 9 x 1.414, or 12.7 mbar.

-10

0

10

20

30

40

50

1 10 100 1000 10000

PRESSURE, PASCALS

KM

AB

OV

E S

UR

FA

CE

Figure 5. Pressure as a function of altitude at Hellas from MGS

Radio Occultation. Pressure extrapolated to lowest altitude of

about -8 km is about 1400 Pascals, equal to 14 mbar.

(http://nova.stanford.edu/projects/mgs/late.html)

Approach 2 uses independent pressure measurements

from MGS radio occultation data. Radio occultation

employs microwave radiation transmitted through the

Martian atmosphere by MGS prior to entry and just after

exiting the backside orbit where loss of signal (LOS)

occurs (hence the name occultation or hidden). Analyzing

the phase shift of these waves provides pressure for

specific longitudes, latitudes and time of day. Data for

Hellas at a depth of –6.86 km below datum was recorded

at 11.4 mbar, for example (see Table 1 and Figure 5).

Approach 3 simply uses the trend line of Figure 5 to

predict pressures at lower elevations by extrapolating the

trend line, in this case, 8 km below datum at Hellas as

shown in the Figure.

Pressures deduced from the above analyses predict ~10

mbar 5 km below datum level at Candor Chasma and ~13

to 14 mbar at the low point of Hellas (-8 km). It should be

noted that pressures in Approach 1 have a 10% inherent

error due to uncertainties in scale height and atmospheric

mass distribution versus latitude and longitude in a given

season. Errors in Approach 2 associated with MGS Radio

Occultation near the surface are ~ 1% for large areas and

increase at abrupt topographical features such as Candor

due to vertical resolution of the MGS oscillator. Errors in

Approach 3 result from assumptions of constant density

and curve extrapolation.

Table I. Martian Weather Observations at Hellas

(http://nova.stanford.edu/projects/mgs/late.html)

Date of Measurement: 04-01-2004

Time of Measurement: 09:23 GMT

Local Time on Mars: 03:41

Latitude: 39.9 degrees S

Longitude: 66.9 degrees E

Elevation: -6860 meters

Surface Temperature: -77.4 Celsius (-107.3

Fahrenheit)

Surface Pressure: 11.40 millibars

Martian Season: Early Fall

DISCUSSION AND SUMMARY

Mars is a fascinating planet in many respects, one of them

thermodynamically. A glass of water there could

sublimate, freeze, boil or remain liquid for hours, perhaps

days on end. Depending on location, all four processes

could occur at the same place over the course of a single

day. MGS Radio Science Weather Reports have observed

a preponderance of such locations from orbit, typically at

higher elevations above datum level. The Landers, on the

other hand, (VL1, VL2 and Pathfinder) have only seen

pressures above the triple point, probably owing to the

lower elevations at their landing sites.

This study has focused on using the water phase diagram

and data from surface probes and orbiters to identify

landing sites that could maximize the probability of liquid

water, past and present. Sites such as Candor Chasma and

Hellas have been shown to be locations where pressures

of 10-14 mbar could be routinely expected. The heat trap

effect at depressions such as these will also drive up the

temperature. The Inner Gorge of the Grand Canyon, for

example (elevation 670 meters), is substantially warmer

than the 2300-m high South Rim. In mid-April the

daytime temperature in the Inner Gorge exceeds 85oF and

cools only slightly at night, while night-time temperature

on the South Rim fall below the freezing point of water.

By inference, this suggests daily temperature excursions

at Candor and Hellas, taken together with higher

pressures should translate to increased residency time in

the liquid water envelope between freezing and boiling.

The appeal of Hellas and Candor increases with

hypotheses such as Hynek’s (2004), that vast ancient seas

may have covered Hellas in the distant past, and Malin

and Edgett’s (2000a,b) statement that MGS MOC

observations strongly indicate that the deepest parts of

Vallis Marineris were not brecciated but layered. If true,

the bottom of these locales would have been the last

refuge for large amounts of water on Mars; any life that



may have existed at the time, and their fossilized remains

that may exist today. All of this begs the question asked

by physics: Might not these and other places like them be

the most ideal landing sites to look for liquid water and

life on Mars today? If so, identifying and targeting these

areas should be a priority. Equipping future rovers with a

suite of instruments that can “sniff” liquid water should

also be considered. After landing, such a rover might

“predict-correct” over a wide area like a “water witch”,

fine tuning the location of surface or subsurface water

with a suite of instruments such as an alpha X-ray proton

spectrometer, temperature, pressure and water vapor

sensors.

The human exploration program would also benefit from

a high probability water site. Other than the obvious need

for a reliable, low cost water source, the design of a blue

collar space suit may well hinge on higher pressure,

warmer temperature locales. Current suits function by

sublimation, a process that will not work at datum level or

below. They are also far too heavy. A key to cutting mass

and complexity is to design a suit to its climate of intent,

as protective clothing does on Earth. Studies indicate that

a suit designed for Candor or Hellas might be more like

an Arctic coverall than the thermos bottle-like designs

that exist today (Kuznetz, 1989). Pressures above the

triple point and warmer temperatures may not only be the

key to finding environments that support liquid water and

life, but maximize human exploration potential as well.

ACKNOWLEDGEMENTS

The author is grateful to Drs. David Hinson and David E.

Smith of the MGS Radio Science Team for suggesting

approaches to use the MGS Radio Occultation and VL1

data to correlate longitude, latitude and elevation with

atmospheric pressure, and for providing error estimates in

MGS MOLA and RS data.

REFERENCES

Haberle, R.. 2000. On the stability of liquid water on

present day Mars. Proceedings of the First NASA Ames

Astrobiology Conference, NASA Ames Res Center,

Moffett Field, CA.

Hecht, H. 2002. Metastability of liquid water on Mars.

Icarus 156: 373-376.

Holman, J.P. 1990. Heat Transfer, 7th

ed. McGraw Hill,

San Francisco.

Hynek, B. 2004. Opportunities Great Lakes. University of

Colorado report, Astrobiology Magazine.

Ingersoll A.P. 1970. Mars: Occurrence of liquid water.

Science 168: 972-973.

Kuznetz, L. 1991. Space suits and life support systems for

the exploration of Mars. National Science Foundation

Fellowship, NASA Ames Research Center, Moffett Field,

CA.

Kuznetz, L.H. and Gan D. 1999. The hunt for liquid water

on Mars today. Third Mars Society Conference, Mars

Society, Boulder, Colorado.

Kuznetz, L. H. and Gan, D. 2002. On the existence and

stability of liquid water on the surface of Mars today.

Astrobiology 2 (2): 183-195.

Malin, M. and Edgett, K., 2000a. Evidence for recent

groundwater seepage and surface runoff on Mars. Science

288: 2330.

Malin, M. and Edgett, K., 2000b. Sedimentary rocks on

early Mars. Science 290: 5498. 1927

McKay, C. and Stoker, C. 1989. The early environment

and its evolution on Mars: Implications of Mars. Reviews

for Geophysics 27 (2): 189-214.

Moore, S. and Sears, D. 2005. Stability of contemporary

liquid water on Mars. Journal of Geophysical Research

Planets, in review.

Murray, J., Muller, J., Neukum, G. Werner, S., Gassest, S.

2005. Evidence from HRSC Mars Express for a frozen

sea. Nature 434: 352-356.

Quinn, R. and McKay, C. 2000. Mars Analog

Experiment, Space Science Division, NASA Ames

Research Center, Moffett Field, CA.

Weast, R. (Ed.) 1973-74. Water Phase Diagram. Chemical

Rubber Handbook, 54th

ed., pp DD 158-160, Cleveland.


EXTREMOPHILES FOR ECOPOIESIS: DESIRABLE TRAITS FOR AND SURVIVABILITY OF

PIONEER MARTIAN ORGANISMS

David J. Thomas1, John Boling

1, Penelope J. Boston

2, 3, Kathy A. Campbell

1, 4, Tiffany McSpadden

1, Laura

McWilliams1, and Paul Todd

5

1Lyon College, Science Division, 2300 Highland Road, Batesville, AR 72501

2Complex Systems Research, Inc., P.O. Box 11320, Boulder, CO 80301.

3Department of Earth and Environmental Science, MSEC 208, New Mexico Institute of Mining and Technology,

801 Leroy Place, Socorro, New Mexico 87801. 4Newark High School, 1500 N. Hill Street, Newark, AR 72562.

5SHOT, Inc., 7200 Highway 150, Greenville, IN 47124.

ABSTRACT

Humanity is on the verge of having the capability of

constructively directing environmental changes on a planetary

scale. Within the foreseeable future, we will have the

technology to modify Mars' environment, and make it a

habitable planet. However, we do not have enough information

to determine the course of such an event. To our knowledge, no

known terrestrial organism has the capability of living on Mars'

surface under present conditions. However, with some

modification, Mars' environment could be brought into the

survival and growth range of currently known microorganisms.

Using the SHOT Ecopoesis Testbed, we performed

survival/growth experiments to determine the suitability of

potential pioneering life forms for Mars. Included among the

potential pioneers were five genera of cyanobacteria (Anabaena,

Chroococcidiopsis, Plectonema, Synechococcus and

Synechocystis), three partially-characterized Atacama Desert

heterotrophic eubacterial strains, and several desert varnish

isolates. Microorganisms were exposed to a present-day mix of

martian atmospheric gases, but at a pressure of 100 mbar (10

times Mars' current atmospheric pressure). Cultures were

inoculated into samples of JSC Mars-1 soil stimulant and

exposed to full-spectrum simulated martian sunlight. Day/night

temperature cycled from 26°C to -80°C and back. Preliminary

results indicate that both autotrophic and heterotrophic bacteria

can survive in the simulated engineered martian environment.

INTRODUCTION

In relation to terrestrial organisms, Mars' environment is

harsher than any on Earth. The combination of low

temperature, low atmospheric pressure, low moisture,

high atmospheric CO2 fraction and high UV flux is not

experienced by any known Earth organism. However,

Earth organisms may be found in environments that

include at least one of the martian environmental factors

(although Mars is still harsher in most respects). The dry

valleys of Antarctica provide a low temperature, low

moisture, high UV environment. Chile's Atacama Desert

is arguably the driest place on Earth. Some hot springs

and volcanic vents produce anoxic environments with

very high CO2 levels. Alpine and stratospheric

environments possess low pressure, low temperature and

high UV.

Each of these terrestrial environments harbors some form

of life. Organisms living there have adapted to the

extreme conditions, and those adaptations are genetically

encoded. In theory, genes encoding favorable

characteristics from one organism could be transferred to

another organism, which already possesses some other

characteristics for extreme environment survival. With

enough genetic alterations, we could conceivably "build"

a microorganism—a "marsbug"—that could survive,

reproduce and grow in a somewhat modified martian

environment.

Ten years ago, Hiscox and Thomas (Hiscox and Thomas,

1995) detailed some of the characteristics that an ideal

marsbug might possess. Here, we elaborate upon and

update these characteristics, and summarize some initial

experiments in which organisms with some of these

characteristics were tested under approximated martian

conditions.

DESIRABLE TRAITS FOR PIONEER

MICROORGANISMS

For the purposes of this paper, we assume that initial,

non-biological, modification of Mars' environment would

occur before pioneer microorganisms are introduced. At

minimum, Mars would have an atmospheric pressure of at

least 25 mbar, significant periods of above-freezing

temperatures, and significant bodies of liquid water

(Thomas, 1995). At 25 mbar, water boils at 9°C,

providing a narrow temperature window at which water

remains liquid. Also, recent studies suggest that Earth

bacteria do not grow at pressures below 25 mbar

(Schuerger et al., 2006a; Schuerger and Nicholson, 2005).

Even with initial modification, the martian environment

would be at least as harsh as the least hospitable

environments on Earth. Accordingly, pioneer

microorganisms would require many, if not all, of the

following characteristics (summarized in Table I).

Autotrophy. Mars has no known reserves of organic

materials. Pioneer organisms would need to make their

____________________

* Correspondence to: David J. Thomas

Lyon College, Science Division

2300 Highland Road

Batesville, AR 72501


Phone: 870-698-4269

D.J. Thomas — Extremophiles for Ecopoiesis


Table I. Desirable characteristics for pioneer martian microorganisms. The lists of microorganisms and references are examples

only, and are not meant to be exhaustive.

Characteristic Example Microorganisms References

Autotrophy: oxygenic

photosynthesis

Cyanobacteria, algae (Blankenship and Hartman, 1998; Burger-Wiersma

and Matthijs, 1990; Dismukes et al., 2001; Fay, 1983;

Goodwin, 1980; Ho and Krogman, 1982; Kaplan et

al., 1988; Tabita, 1994; Xiong and Bauer, 2002)

Autotrophy: chemosynthesis Nitrosamonas, Nitrobacter,

Thiobacillus, methanogens

(Atlas, 1997; Boston et al., 1992; Chyba and Hand,

2001; Northup et al., 2003; Stevens and McKinley,

1995)

Psychrophily Pyramimonas, Fragilariopsis,

Pseudo-nitzschia, Porosira,

Entomoneis, Nitzschia.

(Chen and Berns, 1978; Christner et al., 2003;

Daugbjerg, 2000; Gaidos et al., 2004; Gilichinsky et

al., 2003; McMinn et al., 2005; Morita, 1975)

CO2 tolerance Anabaena, Plectonema, Cyanidium,

Nannochloris

(Negoro et al., 1991; Seckbach et al., 1970; Seckbach

and Libby, 1970; Thomas et al., 2005)

Hypoxia tolerance Anabaena, Plectonema, Cyanidium (Seckbach et al., 1970; Thomas et al., 2005; Zehnder

and Svensson, 1986)

Carbonate dissolution Matteia, other cyanobacteria and

algae

(Crispim et al., 2003; Friedmann et al., 1993)

Denitrification Pseudomonas, Paracoccus,

Streptomyces

(Baker et al., 1998; Hart et al., 2000; Kumon et al.,

2002; Zumft, 1997)

Nitrogen fixation Anabaena, Chroococcidiopsis,

Cyanothece, Synechococcus,

Trichodesmium

(Almon and Böger, 1988; Berman-Frank et al., 2001;

Böhme, 1998; Fay, 1983; Friedmann and Kibler,

1980; Golden and Yoon, 1998; Klingler et al., 1989;

Mitsui and Cao, 1988; Olson et al., 1998; Potts et al.,

1983; Schneegurt et al., 2000; Wolk, 1988)

Osmotic tolerance,

desiccation resisistance

Chroococcidiopsis, Halomonas,

Klebsormidium, Oscillatoria,

Phormidium, endospore-forming

bacteria, extreme halophiles

(Abyzov et al., 1967; Billi et al., 2001; Billi et al.,

2000; Cockell et al., 2005; de Winder et al., 1989;

Frankenberg-Schwager et al., 1975; Imshenetsky and

Lysenko, 1965; Kraegeloh and Kunte, 2002; Olson et

al., 1998)

UV/ionizing radiation

resistance

Aspergillus, Bacillus,

Chroococcidiopsis, Deinococcus,

Mycobacterium, Rubrobacter

(Battista et al., 1999; Bauche and Laval, 1999; Billi et

al., 2000; Cockell et al., 2005; Ferreira et al., 1999;

Imshenetsky et al., 1967; Imshenetsky et al., 1977)

Hypobaric tolerance Aspergillus, Mycobacterium,

Pseudomonas, Staphylococcus,

Streptococcus

(Frankenberg-Schwager et al., 1975; Hawrylewicz et

al., 1967; Imshenetsky et al., 1977; Imshenetsky et al.,

1970; Silverman and Beecher, 1967)

Switchable genes and

pathways

Cyanobacteria, facultative

autotrophs, genetically modified

agricultural plants

(Garlick et al., 1977; Guay and Silver, 1975; Sarhan

and Danyluk, 1998; Sorokin et al., 2000)

own biomolecules from inorganic constituents via

photosynthesis or chemosynthesis. Photosynthesis would

replace the CO2 in Mars' atmosphere with O2—a key

process in planetary engineering (Graham, 2004; McKay,

1998; McKay et al., 1991; Thomas, 1995). Iron and

manganese-oxidizing chemolithotrophs produce dark-

colored byproducts (Dorn and Oberlander, 1981), which

decrease the albedo of rock surfaces, and would aid in the

heating of Mars (Boston et al., 2004).

Antioxidants. Oxygenic photosynthesis produces a

variety of reactive oxygen species (ROS) as normal

byproducts of electron transfer. In addition, Mars’

surface is currently a highly oxidizing environment,

presumably due to UV-produced ROS (Hunten, 1979;

Plumb et al., 1989; Yen et al., 2000). Currently, we do

not know whether initial engineering efforts would

change the oxidizing nature of Mars' surface. However,

other environmental stresses, such as chilling, increase the

amount of ROS produced by photosynthesis (Clare et al.,

1984; Hodgson and Raison, 1991a; Hodgson and Raison,

1991b; Thomas et al., 1999; Wise, 1995; Wise and

Naylor, 1987). Pioneer microorganisms would require

robust antioxidant systems in order to detoxify both

internally- and externally-generated ROS.

Psychrophily. Even under engineered conditions, Mars

will be cold. Truly psychrophilic organisms have

optimum growth temperatures below 20°C (Atlas, 1997).

The ability to grow at low temperature requires an entire

suite of enzymes that operate well at low temperature as

well as highly unsaturated membrane lipids

(Chattopadhyay and Jagannadham, 2001; Gerday et al.,

2000; Morita, 1975; Russell, 2000; Zecchinon et al.,

2001). Thus, psychrophilic autotrophs may be good

starting organisms to which other characteristics could be

added.

CO2 tolerance. Mars' atmosphere currently contains 95%

CO2, albeit at low pressure. We expect initial engineering



efforts to increase the total atmospheric pressure primarily

through the release of additional CO2 from the polar caps

and from possible carbonate rocks, which would further

increase the proportion of CO2. At high CO2 levels,

decreased pH becomes a factor as well. When CO2

dissolves in water, it forms carbonic acid with a pH of 5.5

- 6.0 at 1000 mbar. Fortunately, several cyanobacteria

and at least one alga survive and grow in >20% CO2 at

1000 mbar, and many microorganisms grow within the

pH range of 5 - 6 (Negoro et al., 1991; Seckbach et al.,

1970; Seckbach and Libby, 1970; Thomas et al., 2005).

Hypoxia tolerance. Mars' atmosphere contains very little

O2. Pioneer microorganisms will have to grow during

extended periods of hypoxia or anoxia. Even though

photosynthesis produces O2, plants require at least 50

mbar pO2 for proper development (due in part to the large

proportion of non-photosynthetic tissues) (Alpi and

Beevers, 1983; Atwell et al., 1982; Barclay and Crawford,

1982; Jackson and Drew, 1984). Most chemoautotrophs

also require O2 in order to oxidize inorganic molecules for

energy production (Atlas, 1997), thus limiting their initial

growth on Mars. Fortunately, most of the CO2-tolerant

photoautotrophic microbes, cited previously, also grow

anaerobically.

Carbonate dissolution. As mentioned previously,

carbonates are a potential source of atmospheric CO2.

Many cyanobacteria and algae cause damage to

limestone-based structures due to carbonate dissolution—

the conversion of carbonate ions into CO2 (Crispim et al.,

2003). If Mars has extensive carbonate deposits, such

organisms could speed the process of thickening Mars'

CO2 atmosphere while also providing oxygen. The

Antarctic cyanobacterium, Matteia, has been specifically

suggested for this purpose (Friedmann et al., 1993).

Denitrification. Although Mars' atmosphere could be

thickened by the addition of CO2, photosynthesis will

remove the CO2 and replace it with O2. An atmosphere of

pure O2 would result in catastrophic combustion. On

Earth, the atmosphere is buffered with N2, which is

largely nonreactive. If Mars possesses significant nitrate

deposits, these can be converted to N2 via the process of

denitrification (Thomas, 1995). Denitrifying bacteria use

nitrate in place of O2 for respiration, and thus are usually

hypoxia tolerant as well. Pseudomonas aeruginosa, a

heterotrophic facultative anaerobe, grows well and

denitrifies under conditions similar to those expected

during ecopoesis (Hart et al., 2000). Additionally, some

chemoautotrophs utilize nitrate in place of oxygen

(Oremland et al., 2002), thus avoiding the problem of

hypoxia and chemosynthesis mentioned previously.

Nitrogen fixation. Later in the process of planetary

engineering, biologically-usable nitrogen will need to be

recycled from the atmosphere. Nitrogen fixation

(Mancinelli, 1996)—the opposite of denitrification—

would be detrimental during the initial stages, but would

be required later in order to stabilize biological

communities (Thomas, 1995). Azotobacter vinelandii and

Azomonas agilis have the capability of fixing nitrogen at

pN2 as low as 5 mbar (Klingler et al., 1989). Many

genera of cyanobacteria also fix nitrogen (Böhme, 1998;

Fay, 1983; Friedmann and Kibler, 1980; Golden and

Yoon, 1998; Mitsui and Cao, 1988; Mulholland and

Capone, 2000; Olson et al., 1998; Potts et al., 1983;

Schneegurt et al., 2000; Zehr et al., 2001).

Osmotic tolerance. The initial bodies of water that form

on Mars probably will contain significant amounts of

dissolved salts; they may also be transient. Pioneer

microorganisms will need to tolerate osmotic stress, and

survive periods of desiccation. A large number of Gram-

positive bacteria form endospores that allow long periods

of dormancy under adverse conditions. Many non-spore-

forming bacteria also survive periods of desiccation. The

period between dormancy and the resumption of

metabolism should be as short as possible for pioneering

marsbugs so that they can take full advantage of periods

of favorable conditions. Some cyanobacteria undergo

seasonal desiccation and quickly become active during

wet periods (de Winder et al., 1989; Hershovitz et al.,

1991a; Hershovitz et al., 1991b; Scherer et al., 1984).

Ultraviolet and ionizing radiation resistance. Mars has

very little free O2 and only a very thin ozone layer that

changes with season and latitude (Barth et al., 1974;

Lefèvre et al., 2004), which would offer little or no

protection to potential marsbugs. Even though Mars

receives about half as much total solar radiation of Earth,

the amount of UV at the surface is much higher. Sun-

exposed surfaces on Mars receive sterilizing doses of UV

radiation. However, presumed planetary engineering

processes would release additional CO2 into the

atmosphere, and reduce the UV flux at the surface. For

example, Mars currently receives approximately 3.5

Watts m-2

UVC at the equator during the Vernal Equinox,

but with an atmosphere of 500 mbar CO2, the UVC flux

drops to 0.9 Watts m-2

(Cockell et al., 2000). Also, Mars

doesn't internally generate a magnetic field like Earth's,

and possesses only patchy remnant crustal magnetic fields

(Acuna et al., 1999; Acuna et al., 1998; Connerney et al.,

2004; Connerney et al., 2001). Although some localized

remnant magnetic fields are up to 30 times stronger than

those of Earth (Connerney et al., 2001), the lack of a

global magnetic field allows the surface of Mars to

receives more cosmic radiation than Earth – 20-30

centiSieverts/year (Cucinotta et al., 2001). A marsbug

would need mechanisms to resist radiation damage, and

repair any damage that occurs. Endolithic cyanobacteria,

which have been suggested as models for martian

microorganisms (Friedmann and Ocampo-Friedman,

1994; Friedmann and Ocampo-Friedmann, 1984; Thomas

and Schimel, 1991), protect themselves from UV by their

habitats (porous rocks) and by the production of

photoprotective pigments (Villar et al., 2005). The

bacterium, Deinococcus radiodurans, survives very high

doses of ionizing radiation due to its efficient DNA repair

mechanisms (Battista et al., 1999; Bauche and Laval,

1999; Levin-Zaidman et al., 2003; Venkateswaran et al.,

2000). Desiccation resistant strains of the



cyanobacterium Chroococcidiopsis also exhibit resistance

to ionizing radiation, presumably due to efficient DNA

repair as well (Billi et al., 2000). Protective pigments and

repair enzymes potentially could be genetically added to

other microorganisms, increasing their abilities to live in

the martian environment.

Hypobaric tolerance. Even after initial engineering

efforts, the atmospheric pressure of Mars would be far

lower than that of Earth. While many bacteria can

survive the desiccation that may occur at low pressure, a

marsbug would have to actively metabolize and grow. As

mentioned previously, bacteria may have a 25 mbar lower

limit for active growth (Schuerger et al., 2006a; Schuerger

and Nicholson, 2005). Bacteria have been isolated from

the upper atmosphere; however, their metabolic state

remains controversial (Imshenetsky et al., 1977;

Wainwright et al., 2003; Wainwright et al., 2004). They

may be actively growing without ever "touching ground,"

or they may be dormant while being transported through

the air. If these bacteria do indeed remain active while in

the upper atmosphere, they may have physiological

characteristics that could be useful on Mars.

"Switchable" genes. While the introduction of new

genes into organisms is relatively easy, their regulation is

more problematic. Many of the characteristics described

here are metabolic opposites (e.g., respiration and

photosynthesis, denitrification and nitrogen fixation). The

ability to control pioneer microorganisms over large areas

with the application of a very dilute controlling agent

would be highly desirable. Large numbers of genes are

switched on and off in organisms that have multiple

energetic pathways (Garlick et al., 1977; Guay and Silver,

1975; Sorokin et al., 2000). "Switchable" genes are also

desirable in genetically engineered agricultural crops to

make them resistant to environmental stresses (Sarhan

and Danyluk, 1998); research in this area may also benefit

planetary engineering.

A relatively small number of experiments have examined

the effects of parts of Mars' environment on potential

pioneer organisms, including UV radiation (Cockell et al.,

2005; Hansen et al., 2005; Nicholson and Schuerger,

2005; Schuerger et al., 2003), high CO2 (Hart et al., 2000;

Kanervo et al., 2005; Thomas et al., 2005), low pressure

and hypoxia (Boston, 1981; Kanervo et al., 2005; Paul et

al., 2004). However, these experiments only integrated 2-

5 of the variable conditions that would occur during

ecopoeisis on Mars. To address this problem, SHOT, Inc.

built a simulator capable of specifically replicating most

of the environmental parameters (with the notable

exceptions of gravity and cosmic radiation) on Mars

during all phases of planetary engineering (Thomas et al.,

in review).

ECOPOESIS SIMULATIONS

The SHOT Martian Environment Simulator has been

operational since May 2005. As of October 2005, six

experiments have been performed with durations of 24

hours to six weeks (Thomas et al., accepted for

publication). The initial experiments were primarily to

determine logistical and analytical needs for longer-

duration research, but we also determined short-term

survival and growth characteristics for a variety of

heterotrophic and autotrophic bacteria.

A 24-hour day was used in all experiments. Illumination

was provided by a xenon arc lamp (Sylvania 69263-0

Short Arc Lamp, XBO, 1000 W/HS OFR) fitted with a

solar filter that provided a close approximation of solar

radiation. Photosynthetically active radiation at sample

level ranged from 15 µmol photons m-2

s-1

in the shaded

region to 1000 µmol photons m-2

s-1

in direct light. Total

ultraviolet radiation (250-400 nm) was 1.7 µmol photons

m-2

s-1

in the shaded region, and 50 µmol photons m-2

s-1

in direct light. The temperature regime cycled from a low

of -80°C to a high of 26°C, diurnally. This approximated

the lower latitudes of Mars during the vernal equinox

(Carr, 1996), but probably is more extreme than what

would occur after the initial stages of planetary

engineering. As of October 2005, all of the experiments

were performed at an atmospheric pressure of 100 mbar—

10 times Mars' current highest pressure, but only 10% of

Earth's atmospheric pressure. For experiments of 14 days

or less, we used simulated atmosphere of 95% CO2, 2.7%

N2, 1.6% Ar and 0.13% O2 (Owen et al., 1977). For

longer-duration experiments, we used an atmosphere of

100% CO2. In most of the experiments, water was added

(1 mL per day or less) in order to maintain atmospheric

water saturation.

Cyanobacteria stock cultures were grown in liquid BG-11

medium, and diluted to A720 = 0.25 with fresh BG-11.

Heterotrophic bacteria stock cultures were grown in

trypticase soy broth (TSB), and diluted to A720 = 0.25 with

fresh TSB. Several sample configurations were tested,

including open trays, multi-well tissue culture plates and

arrays of individual sample containers. Individual liquid

cultures or mixtures of cultures were added to sterile JSC

Mars-1 simulant (5-15 g, depending on the container) to

the point of saturation. Desert varnish and cave

microorganisms were grown on BG-11 agar. Agar

cultures were than macerated and mixed with JSC Mars-

1. While samples were in the simulator, parallel control

samples were kept at 4°C in darkness. In cases where

samples could not be analyzed at the SHOT facility, the

samples were placed on ice and transported by car or by

overnight courier.

Samples were analyzed for esterase activity via an assay

of fluorescein diacetate (FDA) hydrolysis (Adam and

Duncan, 2001; Schnürer and Rosswall, 1982) at the

beginning and end of each experiment. The FDA

hydrolysis assay indicates microbial metabolism across a

wide variety of taxa, and correlates well with assays of

respiration. Subsamples of 0.5 - 1.0 g were taken from

each sample before and after each experiment and

transferred into 15 mL centrifuge tubes. 5 mL of 60 mM

K2PO4 buffer (pH 7.6) was added to each tube and briskly

shaken for 10-20 seconds. Ten µL FDA in ethanol (5 mg



mL-1

) was added to each tube, and then all tubes were

incubated for 3-5 hours at 25°C on a rocker table.

Following incubation, the samples were extracted by

adding 5 mL 2:1 chloroform:methanol. The samples were

centrifuged for 10 minutes at 1000 x g, and the

supernatant was measured spectrophotometrically at 490

nm.

Chlorophyll a extractions were also used to determine the

relative abundance of photosynthetic organisms (Bowles

et al., 1985; Myers et al., 1980). Subsamples of 0.5 - 1.0

g were taken from each sample before and after each

experiment and transferred into 15 mL centrifuge tubes. 5

mL of 80% ethanol was added to each tube. Tubes were

placed in a -20°C freezer overnight, and then were

centrifuged for 10 minutes at 1000 x g. The supernatant

was measured spectrophotometrically at 664 nm.

For the first two experiments (24 hours and 14 days),

plate counts (Atlas, 1997) and trypan blue live-dead stains

(Sigma-Aldrich Co., 2005) were also used to determine

survival. However, the tests were very time-consuming

with ambiguous results, and were discontinued in favor of

the FDA and chlorophyll assays.

The full results of the initial experiments are reported

elsewhere (Thomas et al., accepted for publication).

Here, we summarize the most relevant results.

In experiments of seven days or longer duration, a "water

cycle" became evident within the test chamber as water

tended to condense at the ends as the chamber cooled at

"night," and the samples at the ends of the chamber

tended to be moister than samples in the middle. While

this was disconcerting at first, it probably approximates

the conditions on a planet-wide scale—some areas of

Mars will be wetter than others, and life will become

distributed according to moisture levels.

Live-dead microscopic assays and plate counts showed

survival of all of the organisms tested in the overnight and

14-day trials. However, because of the small sizes of the

cells tested, the live-dead assays were prone to large

errors. Non-cellular material was sometimes counted as

cells, and some cells were incorrectly counted as debris.

Plate counts of cyanobacteria took 1-2 weeks in order to

grow countable colonies. In addition, Plectonema and

Anabaena, being filamentous, gave underestimated results

from plate counts. Further use of these assays was

discontinued.

Figure 1. FDA hydrolysis by eubacteria and cyanobacteria after a 14-day ecopoesis trial. Hydrolysis rates are per gram of total soil

simulant. Samples were placed in two 24-well tissue culture plates—one in direct light, the other in shadow. "Atacama," "Rock Garden,"

and "Yungay" refer to partially-characterized, heterotrophic Atacama Desert isolates. "Atacama 2002" is a strain of Klebsiella oxytoca;

"Rock Garden-2" is a strain of Bacillus licheniformis; and "Yungay-2" is another strain of Bacillus. The other "Rock Garden" strains

appear to be species of Staphylococcus (but not S. aureus). All heterotrophic bacteria were exposed in JSC Mars-1 simulant amended with

trypticase soy broth. The cyanobacteria (five organisms on the right side of the graph) were grown in JSC Mars-1 amended with BG-11

medium. Each bar represents a single sample.



The heterotrophic bacteria, Bacillus and Klebsiella, grew

very well during short-duration experiments in which they

were supplied organic nutrients (Figure 1). These results

indicate the hardiness of both sporogenic (Bacillus) and

non-sporogenic (Klebsiella) bacteria. These organisms

were discontinued in later experiments since organic

nutrients are not expected to be available during the early

ecopoeisis of Mars. However, since both genera are

commonly associated with humans, these results have

relevance for planetary protection issues associated with

both robotic and human exploration of Mars. In our

experiments, heterotrophic bacteria survived at least 14

days in the simulator. Although Bacillus spores are

quickly inactivated in the presence of Mars levels of UV

(Newcombe et al., 2005; Schuerger et al., 2003;

Schuerger et al., 2006b), spores beneath the regolith

would be protected. Similar experiments to ours (without

UV) have shown even longer survival times of Bacillus

under present day martian conditions (Nicholson and

Schuerger, 2005). While these results are encouraging

from a planetary engineering perspective, the presence of

terrestrial "hitch-hikers" in the martian regolith could

interfere with astrobiological research on Mars.

Several genera of cyanobacteria have been tested with a

wide range of survival (Figures 1 and 3). Cyanobacterial

survival in the simulator was somewhat correlated with

CO2 tolerance (Thomas et al., 2005), but desiccation

resistance also had a role. Anabaena, Plectonema and

Chroococcidiopsis appear to be very good candidates for

further study. One strain of Chroococcidiopsis in

particular (CCMEE 029), has been shown previously to

have higher UV resistance than Bacillus (Cockell et al.,

2005). UV resistance is especially important for

cyanobacteria on Mars since they need exposure to light

for photosynthesis, but that same exposure causes damage

from UV radiation. A fine covering of regolith may

provide UV shielding while still allow enough

photosynthetically active radiation to reach the cells.

Further research in this area will prove beneficial both for

ecopoeisis research and in understanding niches for

possible extant life on Mars.

Of the desert varnish and cave bacteria (Figure 2),

Pedomicrobium had very high esterase activity after five

weeks of exposure. Even though all of these strains have

long generation times, Pedomicrobium may also be a

good candidate for further study. However, at this time,

most of these strains have only been partially

characterized and more information is needed about their

metabolism and environmental limits.

Figure 2. FDA hydrolysis by desert varnish and cave bacteria after a 5-week ecopoesis trial. Pure cultures from agar plates were mixed

with BG-11 amended JSC Mars-1 simulant and placed in individual polypropylene containers in direct light. Strain 1 has been identified

as Pedomicrobium manganicum isolated from desert varnish. The other strains are partially-characterized isolates from cave and desert

varnish environments. Each bar represents the mean of three samples; error bars = s.d.



Figure 3. FDA hydrolysis within a simulated soil community after a 5-week ecopoesis trial. A simulated soil community was formed by

mixing cultures of Anabaena sp., Chroococcidiopisis CCMEE171 and CCMEE662, Plectonema boryanum, Klebsiella oxytoca, Bacillus

licheniformis and Bacillus sp. in JSC Mars-1 simulant amended with BG-11 medium. The soil was spread over a polystyrene tray, which

was placed half in light and half in shadow. After the experiment, the soil was divided into three regions: direct light, transition, and

indirect light. Composite samples were mixtures of soil from all three regions. Three samples were obtained from each region and

averaged; error bars = s.d.

CONCLUSIONS

With the increasing availability of martian environment

simulators, the science of planetary engineering is moving

from the theoretical to the experimental realm. Although

planetary-scale ecopoesis is still in the distant future,

population and community level experiments are possible

and are currently in progress. In addition to providing

insight about possible future life on Mars, these

experiments also tell us about the survivability of Earth

organisms in the present martian environment.

As additional simulators become available, voluntary

standardization of environmental parameters will be

desirable. We envision a consortium that would set

guidelines for planetary simulators so that experiments

undertaken with different simulators would still be

directly comparable. Efforts to form such a consortium

are currently underway.

Ecopoeisis also provides an exciting opportunity to

engage students in science. While many students have

heard of terraforming through Star Trek and other science

fiction stories, they usually do not realize that serious

science underlies the fiction. Ecopoesis can be used as a

focal point for discussions of geology, environmental

science, microbiology, ecology and other disciplines to

show the interdisciplinary nature of planetary science—

whether that planet is Earth, Mars or one that has not been

discovered yet.

ACKNOWLEDGEMENTS

This research was funded under subcontract agreement

07605-003-026 with NASA's Institute for Advanced

Concepts (NIAC). Additional support was provided by

the Arkansas Space Grant Consortium. KAC's

participation was supported in part by the Arkansas

Supporting Teachers Research Involvement for Vital

Education (STRIVE) Program.

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PLANETARY ECOSYNTHESIS AS ECOLOGICAL SUCCESSION. James M. Graham

Department of Botany, University of Wisconsin-Madison, Madison, WI. 53706

ABSTRACT

Terraforming is the process of applying global

engineering techniques to transform the climate of a

planet into one that is habitable for terrestrial organisms.

Ecosynthesis is the process of introducing a succession of

ecosystems to such a terraformed planet. The process of

introducing terrestrial ecosystems to Mars can be

compared to a descent down a terrestrial mountain. Each

drop in elevation results in a warmer, wetter climate and

more diverse biological community. Beginning with a

polar desert, the sequence of ecosystems passes through

tundra, boreal forest, and temperate ecosystems where

moisture determines the presence of desert, grassland, or

forest. Mars is like a very high terrestrial mountain. The

goal of planetary engineering is to bring the climate of

Mars down that mountain to the point where some areas

of the planet have a climate similar to a polar desert At

that point a microbial ecosystem containing bacteria,

cyanobacteria, green algae and lichens can be introduced.

Subsequent engineering and biological feedbacks will

move the climate of Mars further down the mountain

through successively more moderate climates and more

diverse ecosystems. At each stage the organisms will alter

the environment to prepare the way for subsequent stages.

This model provides a logical framework for the sequence

of ecosystems to be established on Mars. The physical

requirements for the introduction of each system on Mars

can be extrapolated from the known limitations on Earth.

The ecosystems define the communities to search for

potential colonizing species based on their physiological

properties.

INTRODUCTION

Mars is a bitterly cold planet. Its average surface

temperature is only –60oC compared to +15

oC for the

Earth. The martian atmosphere has a surface pressure of

only 0.5-1 kilopascals (kPa) or 5–10 mbar, a pressure so

low that liquid water is not stable. Intense ultraviolet

(UV) radiation bathes the surface, and the regolith is

thought to contain oxidants, which would break down any

organic material. Terrestrial organisms cannot survive

under these surface conditions (Clark, 1998). Spacecraft,

however, have returned images of valley systems

resembling dried-up river systems and wide outflow

channels indicating the passage of vast quantities of water

in the past. Mars Global Surveyor (MGS) returned

altimeter data indicating shorelines consistent with the

past existence of a northern ocean (Head et al., 1999). If

water was once stable on the surface and flowed in such

vast quantities, the atmosphere must have been denser and

the climate warmer in the past.

These observations are the basis for the current search for

evidence of past or present life on Mars (McKay, 1997).

They are also the source of speculation that Mars might

be returned to its former warmer climate by some sort of

global engineering techniques (Sagan, 1971; Oberg, 1981;

McKay et al., 1991). The new science of planetary

engineering became known as “terraforming” in which an

extraterrestrial body is modified to make an environment

suitable for habitation by terrestrial organisms. The term

“planetary ecosynthesis” emphasizes the fact that the

result of planetary engineering is not a carbon copy of

Earth but a habitable world with its own unique

properties. Fogg gathered this early work in

Terraforming: Engineering Planetary Environments

(1995), and review articles by Fogg (1998), McKay

(1999), and McKay and Marinova (2001) have

established terraforming as a branch of the field of

astrobiology.

As established by NASA the Astrobiology program

includes the study of planetary ecosynthesis. One of the

six key questions addressed by the Astrobiology Institute

is: What is the potential for survival and biological

evolution beyond the planet of origin? Biological

evolution is a phenomenon of populations of organisms

interacting and genetically changing over long periods of

time. To address this question entire ecosystems

(communities of organisms plus their physical and

chemical environment) would have to be placed on an

extraterrestrial planetary body, and Mars is the only

planet in the Solar System capable of receiving terrestrial

ecosystems after modification with existing technology.

The purpose of this paper is to present a model for the

process of the introduction of terrestrial ecosystems to

Mars. The model provides a framework for the sequence

of ecosystems to be established and the physical

requirements for each system. The individual ecosystems

define the communities from which potential colonists

may be selected based on their biological characteristics.

The potential colonists then define areas for research to be

performed before the actual transplantation to Mars.

A MODEL FOR ECOSYNTHESIS ON MARS

A model for the process of ecosynthesis on Mars can be

found in a diagram often found in introductory biology

texts. As one climbs up a mountain, each 150 m rise in

____________________

* Correspondence to: Dr. James M. Graham

Department of Botany

430 Lincoln Drive

University of Wisconsin-Madison

Madison, WI 53706


Phone: 608-262-0657

J.M. Graham — Ecosynthesis on Mars


Figure 1. The mountain model for ecosynthesis on Mars. The present climate of Mars can be compared to a very high mountain on Earth,

one higher than any real mountain that exists in fact. The prebiotic stage of ecosynthesis employs planetary engineering techniques to

bring the climate of Mars at some latitudes into the range of that in the terrestrial Dry Valleys of Antarctica. At that point the introduction

of the first microbial ecosystems can begin. Continued modification of the planetary climate will permit the introduction of a sequence of

ecosystems from tundra through boreal forests to temperate ecosystems.

elevation produces a decrease in average annual

temperature of about 1oC. Moisture and evaporation also

change with altitude. Therefore anyone climbing up a

mountain passes through a series of ecosystems from base

to top. Depending on the latitude of the mountain, the

base may be surrounded in tropical forest or temperate

forest, a prairie or sagebrush area above that, followed by

a montane or coniferous forest, an alpine tundra at higher

elevations, and finally a polar desert ending in permanent

snow and ice. Since the effect is similar at all latitudes,

mountains in the tropics can be capped in permanent ice if

the mountain is high enough. This is the model of the

ecosynthesis process for Mars (Figure 1). The present

climate of Mars is like that of a very high mountain on

Earth. The average atmospheric surface pressure on Mars

(6-7 mbar) occurs in the atmosphere of Earth at an

altitude of about 37,000 m (121,000 ft), far higher than

any terrestrial mountain.

The mountain model of ecosynthesis on Mars provides an

order to the sequence of ecosystems that will be

implanted. The initial engineering stage will employ

global techniques to move the climate of Mars from its

present state to one similar to a high arctic barren or polar

desert. The first ecosystem will then be a microbial one

dominated by the kinds of microorganisms found in the

Dry Valleys of Antarctica or polar deserts in the Arctic.

The second ecosystem in the sequence will be similar to

arctic tundra and dominated by plants called bryophytes

(mosses and liverworts) and lichens (organisms formed

by symbioses between algae and fungi). The third

ecosystem will be characterized by the arrival of

flowering plants, initially as small numbers of herbs but

with an increasing role as climate and moisture improve

and ending with the first few stunted trees in favorable

sites. Boreal forest will mark the fourth stage in the

process, which will likely end with the establishment of

temperate ecosystems on Mars. The nature of the

temperate ecosystem will depend on the amount of

moisture available. If moisture is low, a desert may

develop, but if moisture is higher, grasslands and

temperate forests may be possible. The climate of

temperate ecosystems can support a martian agriculture

on the surface.

Each of these ecosystems is a stage in the process of

planetary ecosynthesis. Each stage may be characterized

by certain physical parameters, such as oxygen levels and

UV radiation, and by the dominant organisms, such as

microorganisms or flowering plants. Each stage is part of

a process that may extend over 1,000 years or more. The

entire process of ecosynthesis can be compared to the

terrestrial process of ecological succession. Consider the

retreat of a glacier in a high latitude environment on

Earth. As the glacier melts back within its valley, new

land is exposed. The first pioneer organisms on this

barren land are microorganisms, lichens, and liverworts.

Their activities add organics to the rocky detritus left by



TABLE 1. The physical parameters of Mars and Earth

Parameter Mars Earth

Mean distance from Sun 2.28 x 108 1.49 x 10

8

Orbital eccentricity 0.0934 0.0167

Rotation rate 24.62 h 24.0 h

Year 668.59 sols1

365.25 days

Obliquity 25.19° 23.45°

Surface gravity 0.38 g 1.00 g

Mean surface temperature

-60°C

+15°C

Surface temperature range -145°C to 20°C -60°C to 50°C

Insolation (PAR) 860 µmol quanta m

-2 s

-1 2000 µmol quanta m

-2 s

-1

UV radiation >190 nm >300 nm

Atmospheric pressure 5 – 10 mbar 1013 mbar

Atmospheric composition N2 0.189 mbar (2.7%) N2 780 mbar (78%)

O2 0.009 mbar (0.13%) O2 210 mbar (21%)

CO2 6.67 mbar (95.3%) CO2 0.38 mbar (0.038%)

Ar 0.112 mbar (1.6%) Ar 10.13 mbar (1%)

1686.98 Earth days

Data from Carr (1981), Kieffer et al., (1992), and McKay and Marinova, (2001).

the glacier and begin to erode exposed rocks. Soon

grasses and herbs colonize the area and generate a true

soil. Alders add nitrogen to the soil, and finally spruce

trees form a dense forest. Each stage in the succession

prepares the way for the next stage by altering the

environment. Similarly, on Mars each stage in

ecosynthesis alters the environment such that the next

stage becomes possible. The microbial stage will

transform the martian regolith into a true soil and

facilitate the arrival of bryophytes in the second stage. As

climate improves under global engineering, the bryophyte

stage will replace a pure microbial stage in favorable

areas while the microbial stage will spread into areas that

were not previously habitable. The microbial ecosystem

will persist even on a fully terraformed Mars at high

latitudes and elevations. Planetary geography always

plays a strong role in the locations of ecosystems.



The rest of this paper will examine each of the stages in

the ecosynthesis process. The initial planetary engineering

stage will be presented in less detail because the

engineering processes and their effects on martian climate

have been given previously (McKay and Marinova,

2001). The physical characteristics of each stage will be

considered, and the organisms present will be presented in

relation to their ability to adapt and thrive under those

physical conditions. Where predictable, the ways that

those organisms will alter the environment of Mars to

prepare for later stages will also be discussed.

THE INITIAL ENGINEERING STAGE

The physical parameters of Mars and Earth are given in

Table 1. The rotation rate (and therefore the day length)

and the obliquity of the two planets are very similar.

Surface gravity and solar radiation are lower on Mars than

on Earth, but the year length and orbital eccentricity are

longer. None of these differences creates a problem for

planetary ecosynthesis. Mars can be transformed into a

habitable planet by altering its atmospheric composition

and density. These alterations will raise the mean surface

temperature, reduce the surface temperature range, and

reduce the level of UV radiation at the surface.

The main planetary engineering techniques for Mars are

the manufacture and release of greenhouse gases such as

perfluorocarbons from materials in the regolith and the

potential use of orbiting solar mirrors to raise the amount

of solar radiation at the planet’s surface. If both methods

are used, planetary engineering will raise the surface

temperature of Mars by increasing the amount of solar

radiation at the surface and the retention of that radiation

through the greenhouse effect. The immediate goal of

both methods is the release into the atmosphere of carbon

dioxide frozen at the poles or absorbed in the regolith.

The released carbon dioxide will increase the atmospheric

pressure and further warm the planet by creating a

runaway greenhouse effect. Estimates of the amount of

this carbon dioxide vary from 2 to 200 kPa (20 to 2000

mbar), but for purposes of planetary ecosynthesis an

intermediate amount of 10 to 40 kPa (100 to 400 mbar)

would be adequate for the early stages. In addition to

raising the surface temperature of Mars, a thicker

atmosphere of carbon dioxide would also retain heat

better over the day-night cycle and therefore reduce the

diurnal temperature range. One objective of the

engineering phase will be to raise the mean surface

temperature into the range of the Dry Valleys of

Antarctica (-20°C).

A denser atmosphere around a warmer planet will also

permit liquid water to be stable on the surface. That Mars

once had abundant liquid water running on its surface has

long been established (Carr, 1981; Squyres, 1989). That it

still has abundant water in its regolith has been recently

shown by the Gamma Ray Spectrometer (GRS) aboard

the Mars Odyssey spacecraft. The GRS found evidence of

abundant hydrogen as water ice within one meter of the

surface below 60° south latitude. The percentages of ice

by mass within this one-meter depth varied from 20 to 50

percent. Abundant water ice was also detected above 60°

north latitude. Water is clearly available for ecosynthesis.

Mars has relatively little oxygen in its atmosphere (0.009

mbar), but the surface is highly oxidized. Many of these

oxidants will likely release molecular oxygen when a

warmer wetter climate is established. The net amount of

this oxygen released to the atmosphere would be slight,

probably less than 2 mbar, but the fact that the surface is

oxidized rather than reduced simplifies the ecosynthesis

process. Oxygen produced by photosynthetic

microorganisms can enter the atmosphere to raise the

levels of oxygen and ozone rather than rusting reduced

metals as occurred on the early Earth.

As the initial planetary engineering stage draws to a close,

the atmospheric pressure on Mars will reach 10 to 40 kPa.

The atmosphere is largely composed of carbon dioxide

with small amounts of nitrogen, argon, water vapor, and

oxygen. Average surface temperatures over some areas of

the planet reach –20°C and the daily temperature range is

reduced. The same greenhouse gases that have warmed

the planet also provide a shield from UV radiation. Liquid

water is now stable on the surface. Water will be cycling

through the atmosphere and returning to the surface as

some form of precipitation. It will likely collect in basins

and craters and may resume flowing down ancient

channels during summers. The stage is set for the

introduction of the first ecosystem.

Figure 2. A Dry Valley in Antarctica. The Antarctic Dry Valleys

are the terrestrial analogue of the first ecosystem on Mars.

Photo by Joby Chesnick.

THE MICROBIAL ECOSYSTEM

It is not possible to discuss all possible microorganisms

that would play some role in the first ecosystem on Mars.

This discussion will therefore focus on four groups of

organisms: the bacteria involved in a nitrogen cycle,

cyanobacteria, green algae, and lichens, which are

symbiotic associations between fungi and algae. These

four groups of microorganisms occur in polar deserts such

as the Dry Valleys of Antarctic (Figure 2) and will play a

major role in the first ecosystem on Mars. Other groups of

algae may occur, but they will be restricted to aquatic

habitats and therefore less widespread initially.



Figure 3. The nitrogen cycle on Mars. Initially the nitrogen cycle will be dominated by denitrification of regolith nitrate and nitrite into

molecular nitrogen (N2) and the assimilation of nitrate and nitrite by microbial reductases into ammonia and amino acids. Nitrogen will

cycle among microorganisms through ammonia and amino acids. As the nitrogen level of the atmosphere increases, nitrogen fixation will

begin at a pN2 of about 5 mbar.

Bacteria and the nitrogen cycle

In the present atmosphere of Mars, nitrogen is present at

about 2.7% (0.16 mbar), but Mars should have outgassed

some 300 mbar of nitrogen. If this nitrogen gas were

present initially, it was probably oxidized to nitrate by

lightning and volcanic electric discharges and is now in

the regolith. If nitrate is in the regolith, it is possible to

propose an order in which a nitrogen cycle might be

established. The initial processes will be essentially

anaerobic and will utilize nitrate and nitrite in the regolith

via two main pathways (Figure 3). Denitrification is

carried out by a variety of bacteria that convert nitrate to

nitrite, nitrite to nitric oxide, nitric oxide to nitrous oxide

and finally nitrous oxide to nitrogen gas. This process

could raise the partial pressure of N2 (pN2) in the

atmosphere to 60 to 300 mbar. Experiments with

denitrifying bacteria in a CO2 atmosphere indicate that

nitrate could be rapidly converted to N2 if liquid water

and organic substrates were present (Hart et al., 2000).

The microorganisms performing the second pathway

(assimilatory nitrate reduction) would provide the organic

substrates. Many microorganisms, including algae and

cyanobacteria, can perform assimilatory nitrate reduction

using nitrate and nitrite reductases. Nitrate is reduced to

ammonia that is incorporated into amino acids within

living organisms. When these organisms die and decay,

they release ammonia and amino acids into the

environment where other microbes can take them up by

ammonia assimilation. Ammonia thus cycles through a

closed loop. These two pathways will reduce nitrate in the

regolith as the pN2 in the atmosphere rises.

The remaining process shown in Figure 3 is nitrogen

fixation. Prokaryotic microorganisms such as archaeans,

bacteria, and cyanobacteria can fix nitrogen, but the

present atmospheric levels on Mars (0.16 mbar) are too

low for fixation. Klinger et al. (1989) examined

Azotobacter and Azomonas over a range of pressures of

N2. No growth occurred at a pN2 less than 1 mbar, but

both bacteria grew and fixed nitrogen at a pN2 of 5 mbar.

Limited data suggest that nitrogen fixation could begin on

Mars once the pN2 reaches at least 5 mbar. The

establishment of a nitrogen cycle on Mars will profoundly

change the composition of the atmosphere from one

composed of mostly CO2 at a pressure of around 90-400

mbar to one with an additional 60-300 mbar of N2.



TABLE 2. Mechanisms of ultraviolet radiation resistance in cyanobacteria

Category Mechanism

Avoidance Growth in fissures, under the surfaces of porous rocks (endolithic), beneath translucent

rocks, under water, and under the ice on ice-covered lakes.

Screening Scytonemin, yellow-brown sheath pigment

Phycoerythrin, red intracellular pigment

Mycosporine-like amino acids (MAA), colorless

Quenching Carotenoids, orange pigments

Xanthophylls, yellow pigments

Superoxide dismutase

Repair Enzyme-based DNA repair of UV damage

Polyploid genomes, up to 10 copies

Based on data in Vincent and Quesada (1994) and D. Wynn-Williams (1994)

Cyanobacteria

Cyanobacteria are widespread members of the lake,

stream, soil and lithic communities in Antarctica,

especially in the Dry Valleys (Vincent, 1988). They do

not require oxygen and can carry out both oxygenic

photosynthesis and anoxygenic photosynthesis using

hydrogen sulfide as proton donor. They are highly

resistant to freezing and drying. Filamentous

cyanobacteria in Dry Valley streams remain freeze-dried

and exposed for months until the few brief weeks in

austral summer when the streams flow with water

(Vincent and Howard-Williams, 1986).

Their resistance to UV radiation is varied and versatile

(Table 2). They occupy a number of habitats that permit

them to avoid exposure to full UV radiation. Some

cyanobacteria also occur as epilithic crusts, directly on

rock surfaces, at high latitudes and altitudes. Screening

pigments and quenching agents heavily protect these

filaments.

Phycoerythrin and mycosporine-like amino acids (MAA)

are both intracellular compounds that act to screen out

UV radiation in cyanobacteria. Scytonemin is an UV

screening pigment found in the outer sheaths that

surround cyanobacteria (Vincent and Quesada, 1994). The

level of scytonemin in cyanobacteria increases with

increasing light levels (Garcia-Pichel and Castenholz,

1991). Carotenoids and xanthophylls are quenching

agents that dissipate excess solar energy absorbed by

chlorophyll that would otherwise result in damaging free

oxygen radicals and hydrogen peroxide within cells.

Superoxide dismutase scavenges free oxygen radicals (O2-

) and converts them to hydrogen peroxide. The hydrogen

peroxide is then converted to water and oxygen. In

Antarctica mats of cyanobacteria typically contain high

concentrations of these substances as photochemical

protection (Vincent and Quesada, 1994).

As a final

defense against UV radiation, cyanobacteria have

mechanisms for repair of damage to their DNA (Levine

and Thiel, 1987) and are polyploids, meaning they have

multiple copies (as high as 10) of their genome. Damage

to a single strand of DNA is therefore less likely to

disrupt the cell. All these UV radiation adaptations to the

harsh environment of Antarctica preadapt cyanobacteria

to fill a wide range of habitats in the first stage of the

ecosynthesis.

Green algae

Many green algae are not obligate aerobes, including

members of such common genera as Chlorella,

Chlamydomonas, Scenedesmus and Selenastrum (Spruit,

1962). There is one genus of green algae called

Pyrobotrys found only in anaerobic soils (Nozaki, 1986).

In the dark these algae will initially carry out fermentation

and produce carbon dioxide and organic acids, as do



many plants. But after some time they will evolve

hydrogen gas. If anaerobic conditions persist in the light

and levels of carbon dioxide and hydrogen are adequate,

these algae may reduce carbon dioxide to simple

carbohydrates using molecular hydrogen as electron and

proton donor. Thus green algae possess a more diverse

range of metabolic processes than just photosynthesis and

aerobic respiration. Certain species have the ability to

grow under low pH levels (Sheath et al., 1982), low

temperatures, and high salinity (Vincent, 1988). The

major adaptation of Antarctic green algae is the ability to

survive repeated freezing and thawing (Holm-Hansen,

1963).

The ability to withstand repeated freeze-thaw

cycles would have considerable value during early

planetary ecosynthesis on Mars.

Green algae may be as resistant as cyanobacteria to UV

radiation. Green algae share the same range of habitats as

cyanobacteria in Antarctica (Vincent, 1988). In the Dry

Valleys of Antarctica, cryptoendolithic lichens often

occur as a layer beneath the surfaces of translucent porous

rocks, and the green alga Trebouxia is often the algal

symbiont in the lichen. Beneath this lichen layer the green

alga Hemichloris, which is endemic to Antarctica, may

form another distinct layer. Flavonoids and phenolics are

UV screening agents known to be present in some green

algae. The level of quenching agents such as carotenoids

in green algae is related to the level of exposure to direct

sunlight and UV radiation (Thomas and Duval, 1995).

Lichens

Lichens are generally slow growing, but they grow faster

under elevated carbon dioxide levels, at least up to about

2 mbar of carbon dioxide (Nash et al., 1983). It is

possible therefore that lichens would perform better on

Mars during early stages of ecosynthesis than they do on

Earth because the atmosphere would be largely carbon

dioxide. Antarctic lichens are extremely tolerant of

desiccation and cold. Lange and Kappen (1972) found that

several Antarctic lichens could survive and recover even

after being cooled to –196°C, a temperature below levels

that currently occur at the martian poles. Some lichens

were able to assimilate carbon dioxide at temperatures as

low as –12.5°C to –18°C.

Lichens are extremely resistant to UV radiation. Siegel

and Daly (1968) exposed the arctic lichen Cladonia

rangiferina to UV radiation of 3.6 x 109 ergs

.cm

-2 over 24

hours. This is equivalent to 4.16 x 104 ergs

.cm

-2.s

-1. By

comparison the UV radiation at the equator of Mars

averages 7 x 103 ergs

.cm

-2.s

-1. Cladonia rangiferina covers

large areas of the arctic and presumably could also do so

on Mars. Table 3 lists some of the UV radiation

resistance mechanisms in lichens. Endolithic lichens lie

under the surfaces of translucent porous rocks in the

Antarctic Dry Valleys. The overlying rock layers shield

the lichens from direct solar and UV radiation. Many

lichens, however, grow as crusts on rocks where they may

be colored black, brown, orange or yellow. The black and

brown colors are due to melanins that screen the lichens

from excess solar and UV radiation and also absorb heat

to raise the temperature of the lichen above that of the

surrounding air. The orange and yellow colors are due to

carotenoids that are quenching agents.

TABLE 3. Mechanisms of UV resistance in lichens and mosses

Category Mechanism

Avoidance Endolithic lichens

Screening Lichens: black melanins in endolithic and surface lichens

Mosses: black and brown pigments and flavonoids.

Phenolics in the walls of the moss Andreaea.

Quenching Lichens: carotenoids

Mosses: carotenoids such as violaxanthin (purple)

Repair Mosses: polyploid genomes

Based on data in Post (1990) and D. Wynn-Williams (1994).



Although lichens are slow growing on Earth, their role in

the ecological process of succession makes them

important to include in the early stages of ecosynthesis on

Mars. On Earth lichens are the first pioneer species to

colonize bare rock. They excrete organic acids that slowly

dissolve rock and free minerals to join the accumulation

of organic material that slowly creates a true soil. Their

growth opens the way for the establishment of other

plants.

The microbial stage of ecosynthesis will begin the process

of adding organic matter to the martian regolith, binding

together regolith particles, and transforming the

atmosphere by increasing the amount of free oxygen and

converting nitrates to free nitrogen. The climate will

begin as frigid-polar with average temperatures in the

warmest “month” less than 0°C (Longton, 1988). On

Earth a frigid-polar climate is found only in continental

Antarctica. Toward the end of the first stage, the climate

should warm to cold-polar where the average temperature

in the warmest month falls in the range of 0° to 2°C.

Maritime Antarctica and the arctic polar deserts have a

terrestrial cold-polar climate. Microbial life will thrive in

the regolith, on the surfaces of rocks, under the surfaces

of rocks in the pores between mineral grains, and in

gathering surface waters as plankton and as filaments

attached to surfaces.

THE BRYOPHYTE ECOSYSTEM

Bryophytes consist of relatively simple, usually small,

green plants called mosses, liverworts, and hornworts.

They lack roots but form cellular extensions called

rhizoids for attachment. Bryophytes are extremely

important in terrestrial polar and alpine ecosystems where

the severe climate prohibits most flowering plants (Figure

4).

Figure 4. Alpine tundra. The terrestrial equivalent of the second

stage in ecosynthesis on Mars. Photo by Lee Wilcox.

In the bryophyte stage of ecosynthesis the atmosphere

will be mainly carbon dioxide and nitrogen with small but

increasing amounts of oxygen. Mosses show an unusual

capacity to utilize carbon dioxide at high external levels,

far more so than flowering plants. Silvola (1990) reported

that Sphagnum fuscum, a widespread moss in northern

peatlands, exhibited increased rates of photosynthesis up

to a carbon dioxide pressure of 9 mbar, the highest level

tested. Grimmia antarctici, a common carpet-forming

moss in East Antarctica, reached its maximum rates of

photosynthesis at CO2 levels of 10-20 mbar (Tarnawski

et al., 1992). Mosses photosynthesize more slowly than

flowering plants at terrestrial ambient carbon dioxide

levels of 0.38 mbar. At higher levels of CO2 the

photosynthetic rates of mosses approach those of

flowering plants. Mosses are also capable of carrying out

photosynthesis at lower light levels and lower

temperatures, even under snow, than flowering plants

(Oechel and Sveinbjornsson, 1978; Collins and

Callaghan, 1980). Mosses do require oxygen for aerobic

respiration, but how much oxygen they require is

unknown. The moss Hypnum cupressiforme showed an

increase in photosynthetic rate as oxygen was reduced to

3% (30 mbar) (Aro et al., 1984).

Mosses are not thought to have a high degree of resistance

to UV radiation, but this may be due to lack of

experimental data. Mosses occur in the same habitats as

lichens and cyanobacteria, including on the surfaces of

rocks exposed to full solar radiation at polar latitudes and

alpine locations. Like lichens, mosses in polar and alpine

environments may be dark brown or black in color. These

pigments screen out harmful UV radiation and excess

visible light while warming the body of the moss (Table

3). Phenolics such as flavonoids occur in the walls of a

number of mosses, and these phenolics act to screen out

UV radiation and possibly also reduce desiccation

(Graham et al., 2003). Markham

et al. (1990) reported

UV protective flavonoids in the Antarctic moss Bryum

argenteum. The lower the level of ozone over the field

sites the higher the level of protective flavonoids. Mosses

also possess quenching agents such as carotenoids. The

Antarctic moss Ceratodon purpureus produces the

carotenoid violaxanthin as a photoprotective pigment

(Post, 1990). Mosses are generally polyploid.

Mosses will likely play an important role in planetary

ecosynthesis by sequestering large amounts of carbon

dioxide in the form of decay resistant organic compounds

in martian peatlands. At the same time these peatlands

will add large quantities of oxygen to the atmosphere of

Mars. Martyn Fogg (1995) estimated that a cover of

peatlands on Mars could produce 20 mbar of atmospheric

oxygen in about 700 years. Since mosses photosynthesize

faster in a CO2 atmosphere than on Earth, martian

peatlands might be able to produce this amount of oxygen

in less time, depending on the degree of surface cover and

moisture availability. For bryophytes to sequester large

amounts of carbon in peatlands, planetary engineering

must raise the climate of significant areas on Mars to the

level of mild-polar environments on Earth (Longton,

1988). Mild-polar habitats have average monthly

temperatures in the warmest month of 7° to 12°C. On

Earth they occur in Alaska and northern mainland

Canada. These conditions permit Sphagnum moss to form

the extensive peatlands that sequester so much carbon on

Earth.



With the full development of the bryophyte ecosystem,

the surface of Mars will appear vastly different from that

shown in the cameras of Spirit and Opportunity. Rocks

will be dotted with lichens and mosses. In areas where

water collects, Sphagnum moss may form extensive

peatlands. Mars will assume a distinctly green aspect

interrupted by bright spots of yellow, orange, and red

lichens. At some point in this stage the partial pressure of

oxygen will surpass 20 mbar, a value that is significant

for the next stage in planetary ecosynthesis.

THE FLOWERING HERBS ECOSYSTEM

The crucial factor in determining the transition to the third

stage in martian ecosynthesis, which is characterized by

the arrival of flowering plants, is the level of atmospheric

oxygen. In most terrestrial environments only two types

of flowering plant structures risk low oxygen levels: seeds

and underground organs such as roots, rhizomes and

tubers (Crawford, 1992). Rhizomes and tubers are

underground stems. Many seeds show reduced

germination if oxygen falls below 10 kPa or 100 mbar.

On Earth oxygen always limits to some extent the depth

to which roots can penetrate. Armstrong and Gaynard

(1976) found that 20 to 25 mbar was the critical oxygen

pressure to maintain root respiration in rice (Oryza sativa)

and the aquatic sedge Eriophorum augustifolium. Fogg

(1995) used this data when he proposed that the minimum

pO2 for plants would lie around 20 mbar or 2% O2 in the

martian atmosphere. Higher values, however, have been

reported for other plant species. Corn (Zea mays) had a

critical oxygen pressure of 6kPa (6% or 60 mbar) (Saglio

et al., 1984). A third way in which low oxygen can

constrain flowering plants is through restricting animal

pollinators. Many plants require animal pollinators to

produce seeds. Hard data are lacking but a pO2 of 20 mbar

is unlikely to support pollinators such as flying insects

and certainly not bats and birds. Cockell et al. (1999)

examined a number of insects from different orders and

found that all species seemed to function normally down

to a pressure of 200 mbar (about 40 mbar of oxygen) for

up to 24 h. Low oxygen levels will likely prohibit certain

plants from colonizing Mars until oxygen levels rise

enough to permit their animal pollinators to thrive also.

The first flowering plants on Mars will have anoxia-

tolerant seeds and underground organs and mechanisms

for reproduction that do not require animals.

Seeds, rhizomes, and whole plants under low oxygen

Seeds may store food reserves as lipids, proteins or starch.

The germination rate of seeds that store lipids or proteins

begins to decline if external oxygen levels fall below

10kPa or 100 mbar. Starch seeds, however, can maintain

50% germination rates when the pO2 drops below 1kPa or

10 mbar and can germinate at about 20% even at 0.1kPa

(1 mbar). Rice is an aquatic monocot, and its seeds can

still germinate when pO2 falls below 0.01kPa (0.1 mbar)

and even extend their shoots above water (Al-Ani et al.,

1985). Starch fermentation powers germination.

Although roots cannot survive more than a few days of

anoxia, the underground rhizomes of a number of aquatic

plants can survive under total anoxia for two to three

months (Crawford 1992). Survival depends on a large

reserve of carbohydrates that the rhizome ferments into

ethanol to generate energy in the form of adenosine

triphosphate (ATP). The ATP from fermentation

maintains the rhizome in winter and also powers the

extension of a new shoot in spring (Braendle and

Crawford, 1987). Once the shoot has elongated and the

leaves expanded, aquatic plants develop another

mechanism to thrive in an oxygen-poor environment–a

special tissue called arenchyma. Aerenchyma consists of

interconnected gas-filled channels through stems and

roots that provide a pathway for the movement of oxygen,

carbon dioxide, and ethanol. Aquatic plants are also

tolerant of high levels of carbon dioxide. A number of

aquatic plants should be good candidates for colonizing

Mars early in the third stage.

One other group of flowering plants possesses

considerable tolerance of anoxia. A number of vascular

plants from Spitsbergen in the High Arctic have an

exceptionally high level of anoxia tolerance (Crawford et

al., 1994). This anoxia tolerance extends to the entire

plant including the leaves. The anoxia tolerance of these

high arctic species appears to be an adaptation to ice-

encasement, which may last as long as 8 to 10 months.

The extraordinary anoxia tolerance of these plants makes

them prime candidates for colonization on Mars.

Reproduction without animals at low oxygen

Flowering plants have a number of reproductive

mechanisms that do not require animal pollinators. These

mechanisms include vegetative reproduction, apomixis,

autogamy, and anemophily. In vegetative reproduction

certain somatic (body) cells divide to produce structures

called runners, rhizomes, or bulbils. Runners are

aboveground stems that spread out from the parent plant

and establish new roots and leaves. Strawberry plants are

a familiar example of a plant that spreads by runners.

Rhizomes are underground stems that spread out from the

parent plant. The crab grass in your lawn and the Iris in

your garden both spread by rhizomes. The final type of

vegetative reproduction is less familiar. Bulbils are small,

bud-like plants born on the parent plant. The common

houseplant Kalanchoae produces bulbils along the

margins of its leaves, from which the bulbils drop off and

root in the ground.

Apomixis is asexual seed formation, but there are a

number of different types of apomixis (Asker and Jerling,

1992). The type most suitable for colonizing Mars is

called autonomous apomixis. Autonomous apomictic

plants reproduce independently of pollination. The

familiar lawn and garden dandelion (Taraxacum

officinale) is an example of a common autonomous

apomictic flowering plant. The flowering plant family

Asteraceae, which contains daisies, sunflowers, and



TABLE 4. Flowering plants growing in the cool-polar regions of Earth that reproduce

without animal pollinators.

Genera and species Common name Reproductive mechanism(s)

Cassiope tetragona Arctic bell heather Autogamous

Dryas integrifolia Mountain avens Autogamous

Epilobium latifolium Dwarf fireweed Autogamous

Erysimum pallasi Apomictic

Lesquerella arctica Autogamous

Papaver radicatum Arctic poppy Autogamous

Pedicularis arctica

P. hirsute

P. lapponica

P. sudetica

Arctic lousewort

Hairy lousewort

Lapland lousewort

Sudetan lousewort

Autogamous

Autogamous

Autogamous/vegetative

Autogamous

Potentilla nivea Snow rose Autogamous/apomictic

Saxifraga tricuspidata

S. flagellaris

S. cernua

Three-pointed saxifrage

Whip saxifrage

Nodding saxifrage

Autogamous

Apomictic/vegetative

Apomictic/vegetative

Silene acaulis Cushion pink Autogamous

Taraxacum arctogenum Arctic dandelion Apomictic

Some may use insects if available. Based on data from Kevan (1972) and Williams

and Batzli (1982).

dandelions, has many autonomous apomictic species and

therefore many potential martian colonists (Graham,

2003).

Autogamy means literally self-marriage. An autogamous

plant acquires pollen for fertilization of its egg cells from

itself. Autogamy is a form of sexual reproduction, and it

is widespread among flowering plant families.

Anemophily (wind loving) or wind pollination is normal

sexual cross-pollination where the agent transporting the

pollen is wind rather than animals. Wind pollination

dominates the gymnosperms and prevails in the flowering

plant families Poaceae (grasses), Cyperaceae (sedges),

and Juncaceae (reeds)(Faegri and van der Pijl, 1979).

These three families dominate terrestrial wetlands. Wind-

pollinated plants form a fourth group of potential martian

colonists.

These alternative mechanisms of reproduction occur in

many different groups of flowering plants and in many

environments, but the flowering plants of polar and alpine

environments utilize them to an extreme degree. In these

harsh environments populations of animal pollinators are

often severely restricted by low temperatures, and

population size may vary widely from year to year. If

animal pollinators are scarce and unreliable, then

reproduction is best insured by having some mechanism

that is not dependent on them. The maritime Antarctic is a

cold-polar environment where the mean temperature in

the warmest month is only 0-2°C (Longton, 1988). There

are only two native species of flowering plants: the grass

Deschampsia antarctica and the carnation Colobanthus

quitensis. Colobanthus is autogamous and Deschampsia

is apomictic.

Cool-polar environments, such as occur in Greenland and

the Canadian Islands in the High Arctic, and isolated



islands such as South Georgia and Macquarie around

Antarctica, are the first polar environments with

significant flowering plant communities. In a cool-polar

environment the average temperature of the warmest

month is 3° to 7°C. Table 4 lists a number of cool-polar

plants that are known to be capable of reproducing

without animal pollinators. Dryas integrifolia has roots

that harbor nitrogen-fixing bacteria. The whip saxifrage

(Saxifraga flagellaris) has yellow apomictic flowers,

which form seeds for long-distance dispersal, and long

whip-like runners to spread locally. During the stage of

flowering herbs the level of atmospheric oxygen should

continue to rise and exceed 60 mbar. Beyond this point

the level of oxygen should no longer be a significant

constraint on the subsequent ecosystems on Mars. The

remaining constraints will be the same as those that

operate on Earth, namely temperature and moisture. The

next stage begins when the climate of parts of the martian

surface reaches that of a boreal ecosystem.

THE BOREAL FOREST ECOSYSTEM

During the later part of the third stage of ecosynthesis,

sheltered sites insulated by martian winter snows could

harbor dwarf trees of birch (Betula) and willow (Salix) up

to one meter tall and stands of open, stunted trees

consisting of birches, larches (Larix) and spruces (Abies)

could be common in sheltered valleys where water flows.

But the development of true forests on Mars will first

occur when the climate of some regions reaches that of

the terrestrial taiga or boreal coniferous forest (Figure 5).

The climate of the boreal forest can be defined by the

average temperature of the warmest month of the year,

which is 13 to 18°C in July (Elliott-Fisk, 2000).

Alternatively, the boreal climate can be defined by the

length of its growing season, which on Earth is 30 to 120

days with a daily mean temperature above 10°C (Walter,

1973). The lower figure represents the northern boundary

of the boreal forest and the higher figure the southern

boundary. On Mars these numbers should be revised

upward to 54 and 218 sols to account for the longer

seasons. On Earth annual precipitation in a boreal forest

varies from 30 to 50 cm (Elliott-Fisk, 2000). The revised

values for Mars would be 55 to 90 cm per martian year.

These temperature and precipitation values define the

requirements for establishing a boreal coniferous forest on

Mars.

On Mars as on Earth a boreal coniferous forest is a

spruce–fir forest. White spruce (Picea glauca), black

spruce (Picea mariana) and balsam fir (Abies balsamea)

are the most widespread North American dominants.

Black spruce and tamarack (Larix laricina), a deciduous

conifer, are common in bogs. Jack pine (Pinus banksiana)

and two deciduous flowering plants, paper birch (Betula

papyrifera) and quaking aspen (Populus tremuloides)

dominate disturbed areas (Vankat, 1979). The deciduous

flowering trees are wind pollinated. The seeds of all three

disturbance-site species are adapted for wind dispersal, a

feature that will aid their spread across the surface of

Mars. Willows, dwarf birch (Betula glandulosa), green

alder (Alnus crisps), and members of the heath family

(Ericaceae) make up most of the shrubs in the boreal

coniferous forest. The understory layer is dominated

either by mosses in wet areas or lichens in drier

environments. Bogs dominated by Sphagnum moss are

the most common form of wetland within the boreal

coniferous forest zone.

Figure 5. Boreal forest. The terrestrial analogue of the fourth

stage in martian ecosynthesis. Photo by the author.

The North American boreal coniferous forest is the most

diverse boreal forest on Earth, but its diversity is still the

lowest of any forest in North America. For additional

conifer species to use in ecosynthesis, the best sources are

montane coniferous forests. Montane coniferous forests

occur in the Appalachian Mountains, the Rocky

Mountains, and the Sierra Nevada and Cascade

Mountains of North America. Another potential source of

martian forests might be the high altitude montane forests

of the tropics. With a timberline at or above 4000 m in

Mexico (Beaman, 1962; Lauer, 1978), these montane

forests are adapted to grow and reproduce at lower

atmospheric pressures, higher levels of radiation, and

lower levels of moisture than sea-level forest species.

TEMPERATE ECOSYSTEMS

Temperate ecosystems represent the culmination of

planetary ecosynthesis on Mars. On Earth the growing

season ranges from 120 days to 250 days with mean daily

temperatures above 10°C. The amount of annual



precipitation determines whether a temperate ecosystem is

a forest, grassland, desert grassland, or desert. The eastern

temperate deciduous forest of North America varies in

annual precipitation from a maximum of 150 cm along

the Gulf Coast to a minimum of 75 cm around the Great

Lakes (Vankat, 1979). Annual precipitation in temperate

grasslands varies from less than 75 cm to 25 cm, where

desert grasslands may occur. Deserts generally receive

less than 20 cm in precipitation per year. On Mars these

temperate zone figures would be a growing season of 220

to 455 sols. A martian deciduous forest would receive

from 135 to 275 cm in annual precipitation, grassland

from 45 to 135 cm, and desert less than 45 cm in

precipitation per year. Temperate ecosystems would

permit a widespread open surface agriculture on Mars,

just as they do on Earth.

Because the type of temperate ecosystem depends so

strongly on the amount of annual moisture and on Mars

moisture levels are likely to be lower initially in a stage,

the first temperate systems to consider are arid ones,

deserts and desert grasslands. A good terrestrial analogue

of a martian temperate desert would be the Great Basin

Desert. The soil is low in organic matter and high in

salinity. A sagebrush (Artemisia tridentata) community

dominates lower salinity soils, and a shadscale (Atriplex

tridentata) community occurs on higher salinity soils.

Sagebrush is apomictic, and shadscale is autogamous.

Grasses such as grama grass (Bouteloua spp.) and wire

grass (Aristidia spp.) dominate desert grasslands. The

grasses are all wind-pollinated. Both arid systems should

be capable of colonizing Mars.

TABLE 5. Genera of wind-pollinated flowering trees north of Mexico.

Genus Common name Genus Common name

Ulmus Elm Lithocarpus Tanoak

Fagus Beech Celtis Hackberry

Quercus Oak Morus Mulberry

Corylus Hazel Maclura Osage orange

Alnus Alder Planera Planertree

Betula Birch Trema Trema

Juglans Walnut Platanus Sycamore

Carya Hickory Liquidambar Sweetgum

Fraxinus Ash Populus Poplar

Castanea Chesnut Ostrya Hophornbeam

Castanopsis Chinkapin Carpinus Hornbeam

When the genera of conifers are added to the above list, the groups represent 240 species

or 30% of 787 native trees in the region. Data from Regal (1982).

Grasslands are especially important to establish on Mars

because they generate some of the most fertile soils for

agriculture on Earth. Grasses are wind-pollinated but they

also reproduce by vegetative growth, spreading by

underground stems (rhizomes) or above ground stems

(runners) to form mats. Grasslands also include many

species from the sunflower (Asteraceae) and pea families

(Fabaceae) that contain numerous apomictic and

autogamous species.

Temperate deciduous forests can also generate good to

excellent soils for agriculture as well as timber for wood

and paper products. Many of the genera of trees that

dominate the temperate deciduous forest are wind-

pollinated (Table 5). The genera listed include some 240

species or about 30% of the 787 native species of trees in

the region (Regal, 1982). A temperate deciduous forest

contains five vertical strata: two tree layers, a shrub

stratum, herb layer and surface layer. The surface layer



includes lichens, mosses and liverworts. The herb layer in

cool, wet forests may have a number of ferns and species

of Lycopodium (ground pine). None of these plants

requires animal pollinators. The flowering plants of the

herb layer contain many species that are autogamous or

apomictic. The rose family (Rosaceae) contains a number

of genera and species of shrubs that are apomictic,

including Crataegus (hawthorn), Cotoneaster, Rubus

(blackberries) and Sorbus. Some species of Sorbus are

subcanopy trees. It should be possible to assemble a

temperate deciduous forest on Mars using species that do

not require animal pollinators.

The establishment of temperate ecosystems on Mars

permits the eventual development of agriculture on the

open surface, even if animal pollinators are still restricted

by low levels of oxygen. All our major cereal crops such

as wheat, oats, barley, corn, rice, and rye are wind-

pollinated cereal grasses. Many other crops, such as

tomatoes, some potatoes, beans, lettuce, and strawberries

are autogamous. Agriculture on the open surface would

permit a large, self-sufficient martian civilization.

DISCUSSION

The data used to develop this manuscript came from basic

research on biological phenomena that were not directly

related to astrobiology. The parameters of temperature

and moisture that define different types of ecosystems and

the zonation of ecosystems along an altitudinal gradient

are a long established part of the ecological literature. The

discovery of an ozone hole over Antarctica prompted

research on the effects of UV radiation on various

organisms, particularly microorganisms. This research

was very important in assessing organisms for planetary

ecosynthesis. The effects of elevated levels of carbon

dioxide on growth of photosynthetic microbes and plants

had been largely unexamined until rising levels of CO2

were detected in the terrestrial atmosphere. Most studies,

however, focused on levels of CO2 less than 5 mbar. It is

still not known how well plants can adapt to higher levels

of carbon dioxide, as occurred on the early Earth or will

occur during planetary ecosynthesis. Relatively little is

known about the minimum oxygen requirements of many

terrestrial plants, especially bryophytes, yet this

information is crucial to the transitions to both the second

and third stages in the ecosynthesis process. The

discovery of anoxia tolerant plants on the island of

Spitsbergen was especially important to the arrival of

flowering plants on Mars (Crawford et al., 1994). Even

less is known about the oxygen requirements of terrestrial

insects that might act as pollinators in transplanted

ecosystems. If some insects can function as pollinators at

low ambient oxygen levels, then the dynamic process of

planetary ecosynthesis would be altered considerably.

New discoveries will continue to be made, but until there

is actual funding for ecosynthesis research, these

discoveries will be made for other reasons than their

significance to astrobiology.

This manuscript has attempted to describe the process of

establishing functional ecosystems containing

communities of terrestrial organisms on the surface of

Mars. The process is likely to take from 500 to 1000 years

to approach the final stage. Given the vast scope of the

process, it has been necessary to describe it in somewhat

broad terms. Although the data upon which this

description is based are incomplete, the broad picture

should be correct. Planetary ecosynthesis on Mars will be

a long and great adventure. It will yield a vast amount of

new knowledge and become one of the great acts of

positive creative energy by the human species.

ACKNOWLEDGMENT

I wish to thank Kandis Elliot, the staff artist at the

Department of Botany, UW-Madison, for preparing the

illustrations for this manuscript and for her interest in

astrobiology and planetary ecosynthesis.

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(Volvocales, Chlorophyta) in anaerobic pure culture.

Phycologia 25, 455-468.

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Oechel, W.C. and Sveinbjörnsson, B. (1978) Primary

production processes in arctic bryophytes at Barrow,

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Alaskan Arctic Tundra, edited by L.L. Tieszen, Springer,

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Short Papers

Advanced Life Support and Biotechnology (page 123)

Animal Development, Physiology and Gravity Response (page 133)

Cell Biology (page 143)

Plant Development and Gravity Response (page 155)

Gravitational and Space Biology Bulletin 19(2) August 2006 123

DEVELOPMENT OF A MICROFLUIDIC ION SENSOR ARRAY (MISA) TO MONITOR GRAVITY-

DEPENDENT CALCIUM FLUXES IN CERATOPTERIS SPORES

A.R. De Carlo1, M. Rokkam

2, A. ul Haque

3, S.T. Wereley

3, P.P. Irazoqui

4, H.W. Wells

5, W.T. McLamb

6, S.J.

Roux7, D.M. Porterfield

1,8

1Dept. of Ag. & Bio. Eng.,

2Dept. of Elec. & Comp. Eng.,

3Dept. of Mech. Eng.,

4Weldon School of Biomed. Eng.,

Purdue University, West Lafayette, IN, 5Bionetics Corporation, Kennedy Space Center, FL,

6Dynamac

Corporation, Kennedy Space Center, FL., 7Molecular, Cellular & Devel. Bio., Univ. of Texas, Austin, TX,

8Dept.

of Hort. & Landscape Arch., Purdue Univ., West Lafayette, IN.

Early development of the germinating Ceratopteris fern

spore involves the establishment of cellular polarity based

on gravity sensing in the single cell system (Edwards and

Roux; 1994). This process of cell polarization culminates

in directional nuclear migration which determines the

plane of the first cell division. The product of this first

mitotic division is a prothalial cell (top) and a rhizoid

(bottom).

Previous experiments have shown that polar Ca2+

currents are involved with gravimorphogenic

development in Ceratopteris fern spores (Chatterjee et al.,

2000). In a series of experiments that covered the time

period of polarity development that leads up to spore

germination, it was possible to measure calcium flux at

the top, bottom, and sides of the spore using a self-

referencing Ca2+

electrode (Porterfield, 2002). Using the

single sensor we were able to determine that there is a

transcellular Ca2+

current (into the bottom and out of the

top) that correlates with responsiveness of the cell to

gravity. More recently we have attempted to identify the

differential role of ion channels and pumps in this process

of cell polarity development using both molecular and

electrophysiological methods. We used a microsystem to

rotate individual cells 180° and measure changes in Ca2+

ion currents after reorientation using a single Ca2+

-

selective microelectrode operated as a self-referencing

sensor. This approach was limited as we could only

effectively measure before and after signals from a single

position, and was only able to do these experiments in a

40-second time frame (Stout et al., 2003). These

experiments were not able to measure how fast the

transcellular calcium current reorients, because of the

basic limitations in the sensor technology used.

In order to overcome the technical limitations to

studying this system associated with the current sensor

technology we have been working towards developing

new sensor systems based on adaptation of MEMS

fabrication techniques. The goal is to fabricate and test

dynamic sensing systems for advanced-throughput

physiological measurements of calcium signaling events

at the cellular level. Using silicon micromachining we

have constructed and tested prototype versions of the

Microfluidic Ionic Sensor Array (MISA) lab-on-a-chip

device for conducting real-time multidimensional calcium

flux measurements around 16 fern spores arranged in a

vertically positioned 4x4 matrix. A microfluidic pore

sustains each fern spore in basic growth media while four

solid-state calcium-selective electrodes measure the

calcium concentration differential pairwise between four

positions around the cell (1 top, 1 bottom, and 2 sides).

The microfluidic system and sensor array are fabricated

on a silicon substrate. The pyramidal pores which hold the

fern spores in place are etched at an angle of 54° using

KOH etching, and the electrodes and bonding pads are

defined by depositing silver and gold on top of photoresist

and lifting the photoresist off. Chloride plating of the

electrodes is accomplished through immersion in 6%

(w/w) NaClO. SU-8 photoresist (MicroChem, USA)is

applied to the chip as a structural and insulating layer, and

Ca++

-selective PVC membrane made with ETH-5234

ionophore (Sigma-Aldrich, St. Louis MO) is spin coated

onto the electrode surfaces. The dimensions of the whole

chip are 9.7mm x 11mm, with 250µm x 1000µm

silver/gold composite bonding pads on all sides and 20µm

x 20µm electrodes. For a single wafer there are 41 MISA

chips (Figure 1), of which the current process yields

approximately 80-90%.

Figure 1. Picture of the 9x11 mm silicon fabricated MISA chip.

The MISA chips are mounted and wire bonded

in the center of a printed circuit board (PCB) that contains

an array of 64 non-inverting amplifiers, using low-noise

(~3nV/√Hz), low-drift (~0.8µV/°C) operational

amplifiers, corresponding to the 64 electrodes. Each of the

amplifiers provides a signal gain of 30 and filters out

noise above 50Hz. The PCB is connected to four data

cables through 16-pin connectors with 2mm spacing. The

total size of the board is 30.25in2. The output from the

PCB is then multiplexed by a 512-point crosspoint matrix,

then processed and stored using a 32-input 18-bit data

acquisition (DAQ) card in differential mode. MISAPlot

2.0 software was written in LabVIEW (National

Instruments, Austin TX) for the DAQ. There are four

main modules in the software to facilitae experimental

functionality. The software also provides accesss

tomoothing and compression functions are written in, as

well as functions for logging acquired data, switching

data and notes from the user. Data extraction and rapid

calibration (Figure 2) are also possible.

AR DeCarlo et al. – Microfluidic Ion Sensor Array

124 Gravitational and Space Biology Bulletin 19(2) August 2006

We have also developed a novel coupling

method, dual-electrode differential coupling (DEDC), so

that the differentials between two working electrodes

could be amplified and digitized directly without the use

of reference electrode. This method was validated in tests

(Figure 3) that involved placing the two ion-selective

probes in separate Ca++ solutions, connected by a 3M

KCl salt bridge. One probe was calibrated in 100µM,

1mM and 10mM solution while the second electrode

remained in a standard solution. Each calibration was

done three times in each of the standard solutions.

Calcium Concentration (mM)

0.1 1 10

Raw

Ele

ctr

ode O

utp

ut (m

V)

-160

-140

-120

-100

-80

-60

Figure 2. Calibration (Average +/- SD) of a planar array of 64

separate calcium sensors using the ETH-5234 calcium-selective

membrane (Anker et al. 1981, Bühlmann et al. 1998). After spin

coating to form the membrane, the sensor array was left to sit

overnight in a dust-free container, then soaked in a solution of

10µM CaCl2 + 10mM NaNO3 (Konopka et al., 2004). Potentials

for the electrodes were measured in calibration solutions of

100µM, 1mM and 10mM CaCl2. The potentials were normally

distributed around a slope of 27mV which is in good agreement

with the theoretical slope predicted by the Nernst equation.

Calcium Concentration (mM)

0.01 0.1 1 10 100

Raw

Ele

ctr

ode o

utp

ut (m

V)

-60

-40

-20

0

20

40

60

10.0mM

1.0mM

0.1mM

Figure 3. The DEDC (dual-electrode differential coupling)

method was developed so that transcellular ions concentration

differentials could be measured without need for system offset

settings. Two glass-bodied probes were placed in separate

Ca++ solutions, connected by a salt bridge. Each series was

replicated using each of three standard solutions as a base

solution (figure legend). For each series the Nernst slope was

approximately 27mV, with the potential equal to zero when both

electrodes are in the same solution.

These basic tests have validated the functionality

of the technology. The MISA chip will be able to measure

the calcium concentration differentials associated with

transcellular currents from individual fern spores in real

time. The array of 64 electrodes is organized in quads

within 16 fluidic pores, enabling simultaneous

measurement of calcium concentrations differentials

around sixteen spores. The MISA system will be used in

parabolic flight/microgravity based physiological

experimentation, to study the role of polar calcium

currents in gravity-dependent cellular development.

Future uses of this in silico cell physiology lab-on-a-chip

technology include biomedical applications such as

advanced through-put cell viability and cancer detection.

Possible pharmacological applications include testing the

effectiveness and dose-response curves of drugs. We

anticipate adapting this base system by development and

adaptation of ionophores for other ions. We are also

developing different versions of this system for

amperometric electroanalytical chemistry, which will be

used for analytes like oxygen, nitric oxide, and various

neurotransmitters to name a few. This will also provide

the technical foundation for adapting enzyme based

biosensors for use on the system.

REFERENCES

Anker P, Wieland E, Ammann D, Dohner R, Asper R,

Simon W. 1981. Neutral carrier based ion-selective

electrode for the determination of total calcium in blood

serum. Analytical Chemistry 53: 1970-1974.

Bühlmann, P., Pretsch, E., Bakker, E. 1998. Carrier based

ion-selective electrodes and bulk optodes. 2.

ionophores for potentiometric and optical sensors.

Chemical Reviews. 98: 1593-1687.

Chatterjee, A., Porterfield, D.M., Smith, P.J.S., Roux, S.J.

2000. Gravity-directed calcium current in germinating

spores of Ceratopteris richardii. Planta 210: 607-610.

Edwards, E.S., Roux, S.J. 1994. Limited period of

graviresponsiveness in germinating spores of Ceratopteris

richardii. Planta 195: 150-152.

Konopka, A., Sokalski, T., Michalska, A., Lewenstam,

A., Maj-Zurawska, M. 2004. Factors affecting the

potentiometric response of all-solid-state solvent

polymeric membrane calcium-selective electrode for low-

level measurements. Analytical Chemistry 76: 6410-6418.

Porterfield, D.M. 2002. The use of microsensors for

studying the physiological activity of plant roots. In

Waisel, Y., Eshel, A., Kafkafi, U. (eds) Plant roots the

hidden half, 3rd Edition. Marcel Decker, New York. 333-

347.

Puglisi, J., Bers, D. 2001. LabHEART: An interactive

computer model of rabbit ventricular myocyte ion

channels and Ca transport. American Journal of

Physiology – Cell Physiology 281: C2049-C2060.

Stout, S.C., Porterfield, D.M., Roux, S.J. 2003. Calcium

signaling during polarity development. Annual Meeting

of the American Society for Gravitational and Space

Biology Abstract #39.


USE OF AN INTEGRATED FLOW-CHAMBER ADHESION ASSAY FOR MEASURING LEUKOCYTE

ADHESION PROPERTIES IN SIMULATED AND ACTUAL MICROGRAVITY

Dennis F. Kucik1,2,3,4

, Robyn L. Rouleau3, Lisa W. Smith

3, Xing Wu

1 and Kiran B. Gupta

1

Departments of 1Pathology and

2Biomedical Engineering, and

3Center for Biophysical Science and Engineering,

University of Alabama at Birmingham; and Birmingham VA Medical Center, Birmingham AL, 35233, USA

It has been known for many years that space flight

results in altered immune performance. After long-

duration missions, astronauts have increased leukocyte

counts, decreased T cell counts, and decreased production

of certain inflammatory mediators. Even after short-

duration flights, astronauts have minor immune-system

abnormalities. Although potential causes of space flight-

related immune dysfunction include physical stress and

cosmic radiation, microgravity alone has been shown in

both ground-based and flight experiments to have an

independent effect on white blood cells (Cogoli, 1993)

A key component of leukocyte function that may be

affected by microgravity is leukocyte adhesion to blood

vessel walls. This has been difficult to approach

experimentally, however, since adhesion is a functional

assay requiring live cells, skilled personnel and

sophisticated equipment, and cannot be readily performed

in flight.

To simulate microgravity on Earth, rotating wall

vessels (RWV) are commonly used. However, adhesion

assays as currently conducted require that rotation be

stopped, a sample removed, and cells washed and labeled

before an assay can be performed. The entire process

from cell sampling to completion of an adhesion assay

can take hours, giving the cells time to recover at 1g and

complicating interpretation of results.

To solve this problem, we have designed and built a

highly automated, integrated system that consists of a

modified RWV coupled to an adhesion assay capable of

real-time bio-imaging and recording. Our integrated

RWV/adhesion assay system can measure adhesion of

cells experiencing simulated microgravity within seconds

of returning to 1g, without stopping rotation of the

chamber.

One of the major challenges to overcome in designing

such a system is efficient, reliable extraction of cells from

a RWV without interrupting its rotation. A sample of

uniform cell concentration is essential for cell adhesion

assays, where adhesion is typically expressed as cells

adhering/minute (which depends on the concentration of

cells entering the chamber per minute). Theory predicts

that the distribution of the cells in the vessel may not be

uniform, but will depend on cell density (buoyancy) and

rotation speed, and that the cells will be subject to forces

of gravity, centrifugal force, and Coriolis-induced motion

(spiraling) (Hammond et al, 2001). Our current system is

apable of delivering leukocyte samples of constant

concentration for prolonged periods (Table 1).

Table 1. Uniformity of concentration of cell sample

extracts. 90 µl samples were withdrawn from the

integrated RWV (without stopping rotation) at the time

points indicated (minutes). These aliquots, which would

normally be perfused through the flow adhesion assay

chamber, were saved and cell concentration (X 106

cells/ml) was determined. Concentrations are the mean

of three measurements + SEM. The system reaches a

steady state within 10 minutes of initiation of rotation,

and delivers samples of uniform concentration thereafter.

Time 1 10 20 30 40 50 60

Conc.

(SEM)

0.7

+.1

1.3

+.1

1.3

+.2

1.3

+.1

1.4

+.1

1.3

+.1

1.3

+.1

The adhesion assay in this system is a laminar flow

chamber, which mimics the conditions in the blood vessel

under which leukocytes actually adhere to vascular

endothelial cells in vivo. Besides being more

physiological, there are additional advantages to using a

flow adhesion assay (as opposed to static assays) to

measure the effects of microgravity. First, the assay can

be completed in seconds, minimizing the recovery time at

1g. Second, a flow chamber assay is more sensitive to

weak adhesion forces than static assays In addition,

some adhesive events (such as adhesion mediated by

selectins, an important class of adhesion molecule) occur

only in the presence of shear, and, thus, cannot be

characterized under static conditions (Finger et al, 1996).

Finally, adhesion to blood vessel walls takes place in a

series of steps, or sub-processes. The flow cell assay

allows visualization of these sub-processes of adhesion,

including rolling, firm arrest, adhesion strengthening,

spreading, and migration.

In order to minimize the time between extraction of

cells and delivery to a microscope-based assay (which

depends on the length of the intervening tubing), an

integrated bio-imaging module was designed that was

small enough to be permanently mounted a few inches

from the RWV. A custom optical train was constructed,

with integrated illumination designed to efficiently

visualize unlabeled, live leukocytes as they were perfused

through a laminar flow chamber, and this was coupled to

a videocamera. Images of sufficient quality to permit

detailed computerized motion analysis were obtained with

this system, as illustrated in figure 1.

D.F. Kucik et al – Integrated RWV adhesion assay


0.0 0.3 1.0 3.00.0

2.5

5.0

7.5

10.0

Cyto D (µµµµg/ml)

Cell

s a

dh

ere

ing

/min

ute

Figure 1. Leukocyte adhesion to vascular endothelial

cells as visualized in the integrated system. Endothelial

cells were cultured for 6 days and incorporated into a

flow-chamber assay system as previously described

(Kevil, Chidlow, Bullard, and Kucik, 2003). Adhesion of

HL-60 monocytic cells to the endothelial cell monolayer

was then observed and analyzed. Arrows indicate

monocytic cells adhering to a monolayer of vascular

endothelium.

Both integrin- and selectin-mediated adhesion were

assayed with this system and compared to results obtained

using a conventional microscope-mounted shear-flow

adhesion assay system. Our data show that the new

integrated assay can detect defects in both rolling and

firm adhesion with sensitivity equal to that of large,

microscope-based flow chamber adhesion assays. Figure

2 shows the dose-dependent effect of cytochalasin D on

HL-60 monocytic cells rolling on purified E-selectin and

ICAM-1 as assayed with the integrated system. This is a

test for modulation of β2-integrin-mediated adhesion that

occurs in response to rearrangement (clustering) of the

integrins. As demonstrated earlier (Kucik et al, 1996),

low-dose cytochalasin D releases β2 integrins from

cytoskeletal constraints, and the resulting rearrangement

into clusters (van Kooyk et al, 1999) is pro-adhesive. At

higher cytochalasin D concentrations, the morphology of

the cell is altered, and adhesion becomes less effective (Ni

et al, 2003). The integrated system was sensitive enough

to detect both the increase in adhesion and its dose

dependence, in good agreement with previous studies

using more conventional, commercial microscope-based

assay systems (Ni et al, 2003). Thus, the integrated

system will be suitable for detecting even subtle effects of

simulated microgravity on adhesivity.

This system has now been adapted to measure acute

effects of actual microgravity in parabolic flight

experiments. Lessons learned from development of this

Earth-based system can form the basis for a future space-

flight culture and assay system to allow NASA to monitor

microgravity-inducible physiological events during space

flights.

Figure 2. Detection of integrin rearrangement-induced

effects on adhesion. HL-60 cells were pre-treated with

cytochalasin D at the concentration indicated and

perfused over a substrate of 25 ng/ml ICAM-1 and 0.5

ng/ml E-selectin (SOURCE) at a shear stress of 0.5

dynes/cm2. HL-60 cells/minute adhering to the substrate

during the period of observation (3 min.) were then

quantified. Data are the mean (+SEM) of at least 5

observations for each concentration.

REFERENCES

Cogoli,A. 1993. The effect of hypogravity and

hypergravity on cells of the immune system. Journal Of

Leukocyte Biology 54:259-268.

Finger,E.B., K.D.Puri, R.Alon, M.B.Lawrence, U.H.Von

Andrian, and T.A.Springer. 1996. Adhesion through L-

selectin requires a threshold hydrodynamic shear. Nature

379:266-269.

Hammond,T.G. and J.M.Hammond. 2001. Optimized

suspension culture: the rotating-wall vessel. American.

Journal of Physiology. - Renal Physiology. 281. (1):F12. -

25. , 2001. Jul.F12-F25.

Kevil,C.G., J.H.Chidlow, D.C.Bullard, and D.F.Kucik.

2003. High-temporal-resolution analysis demonstrates

that ICAM-1 stabilizes WEHI 274.1 monocytic cell

rolling on endothelium. American Journal of Physiology -

Cell Physiology. 285:C112-C118.

Kucik,D.F., M.L.Dustin, J.M.Miller, and E.J.Brown.

1996. Adhesion activating phorbol ester increases the

mobility of leukocyte integrin LFA-1 in cultured

lymphocytes. J. Clin. Invest. 97:2139-2144.

Ni,N., C.G.Kevil, D.C.Bullard, and D.F.Kucik. 2003.

Avidity modulation activates adhesion under flow and

requires cooperativity among adhesion receptors.

Biophysical Journal. 85:4122-4133.

van Kooyk Y., van Vliet SJ, and C.G.Figdor. 1999. The

actin cytoskeleton regulates LFA-1 ligand binding

through avidity rather than affinity changes. J. Biol.

Chem. 274:26869-26877.


DEVELOPMENT OF THE EMCS HARDWARE FOR MULTIGENERATIONAL GROWTH OF

DROSOPHILA MELANOGASTER IN SPACE

M. E. Sanchez1, M. Shenasa

2, A. Maldonado

1, A. Kakavand

1, D. Leskovsky

1, E. Houston

3, A. Howard

3, M. K.

Steele1, and S. Bhattacharya

4.

1Lockheed Martin Space Operations,

2 NASA/Ames Internship Program,

3H&H Engineering, and

4NASA

Ames Research Center, Life Sciences Division, Moffett Field, CA 94035

Understanding the changes that occur in living

organisms to bring about adaptation to the space

environment is essential to supporting future plans for

long-term missions to the Moon and Mars. The European

Modular Cultivation System (EMCS), a life science

research facility developed by the European Space

Agency (ESA), can serve as a habitat for culturing

multiple generations of Drosophila melanogaster. Based

on the results obtained from previously tested prototype

hardware, a new Prototype Container III (PIII) was

developed (Figure 1-A) and tested. Data from Prototype

containers I and II were published previously (M.E.

Sanchez, et al., 2004).

Figure 1. Figure 1-A depicts the EMCS Drosophila

Prototype Container III and its food compartment. Figure

1-B is a close-up of an Experiment Container containing

two Drosophila Prototype Containers and the staggered

configuration of the prototype containers within the

Experiment Container allow optimal lighting and imaging

of the flies.

The primary differences between the new design and

the previous models were: A. Increase in growth chamber

volume, B. Modification of air holes in order to fit the

holding frame for the Experiment Container or EC

(Figure 1-B), C. Addition of stainless steel membranes to

cover air holes, and D. Addition of a rubber band

covering the junction between the growth chamber and

food cylinder in order to prevent larval escape.

The goal of these experiments was to optimize growth

of Drosophila cultures and to ensure biocompatibility of

the cultures within the containers. During the

development phase, modifications to the containers were

made as required to resolve any suboptimal performance

issues. Fly behavior, humidity levels, and hardware

mechanics were assessed. Flies were grown in the

containers using standard techniques for fly handling and

videotaped using the video capabilities of the EMCS

ERM (Experiment Reference Module) camera system.

Assessing optimal humidity levels within the growth

chamber is critical for culture health. Adults could adhere

to container surfaces and die if the humidity levels

dramatically increase, while larvae might dessicate if

humidity levels decrease. In order to find an optimal

material that allows sufficient airflow and maintains

normal levels of humidity, we grew flies in the containers

using three different types of breathable membranes:

stainless steel mesh, Nytex™ (nylon mesh), and a paper

membrane. Growth in these conditions was compared to

standard culture vials covered with the three different

types of membranes (Figure 2).

Figure 2. Population densities of flies in containers that

were covered with stainless steel, Nytex, and paper

membranes. All three membranes allowed air exchange.

Due to shortage of hardware, results of the containers are

representative of a single experiment with duplicates for

each condition. Results of the control-covered vials were

representative of three independent experiments.

While all three membranes allowed air exchange

(Figure 2), only the Nytex membrane covered prototype

containers showed a reduction in progeny size (Figure 3).

Since the integrity of the paper and Nytex membranes are

affected by the humidity levels, the stainless steel

membrane was selected for further experiments.

Selecting a food media that can withstand long-term

storage without losing its nutritional value is also critical

for space flight experiments. Making fresh media on orbit

is impractical and would require significant crew time,

therefore pre-prepared fly media will need to be stored for

several weeks prior to conducting multi-generation

experiments in space. In this test, four media recipes

were made and stored in Mylar™ bags (Impak

Corporation PAKVF 3.5M Silver, available from

www.sorbentsystems.com). We tested the following four

media recipes: A. Semidefined (1% Agar, 8% Brewer’s

Yeast, 2% yeast extract, 2% peptone, 3% sucrose, 6%

glucose, 0.05% magnesium sulfate, 0.05% calcium

chloride, 0.6% propionic acid, 1% of 10% p-Hydroxy-

benzoic acid methyl ester in 95% ethanol) B. Semi-

Biocompatibility of Membranes

0

50

100

150

200

250

300

350

400

450

500

PIII Control-covered

Container Type

Stainless steel

Nytex

Paper

A B

M.E. Sanchez et al. – Development of EMCS Hardware


defined with cornmeal (same as semidefined with the

addition of 60g of cornmeal) C. EMCS Fab Feast 1:1

(6.47% dextrose 6.47% molasses, 0.93% agar, 6.12%

cornmeal, 3.24% yeast, and 2.0% Tegosept) D. EMCS

Fab Feast 3:1 (Same as EMCS Fab Feast 1:1, with the

exception of 9.7% dextrose and 3.23% molasses).

The media was prepared and packed under sterile

conditions. In addition, we used proton beams at a range

of 25-33 kgrays to sterilize a number of our samples. This

was done through the company Nutek at Hayward

California. The additional irradiation was conducted in

order to assess if additional sterilization was necessary for

samples that will be stored for long periods of time prior

to use.

Figure 3. Figures A and B show the results from non-

proton sterilized and proton sterilized media respectively.

Overall, the non-proton sterilized media performed better

than the sterilized media with fly cultures. Data shown

here represent four independent studies at room

temperature.

All four media recipes were able to support fly growth

(Figure 3-A and 3-B). Non-proton sterilized media

performed better than proton sterilized media, by

supporting larger fly populations while still showing no

bacterial or fungal contamination. The reason for the

decrease in performance of the proton sterilized food is

unclear, but it may be due to the degradation of

constituents in the media as a result of proton irradiation.

For purposes of supporting growth in the prototype

containers for 2-3 day egg-lays, we found that the

standard Semidefined media performed the best. The

progeny yields from the Semidefined with cornmeal

media were too large and caused overcrowding of the

containers. However, during space flight experiments, if

longer egg lay periods need to be used to accommodate

crew schedules, then Fab Feast media can be used or for

shorter egg lays, Semidefined with cornmeal can be used.

The data shown in Figure 3 shows that the food is usable

for 2 months. We have also tested these media for longer

periods of time, and find that the food can be stored in

this way at room temperature for periods of over a year

(data not shown) and still sustain fly growth.

Having completed a considerable amount of testing

to optimize the Prototype Fly Containers, we proceeded to

test the Prototype Containers within the Experiment

Reference Model (ERM) hardware in order to optimize

performance within the full flight hardware configuration.

The ERM hardware provides the interfaces and

environmental controls similar to the flight EMCS

research facility that will eventually be used for

conducting Drosophila research in space. Temperature

levels were set at 23°C during the light cycle and 25°C

during the dark cycle. This delta of 2°C was to neutralize

the heat generated from the LED board immediately

above the EC. Growth under these conditions was

compared to standard culture vials that were maintained

in our laboratory incubators.

The ERM was able to support fly growth (Figure 4).

Both locations of the EC, top and bottom, produced

nearly the same numbers of flies. These positions of the

hardware were described earlier in Fig 1B. Temperature

and humidity levels were monitored throughout the

experiment (data not shown). About three days before the

1st generation emerged, we removed the parental

population. Figure 4 shows the counts from the 1st

generations that emerged from each container.

Figure 4. Testing of Prototype containers within the

Experiment Reference Model hardware. Both positions in

the holding frame of the EC generated a comparable

numbers of flies. The controls were conducted using

standard culture vials in our laboratory incubators.

Summary A prototype container has been developed for conducting

experiments with Drosophila melanogaster in the EMCS

hardware on the International Space Station. This

container is capable of supporting larger populations of

flies than standard laboratory vials, as depicted in figures

2 and 4. Our studies have helped optimize humidity

levels, food formulations, and container materials in order

to effectively support fly populations in this hardware.

Tests that should continue in the future include

multigenerational growth testing and the development of

sampling devices.

A Non-Proton Sterilized

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Semidefined-cornmeal

EMCS Fab 1:1

EMCS Fab 3:1

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Bottom Container Top Container Controls

Container Location


INVESTIGATING LOCAL IMPACTS OF HEAT-PULSE SENSORS FOR MEDIA MOISTURE

CONTENT

M.A. Ask1, J.J. Prenger

2, D. Rouzan-Wheeldon

2,

V. Rygalov3, J. Norikane

4 and H.G. Levine

5.

1Kennesaw State University, Kennesaw, GA

2Dynamac Corporation, Kennedy Space Center, FL

3University of

North Dakota, Grand Forks, ND 4Universtiy of Kentucky, Lexington, KY

5NASA Biology Division, Kennedy Space Center, FL

Introduction Cultivating plants in space will require automated

water provision systems to both maximize plant

performance and minimize crew operations. We

report here on studies evaluating small (1 cm

diameter) heat-pulse sensors designed to measure

media moisture content (TMAS sensors produced by

Orbital Technologies Inc., Madison, WI).

Calibrating the sensors’ response to the moisture

content of the media is a critical step towards their

effective and accurate use. The sensors were

designed to perform two functions: heat up and take

temperature readings. When heat is applied to an

object, the local environment of that object, relative

to the heat source, is affected. Analyzing the sensor

areas after an extended period of use was important

to determine the sensors’ lasting effects on their

environment. Thermal imaging was used to observe

the heat dissipation during the heating and cooling

processes. In this way we have addressed the

question of whether the sensors’ thermal input could

change their local environment with respect to the

bulk media. Studying these local effects will guide

procedures for calibration and operation.

Materials and Methods Six moisture sensors were prepared for testing.

Pairs of felt capillary mat squares were cut and

stitched together to hold the sensors in place (Figure

1). The mats were designed to fasten to small,

horizontally oriented fixtures. Relative water

content (RWC) of the capillary mats was calculated

as the ratio of the mass of water in the capillary mat

(the medium) to the mass of water capable of being

held within the mats at 100% saturation (measured

gravimetrically). Different RWC levels were

achieved by wetting the capillary mats to 100%

RWC and then allowing them to dry in open air until

the desired RWC was reached. The TMAS sensors

were programmed to provide thermal pulses of 90

second (s) durations, and temperature data were

acquired at 10 s intervals during each heat pulse and

for a 90 s cooling interval.

7 cm

7 cm

1 cm

Sensor

Software Sensor

Board

Capillary Mats

Legend

TMAS Sensors

Stitching

Drawing not to scale

Top View

IR Camera

Capillary Mats

Plastic bases

Standoffs

Side View

7 cm

7 cm

1 cm

7 cm

7 cm

1 cm

Sensor

Software Sensor

Board

Capillary Mats

Legend

TMAS Sensors

Stitching

Drawing not to scale

Top View

IR Camera

Capillary Mats

Plastic bases

Standoffs

Side View

Figure 1. Experimental setup for the TMAS sensor

testing.

Measurement frequency treatments were

administered at TMAS thermal measurement

(sampling) intervals of 10 and 20 minutes (with 3

sensors in each treatment) for 1 week. Mats were

stored in closed containers during the test to reduce

evaporation and control RWC levels. After one

week, thermal imaging was used to observe heat

dissipation. Heat was applied to each mat internally

via TMAS sensors and externally using a heat gun.

Mathematical Modeling

TMAS readings were used to develop models

relating change in temperature and the slope of the

cooling curve with RWC based upon temperature

measurements recorded every 10 s during heating

and cooling intervals. Five sets of TMAS

measurements were taken at similar RWC levels

during mat drying. Three tests were used to create a

correlation between the reference RWC and TMAS

response for two calibration equation methods.

Independent data sets for TMAS response were used

to validate the calibration methods:

Method 1: ( )iif TcTTccRWC 321 +−+=

Method 2: coolingdt

dTccRWC 54 +=

where: RWC is Relative Water Content (%), Tf is

final temperature after heat pulse (°C), Ti is initial

temperature before heat pulse (°C), dT/dt is the slope

of the cooling curve for the first 20 s after heat pulse

(°C/s), and c1 - 5 are empirical constants.

M.A.Ask, et al. – Local Impacts of Heat-Pulse Sensors


Thermal Imaging

Heat dissipation patterns from TMAS sensor thermal

input and from the external (heat gun) source were

observed in the mats using time-lapsed thermal

photography. Spatial temperatures (as shown by

color) were compared before and after thermal

inputs, internal and external, to determine if there

was a lasting difference attributable to sensor

location compared to the rest of the capillary mat.

Results The heating and cooling phases of the treatment

presented two unique mathematical problems. The

correlations between sensor responses and RWC

were used to create calibration equations using two

mathematic approaches, while independent data

were used to validate the calibrations. Table 1

shows the range of error for all sensors with average

errors of 6.3% and 6.1% for Methods 1 and 2,

respectively. There were data points for which the

prediction error was significantly greater than the

average error. Though there was a lower degree of

variation in Method 2, there was still enough error to

warrant further investigation.

Table 1. The root mean square errors (RMSE) of

validation data sets (N=8) are given for two separate

methods used to calibrate the sensors.

Method 1 Method 2

Sensor 1 8.1% 5.5%

Sensor 2 5.1% 7.1%

Sensor 3 5.6% 5.9%

Sensor 4 6.4% 5.2%

Sensor 5 6.1% 6.3%

Sensor 6 6.3% 6.3%

Average RMSE 6.3% 6.1%

Figure 2 shows typical heating/cooling profiles for

one TMAS sensor (colorized version available at

publication website). After 210 s, observable heat

had dissipated across the mat and temperatures had

returned to pre-heating levels for all but the lowest

RWC level. Measurement frequency during week-

long operation prior to thermal imaging did not

affect the results.

Summary and Conclusions Thermal heat-pulse moisture sensors operate by

emitting heat that could alter the sensor’s

environment and possibly bias their accuracy.

Experiments were done to determine the localized

effects of frequent sensor heating. Two methods

were devised to model the sensor’s response with the

RWC of the media. While one method more

accurately modeled the relationship, significant

errors warrant further investigation. Time-lapsed

thermal photography was used to monitor the

media’s response to internal (TMAS) and external

(heat gun) heating. Infrared images showed no

significant long-term (>3 min) localized changes in

thermal characteristics within the substrate from

either internal or external heat sources.

Figure 2. Thermographic images of the experimental

mats with sensors embedded. A. For internal heat

pulses, faster heat dissipation was evident at higher RWC

levels, but for all RWC levels there was nearly complete

heat dissipation after 210 s. NA – image Not Available.

B. For the external heat applications (white represents

the highest temperature) recovery was again nearly

complete at 210 s. The color version of this figure may be

viewed in the online publication of the journal at

http://asgsb.org/publications.html, link to Gravitational

and Space Biology.

Acknowledgments

This research was supported by NASA contract

NCC10-52. We thank the SLSTP program and

Bionetics Corp. for their participation in this

research.

NA

Temp ¡C

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Time (s)

Relative Water Content

60% 75% 90% 100%

0

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Relative Water Content

60% 75% 90% 100%

Heating Cooling0 s 210 sB.

A.

NA


PERFORMANCE EVALUATION OF A LABORATORY TEST BED FOR PLANETARY

BIOLOGY

Nathan A. Thomas1, Paul Todd

1*, G. William Metz

1, Michael A. Kurk

1, David J. Thomas

2,

1SHOT, Inc.,

Greenville, IN 47124, and 2Lyon College, Batesville, AR.*Corresponding author [email protected]

The major issues of planetary biology consist of

searching for life, planetary protection, environmental life

support for human visitors, and terraforming. A

definition of terraforming is the conversion of a planetary

surface to earth-like conditions hospitable to Earth life. A

subset of terraforming is ecopoiesis, the early

development of a living ecosystem (Haynes and McKay,

1992). Despite approximately two decades of public

discussion there has been little or no experimental

research focusing on ecopoiesis. Initially, small

experiments with terrestrial organisms in a planetary

(Mars-like) environment are required. Such experiments

simultaneously address issues of ecopoiesis and planetary

protection. To facilitate such experiments in research

laboratories a laboratory test bed was constructed for the

simulation of planetary environments for biological

experimentation.

The surface of Mars was chosen as the principal

environment to simulate. The requirements for high-

fidelity simulation of the Mars environment are based on

current knowledge or understanding of each of the

following features (Banin and Mancinelli, 1995):

Atmosphere: The atmospheric pressure on the surface of

Mars varies between 7 and 15 mbar, depending on

altitude (mountains vs. valleys) and season. This is about

1% of the pressure on the surface of the Earth. The

atmosphere contains 95% CO2, 2.7% N2, 1.6% Ar, 0.13%

O2, 0.03% H2O and trace amounts of Ne, Kr, Xe and O3.

Since the trace O3 does not contribute to the UV

absorbance of the atmosphere it and the minor inert gases

are not essential components of a simulated Mars

atmosphere. Temperature: The maximum temperature on

the surface of Mars on a cloudless summer day at

equatorial and mid-latitudes is about 26oC. The minimum

night-time temperature at higher latitudes is -135oC. An

average daily cycle under consideration for simulation

purposes is -80oC minimum night-time temperature and

+26oC maximum mid-day temperature (Carr, 1996). This

day-night temperature cycle must be repeated every 24.6

h, the length of the Martian sol (day). Sunlight: The total

intensity of sunlight on the surface of Mars is about 40%

of that at the top of Earth’s atmosphere, so that the

Martian surface receives 590 W-m-2

of the essentially

unattenuated solar spectrum. This spectrum includes

ultraviolet light with wavelength below 300 nm, including

down to 190 nm. This spectral range is photochemically

damaging to nucleic acids and known to kill cells and is

included in the spectral output of xenon arc lamps.

Regolith: The dust on the surface of Mars has a high

content of oxides of iron, some of which were found to be

oxidizing agents in the experiments on the Viking

missions. In elemental composition the regolith is 12.5%

Fe, 21% Si, 5% Mg, 4% Ca, 3.1% S, 3% Al, 2.3% Na,

0.7% Cl, 0.3% P, and contains traces of Mn, Co, Cu, I,

Zn, B, etc. Thus, in addition to the aluminosilicates there

is an ample supply of components required by living

organisms, except nitrogen and carbon, which are

atmospheric (Banin and Mancinelli, 1995).

In order to match these requirements the test bed

consists of the following subsystems: a 1,000 W xenon

arc lamp with an “AM0” filter to simulate solar irradiance

in space, an 8 ft3 cryogenic thermal cabinet capable of

cooling to -135oC and heating to +50

oC using evaporating

nitrogen and resistive heaters, a fused silica cylinder that

transmits the full solar spectrum and can be evacuated to

<10 mbar through stainless steel end caps, a 500-gal

liquid nitrogen storage tank, a cylinder of pressurized

support gas, and a control system that automatically

cycles the interior of the cylinder through a Mars daily

cycle, for example. Biological samples placed in the

quartz cylinder are typically mixed with simulated

planetary regolith. A rendering of the simulator design is

shown in Figure 1.

During initial operation the test bed was used for

preliminary biological experiments designed to test

concepts of ecopoiesis. The light and temperature profiles

matched those of Mars near the equator at the vernal

equinox. The test bed was cycled daily between -80oC

and +26oC following a published temperature curve (Carr,

1996) shown in Figure 2, which includes a demonstration

of maximum heating and cooling rates achievable in the

test bed.

The atmospheric pressure on Mars is 7-15 mbar, and the

day length is 24.6 hours, but internal pressure of 100 mbar

and day length of 24.0 hours were used for early

biological experiments. The gas composition used was

0.139% O2, 1.615% Ar, 2.760% N2 with the balance CO2

or, in some cases, pure CO2. The atmosphere was

saturated with water by the daily injection of a few grams

of (liquid) distilled water into the atmosphere (water boils

at 49oC at 100 mbar). The measured photosynthetically

active photon flux was 1100 µmoles-m-2

-s-1

in direct light

and 12 µmoles-m-2

-s-1

in the shaded interior of the

cylinder. This corresponds closely to values determined

for the Martian surface and is consistent with the 590 W-

m-2

total energy flux. Pressure, illumination, cabinet

temperature and regolith temperature were recorded

during operation of the test bed for 1, 8, 14 and 35-day

periods. An example of a 14-day data set is shown in

Figure 3. Under daily cycling liquid nitrogen was

consumed at approximately 1600 liters/week and was

exhausted safely to the exterior of the laboratory.

N. Thomas et al. – Planetary Biology Test Bed


Figure 1. Laboratory Test Bed design showing components for illumination, temperature control, atmosphere supply, and

specimen chamber (“Mars Jar”).

Figure 2. Temperature profile chosen for simulation in

early test-bed experiments. Left (triangles) and right

(squares) vertical curves represent maximum heating and

cooling rates, respectively, in the environmental chamber.

This research was funded under subcontract agreement

07605-003-026 with NASA's Institute for Advanced

Concepts (NIAC), a program of the Universities Space

Research Association (USRA) funded by NASA contract

NAS5-03110.

Figure 3. A record of physical data for a 14-day

experiment with daily cycling of temperature between -

80oC and +26

oC, 12 h of light and 12 h of darkness,

pressure of 100 mbar. Lower trace is cabinet

temperature, and upper trace is regolith temperature.

Horizontal bar at 0 indicates illuminator on, bar at 10,

off.

REFERENCES

Banin, A. and Mancinelli, R.L. 1995. Life on Mars? I.

The chemical environment. Advances in Space Research

15:163-170.

Carr, M. H. 1996. Water on Mars. Oxford University

Press, New York.

Haynes, R. H., and McKay, C. P. 1992. “The

implantation of life on Mars: Feasibility and motivation.”

Advances in Space Research 12: 133-140.

ENVIRONMENTAL

CONTROL UNIT

1.5kW SOLAR

SPECTRUM

GENERATOR LAPTOP

COMPUTER

SPECIMEN

CHAMBER

FULL SPECTRUM

LIGHT DISTRIBUTION

MIRROR

2 STAGE REGULATOR

LIQUID

NITROGEN

SIPHON TANK

VACUUM

PUMP

LOW PRESSURE

GAS RESERVOIR

MARS ATMOSHPERE

HIGH PRESSUREGAS TANK

STAND

MARS GAS SUPPLY/

VACUUM SYSTEM

MULTI-PANE

WINDOW (UV SAFE)

WITH CURTAIN

MARS

ATMOSPHERE

GAS LINE IN

VACUUM

LINE OUT

INSULATED

DOOR


A STUDY OF THE EFFECTS OF SPACE FLIGHT ON THE IMMUNE RESPONSE IN DROSOPHILA

MELANOGASTER

Thomas F. Fahlen1*

, Max Sanchez1*

, Matthew Lera2, Edina Blazevic

2, Jennifer Chang

1, and Sharmila

Bhattacharya3

1Lockheed Martin Space Operations, NASA Ames Research Center, Moffett Field, CA 94035

2NASA/Ames Internship Program, NASA Ames Research Center, Moffett Field, CA 94035

3NASA Ames Research Center, Life Sciences Division, Moffett Field, CA 94035. *Both authors contributed

equally to this work and should be considered as joint first authors.

Drosophila melanogaster (fruit fly) is a small, highly

tractable and well-characterized organism that is ideal for

the study of molecular, cellular, developmental, and

physiological processes. In Drosophila, many of these

processes, including certain aspects of innate immunity,

are very similar to those in humans (1). There is evidence

that space flight affects the immune system of Astronauts

such that the Astronauts may become

immunocompromised during and after flight (2, 3). In

order to study those possible effects, our laboratory will

be supporting a study to be carried in the mid-deck of the

Space Shuttle to examine the effects of space flight on the

immune system using D. melanogaster as a model.

Our experiment configuration consists of 10 sample

containers (divided into 5 primary and 5 secondary

containers) that support adult Drosophila and allow egg

laying and larval development during flight. During the

scheduled flight, virgin female and male adult flies will be

loaded into the primary containers. The flies will then be

allowed to lay eggs on the food for three days. At the end

of the third day, the food tray in the primary containers

will be switched to the secondary containers. This will

move eggs and larvae produced during pre-flight and

launch to the secondary cassettes. A new food tray will be

placed in the primary container. The sample containers

consist of ventilated aluminum cassettes enclosed by

ventilated type I containers (Fig. 1 A-B). In order to

execute this experiment in space, these aluminum

containers were built specifically to allow Drosophila

growth under the ambient conditions of the shuttle

middeck, without requiring power. This paper describes

biocompatibility tests to show the suitability of the

materials to specimen welfare, and should facilitate the

use of these containers by other investigators in the future.

Figure 1. Figure 1-A depicts one fully loaded Drosophila

container. Figure 1-B depicts the Type I container in

which the cassette will be enclosed.

The individual containers will be housed in one of the

middeck drawers of the Space Shuttle. This consists of a

foam tray holding change-out platform. The foam used in

this drawer is made out of polyurethane foam Pyrell®.

This material is frequently used in the packaging of flight

payloads because it is a low flammability foam. In order

to check if the foam used in the mid-deck drawer had any

adverse effect on the viability of the flies, 1cm cubes of

flight foam were placed in vials containing fly media. 10

virgin females and 10 male flies were added to each of

these vials. The flies were allowed to mate and lay eggs

for two days at 25°C. At the end of this period, the

parental population was removed from the vials. The vials

containing eggs were allowed to develop for 12 days at

25°C. The progeny were counted and the male/female

ratios were scored (Figure 2).

Figure 2. Total progeny produced from eggs laid in the

presence or absence of polyurethane foam Pyrell®. N=5

The results on Figure 2 suggest no significant difference

in the number of progeny produced in the presence or

absence of the flight foam material. Additionally, no

significant death of parental flies was observed (data not

shown). It was concluded, therefore, that Pyrell® foam

was biocompatible with the specimen in this experiment

configuration.

The next study was used to evaluate the effects of

different starting population densities on progeny

production. In order to find the optimal number of starting

parental population, we tested two different

configurations: A. 5 virgin females and 5 males (5F+ 5M)

and B. 10 virgin females and 5 males (10F + 5M). It is

important to optimize the size of the starting population in

order to maximize the number of progeny developed in

space for each experiment. As shown on Figure 3, a

A B

T. Fahlen et al. – A Study of the Effects of Space Flight on the Immune Response in Drosophila

melanogaster


starting population of 10 virgin females and 5 virgin

males is optimal for our 12 day experiment in space. The

total progeny size from 10 females and 5 males was

bigger than that produced by 5 virgin females and 5

males, and yet these containers did not show signs of

overcrowding.

When the samples return from space, a considerable

amount of work will be conducted with blood obtained

from third instar larval samples. An experiment was

performed with the flight containers for the full flight

duration and the number of resulting embryos and larvae

were counted as shown in Figure 4. Large numbers of

third instar larvae were obtained which should be more

than adequate for the hemocyte analyses to be conducted

from the space-flown samples. The embryos and earlier

instar larvae are also present in sufficient numbers for

adequate morphology and gene expression studies

planned for our flight experiment.

Figure 4. Number of animals at each developmental

phase.

Our experiment is designed to assess the immune function

of flies following a shuttle mission. As an assay for

immune function, we have adapted a bacterial clearance

assay, previously described by Brandt et al. (4), to our

post-flight processing. Briefly, this assay involves

infecting the flies with a known quantity of bacteria and

assessing the ability of the flies to clear the infection.

Ground testing in our laboratory shows that we are able to

infect flies with 10,000 E. coli HB101 bacterial cells with

greater than 95% survival of the flies (data not shown).

Following 3 days of infection we find that the flies reduce

their bacterial load by two orders of magnitude (Figure 5).

Our flight experiment will utilize this assay to assess the

function of the immune system of flies that have

developed in space.

Figure 5. Bacterial challenge assay. Flies were injected

with 50 nl of a 2 x 108 cells/ml suspension of E. coli

HB101 in PBS (10,000 cells/fly). A subset of the flies

(Day 0) were immediately assayed for bacterial load by

dilution plating of homogenates prepared in Luria Broth

+ 1% triton X-100 with 3 flies per homogenate. A second

subset of flies were incubated at 29 °C for 3 days and then

assessed for bacterial load in the same manner. Only live

flies were assayed. Error bars show the standard

deviation from the mean. N = 3.

We have developed and tested containers that will sustain

our specimen for an experiment designed to address the

effects of spaceflight on the innate immune system of

Drosophila. These containers should also be appropriate

for use by other investigators needing to use Drosophila

melanogaster as a model specimen in space.

ACKNOWLEDGEMENT

We gratefully acknowledge helpful discussions with Dr.

David Schneider concerning the bacterial infection assay.

REFERENCES

1. Hoffman, J.A., F.C. Kafatos, Ç.A. Janeway Jr.,

R.A.B. Ezekowitz. 1999. Phlogenetic Perspectives in

Innate Immunity. Science. 284:1313-1318

2. Borchers, A.T., C.L. Keen, M.E. Gershwin. 2002.

Microgravity and immune responsiveness:

implications for space travel. Nutrition. 18:889-898.

3. Sonnenfeld, G. and W.T. Shearer. 2002. Immune

function during space flight. Nutrition. 18:899-903.

4. Brandt, S.M., M.S. Dionne, R.S. Khush, L.N. Pham,

T.J. Vigdal, D. S. Schneider. 2004. Secreted Bacterial

Effectors and Host-Produced Eiger/TNF Drive Death

in a Salmonella-Infected Fruit Fly. PLOS. 2:e418.

100

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Figure 3. Comparison of starting parental density on

progeny production in flight cassettes.

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10F + 5M

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Embryos 1st Instar 2nd Instar 3rd InstarDevelopmental Phase

5F + 5M

10F + 5M


EFFECTS OF ALTERED GRAVITY ON IDENTIFIED PEPTIDERGIC NEURONS OF THE CRICKET

ACHETA DOMESTICUS.

Uta Kirschnick1, Hans-Jürgen Agricola

1 and Eberhard R. Horn

2

1 University of Jena, Department of Cell Biology, Philosophenweg 12 , 07743 Jena, Germany

2 University of Ulm, Gravitational Physiology, Albert-Einstein-Allee 11, 89081 Ulm, Germany

The development of organisms requires a permanent

interplay between genes and environment. Identified

neurons that are common in insects are excellent models

to determine the specific contribution of these factors for

the development of the nervous system. The aim of the

study was to investigate effects of microgravity (µg) on

identified peptidergic neurons in a cricket (Acheta

domesticus) (1) if eggs were fertilized in space and (2) if

the period of neuronal proliferation took place in µg.

Proliferation is mainly completed after 50% of embryonic

development. Thus, an 8 days lasting µg-exposure was

sufficient to answer the question. The 10 days lasting

Italian Soyuz taxi flight ENEIDE to ISS in April 2005

was used.

We chose neurons immunoreactive (ir) to allatostatin

(AST), crustacean cardioactive peptide (CCAP), and

perisulfakinin (PSK) because these neuropeptides are

involved in developmental and neuronal processes

(Homberg 1994). AST inhibits the synthesis of juvenile

hormone; AST neurons are usually equipped with short

axons; rarely do they extend to more than 4 segments

(Fig. 1). CCAP has myotrope function with strong effect

on heart activity; it probably contributes also to the

regulation of molting. PSK is a myotrope neuropeptide; in

addition, effects on the central nervous system were

described. Due to its wide projections (Fig. 2), P-PC1

neurons probably exert a modulating effect on neuronal

activity.

We considered these peptidergic neurons as model

structures to study the sensitivity of the topological

organization within the central nervous system to µg

because it is very unlikely that gravity effects on the

development of topological organization depends on

specific neurotransmitters. This assumption might not be

correct if the investigated neurons have mono- or

polysynaptic input from gravity receptors such as the

position sensitive interneuron PSI (cf. Sakaguchi and

Murphey, 1983).

The experiment CRISP-2/ENEIDE was a follow-up

study of the Neurolab experiment CRISP (Crickets in

Space). Then post-flight observations on cricket larvae

included neuroanatomical, neurophysiological, and

behavioral studies of different developmental stages. All

stages as well as embryos used in CRISP/Neurolab had a

fully developed set of neurons at onset of the mission.

The main observations were absence of significant

behavioral modifications and anatomical changes of

cerebral peptidergic neurons, but a significant depression

of the sensitivity of the PSI (Horn, 2003; Horn et al.,

2002).

A special experimental container was developed for

inflight fertilization on ENEIDE. It consisted of an adult

chamber and larval chambers, separated by egg collectors

(Fig. 3). If females inseminated on ground had access to

Figure 1. Allatostatin A-PC2-ir neuron and its primary neurite

within the right hemisphere from a space (middle) and ground

(right) embryo. Post-flight fixation at 60-65% of embryonic

development. The mature A-PC2-ir group consists of 6 neurons

(left). At stage 60-65% embryos, only 4 out of the 6 neurons are

visible in both, flight and ground embryos.

Figure 2. Basic projection pattern of a PSK PC1,2-ir neuron.

Figure 3. Cricket container for in-flight fertilization. On top the

adult chamber (CC-AC), on bottom 2 larval chambers (CC-

LC1,2); in-between 3 egg collectors (CC-EC1,2,3). They were

filled with vermiculite; 2.5 days after launch, females had access

to the vermiculite for 20 hrs to deposite eggs. Inset: individual

egg collector with deposition area on top.

the vermiculite within the egg collectors, they deposited

their eggs. During this process, the eggs were fertilized.

After termination of the 8 days lasting µg-exposure, the

nervous system was taken from embryos or, after hatch-

U. Kirschnick, H. Agricola and E.R. Horn – Effects of altered gravity on identified peptidergic neurons


ing, from the 1st instars for immunocytochemical staining

of neurons. The 1st fixation was done 6 hours after landing

in Kazakhstan, a 2nd

fixation 34 hours later in Moscow,

and a 3rd

fixation after hatching and testing of the geotaxis

of the 1st instars. A total number of 112 embryos and 1

st

larvae from µg-fertilization were available, and 103 from

on-ground fertilization.

The ENEIDE flight revealed that after in-µg

fertilization, AST-ir-, PSK-ir- and CCAP-ir-neurons

developed as after on-ground fertilization with respect to

the location of somata and arborizations. This held not

only for cerebral (Fig. 1), thoracic or abdominal (Figs.

4,5) neurons with short neurites, but also for PSK-ir

neurons that project throughout the whole nervous

system, with cell somata lying in the protocerebrum and

arborizations within the cerebral, thoracic and abdominal

ganglia (Figs. 2,6).

Figure 4. Somata of CCAP-ir neurons C-BM1 in the abdominal

ganglia (AG) from 55% embryos (top and middle) and 1st

instars (bottom). Left: Basic C-BM1-ir projection patterns.

Figure 5. Location of 2 types of CCAP-ir neuron in the

terminal (last) ganglion of 1st instars. Neurons at the rostral

corner of the ganglion: C-BM1+2-ir neuron. - Neurons at the

median caudal location: C-TG neuron. Note the clear similarity

of the arborization and the projection pattern of the flight and

ground embryos. - Calibration 100 µm.

Thirty-three space embryos were reared up until

hatching. They hatched about 1.5 days earlier than the

ground reared embryos. The space larvae revealed no

abnormal geotactic behaviour compared to ground larvae;

however, the quantitative analysis concerning walking

velocity and

curvature of walking trajectories is not yet completed.

These studies demonstrate that developmental

processes within the nervous system of insects are

obviously under a dominant genetic control. This does not

exclude an influence of epigenetic factors on other

developmental processes in insects including the nervous,

motor and sensory systems. The earlier hatching of the 1st

larvae after inflight fertilization makes this hypothesis

very likely.

Supported by DLR: grant 50WB0323 to Horn.

Figure 6. PSK PC1,2-ir neurons from two 1st larvae, after

fertilization in µg (left) or on ground (right). Top: Somata of

PC1,2-ir neurons in the brain. Middle: Projections of the

neurons at the level of the 6th abdominal ganglion. Bottom:

Projections of the neurons within the terminal ganglion (cf. Fig.

5a). Calibration 50 µm (top, middle), 100 µm (bottom).

REFERENCES

Homberg, U. 1994. Distribution of neurotransmitters in

the insect brain. Gustav Fischer, Stuttgart Jena New York.

Horn, E.R. 2003. The development of gravity sensory

systems during periods of altered gravity dependent

sensory input. Adv. Space Med. Biol. 9:133-171.

Horn, E., Agricola, H., Böser, S., Förster, S., Kämper, G.,

Riewe, P., and Sebastian, C. 2002. Crickets in Space:

Morphological, physiological and behavioral alterations

induced by space flight and hypergravity. Adv. Space

Res. 30:819-828.

Sakaguchi, D.S., and Murphey, R.K. 1983. The

equilibrium detecting system of the cricket: physiology

and morphology of an identified neuron. J. Comp.

Physiol. 150:141-152.

Gravitational and Space Biology Bulletin 17(2) June 2004 137

COUNTERMEASURES TO THE EFFECTS OF GRAVITY ON THE SKULLS OF HUMAN INFANTS. R. Lee

1, J. English

1, J. Duke

1 , and J. Teichgraeber

2.

1Dept. of Orthodontics, Dental Branch,

2 Division of Pediatric Surgery, Department of Surgery, Medical School,

University of Texas Health Science Center at Houston, Houston, TX.

The effects of altered gravity on the skeleton have

been studied mainly in weight-bearing bones, so few

gravitational biologists know how Earth’s gravity shapes

the skulls of human infants. Skull deformations due to

prolonged exposure to the g vector in one direction are

well-known to craniofacial experts, and include brachy-

cephaly, a flattening of the back of the skull, and posterior

deformational plagiocephaly (PDP), in which the infant’s

skull, viewed from the top, has the shape of an oblique

oval, which can be enclosed in a parallelogram (Teich-

graeber et al., 2002; Fig. 1A, 1B). This shape results from

a flattening of either the left or right occipito-parietal re-

gion of the skull, causing bossing, or bulging, of the con-

tralateral occipital region and the ipsilateral frontal bone.

The ipsilateral ear and malar eminence, or cheekbone, are

displaced anteriorly as are the eye and the temporo-

mandibular joint (TMJ).

PDP is associated with a number of factors related to

intrauterine constraint: loss of amniotic fluid, premature

and multiple births, the birthing process itself and post-

natal positioning. Some infants have a torticollis, a short-

ened muscle affecting head positioning. Recalcitrant

cases require surgery, and increased levels of TGF β2 and

β3 have been found in affected sutures (Lin et al., 1997).

An infant’s skull is designed to be deformed, being

composed of plates of intramembranous bone connected

by tough fibrous membranes or sutures, a construction

allowing for compression of the infant’s head during birth

and postnatal growth of the bones. In craniosynotosis,

sutures fuse prematurely, resulting in a malformed skull

requiring surgery. Fusion of the lambdoid suture between

the occipital and parietal bones, like PDP, results in

bossing of the contralateral occipital region. The frontal

bossing, however, is on the contralateral side, and the ear

and malar eminence are not displaced (Habal et al., 2003).

PDP includes a facial asymmetry, continuing into

adulthood, due to rotation of the cranial base and the re-

sulting anterior displacement of the TMJ and consequent

asymmetric mandibular growth (St. John et al., 2002).

A. B. C.

Figure 1: A. Normal head shape. B. Parallelogram

shape of head of infant with PDP. C. Thick line indicates

how DOC helmet therapy changes growth of regions of

the skull.

Disturbingly, a number of developmental anomalies are

associated with PDP (Habal, et al., 2003). Delays in both

cognitive and psychomotor development have been noted

and auditory processing disorders affecting speech and

cognition have been found. CT scans have revealed an

accumulation of fluid in the frontal region of the cranium.

In light of these problems in infants with PDP, the

increase in incidence from 1 in 300 to 1 in 10 since 1992

is alarming (Teichgraeber et al., 2002). In 1992, the

American Academy of Pediatrics urged parents to place

sleeping infants on their backs to prevent Sudden Infant

Death Syndrome. The message was reiterated in 1994 in

the AAP’s “Back to Sleep” campaign. As a result, the

incidence of SIDS decreased by 45%, but craniofacial

centers were inundated with parents seeking treatment for

children with misshapen skulls. In 1995, the Division of

Pediatric Surgery at the University of Texas Medical

School in Houson treated two infants with PDP; in 1996,

they treated 54 (Teichgraber et al., 2002).

Some pediatricians advocate a wait and see attitude, but

craniofacial specialists prefer early intervention, while the

skull still has sufficient growth potential to correct the

deformations (Teichgraber et al., 2002). In some cases,

the problem can be corrected with repositioning the infant

during sleep, so that the head does not rest on the

flattened portion of the skull. Increased tummy time, in

which the infant is placed prone under parental

supervision, also alleviates pressure on the occipital

region, and encourages head raising, thus strengthening

the neck muscles. If a torticollis is involved, physical

therapy is required to stretch the infant’s muscles in order

to reposition the head. Time spent in car seats, carriers or

swings should be limited.

Simple countermeasures are not always successful in

skull reshaping, and in these cases a molding helmet such

as Dynamic Orthotic Cranioplasty (DOC) is used (Fig.

1C). The custom molded DOC helmet consists of a semi-

rigid styrene shell and inner polyurethane liner, and

relieves pressure on the flattened part of the skull

allowing growth in that region. Parts of the skull that

developed bossing or bulging are constrained by the

helmet, and growth in those areas thus restricted. DOC

helmet treatment has disadvantages; the helmet must be

worn 23 hrs/day,requires weekly monitoring/adjustment,

and is costly. Sometimes, the helmet does not correct the

deformation, and surgery is required.

A study of 125 patients treated with DOC helmet

therapy (Cranial Technologies, Inc., Tempe, AZ) at the

UT School of Medicine found that asymmetries of the

cranial vault (CV) and the cranial base (CB) were signif-

icantly reduced (41.6% and 40.2%) by treatment (Teich-

graber et al., 2002). Orbitotragial (OT) asymmetry was

improved by 18.7%. Recently, the question of retention

of improvement was addressed by re-examining these

children 5 years after their treatment.

R. Lee et al. – Countermeasures to the Effects of Gravity on the Skulls Of Human Infants


MATERIALS AND METHODS

14 male and 14 female patients from the previous study

were re-examined. 79% had right side flattening. Mean

start age for treatment was 6.7 months, and mean treat-

ment time was 6.2 months. 7.1% were born prematurely,

3.6% were from breech births, and 3.6% from multiple

births. 7.1% had a torticollis. These percentages are

similar to those in the general population. Skull

measurements used anthropometric landmarks (Figure 2):

frontozygomatic point (FZ)-suture of frontal and zygo-

matic bones, midpoint of outer edge of eye orbit; euryon

(EU)-most lateral point on the side of the cranium-from

EU to EU is the widest portion of the skull; exocanthion

(EX)-where eyelids meet in outer corner of eye; tragus

point (TR)-notch in ear cartilage in front of ear; inion

point (IN)- the most prominent point on the external

occipital protuberance in the median sagittal plane. CV

asymmetry was the distance from left FZ point to right

EU point minus the distance from the right FZ point to the

left EU point. Orbitotragial depth asymmetry was the

distance from right EX point to right TR point minus the

left EX point to the left TR point. CB asymmetry was IN

point to right TR minus IN to left TR. Asymmetries were

compared to those immediately post treatment. Facial

asymmetry and malocclusions were also tabulated.

Figure 2: Points for anthropometric measurements.

RESULTS Measurements of asymmetry immediately after treatment

and 5 years later are shown in Table 1. Only the %

change in CB asymmetry was significant.

Table 1: Asymmetry changes 5 years posttreatment.

Measurement Asymmetry (mm) % Change

Cranial Vault 5.57 6.89 +23.7% (n.s.)

Orbitotragical

Depth

2.89 2.25 -22.2% (n.s.)

Cranial Base 3.64 8.11 +122.5%

(p< .004)

In terms of facial asymmetry, 19 of 28 (67.86%) had chin

point deviation to the unaffected side. Deviation of the midline was observed in 67.86%, with upper and lower

jaws not necessarily congruent. Posterior dental crossbites

were noted in 17.89% of the sample. Molar relationships

were examined in 17 patients, and the incidence of Class

II (lower molar posterior to upper) and Class III (lower

molar anterior to upper) relationships was greater than

normal.

DISCUSSION

DOC helmet therapy effectively corrects cranial a-

symmetry, especially CV and CB, but after 5 years,

significant relapse in CB occurs, and may include jaw

asymmetries leading to misaligned teeth, TMJ problems,

and esthetic concerns. Reasons for relapse are unknown,

but likely involve skull growth patterns. CB asymmetry is

based on IN, the middle of the back of the head, and

improves with DOC helmet treatment because cranial

flattening improves. The ear (TR) remains forward due to

the rotated CB, the anterior portion of which continues

growing, increasing the posttreatment asymmetry. Less

than 5% of the general population has noticeable facial

asymmetry.

Since CB asymmetry recurs, producing facial asym-

metries and malocclusions, greater efforts at prevention of

PDP are needed, including increased awareness on the

part of pediatricians, family members and other care-

givers. Parents must realize that the shape problem of

their baby’s skull may not resolve with time, and needs to

be addressed with repositioning from an early age.

The AAP 1992 recommendations on sleep position

allowed side sleeping, but in October, 2005, side sleeping

was recommended against, a change that is likely to cause

a further increase in PDP and makes the need for ed-

ucation even more urgent. The cure for PDP is preven-

tion, and the key to prevention of PDP is education.

ACKNOWLEDGEMENTS

Supported by UTDB Dept of Orthodontics.

REFERENCES

Habal MB. Leimkuehler T. Chambers C. Scheuerle J.

Guilford AM. Avoiding the sequela associated with defor-

mational plagiocephaly. Journal of Craniofacial Surgery

14: 430-7, 2003.

Lin KY. Nolen AA. Gampper TJ. Jane JA. Opperman LA.

Ogle RC. 1997. Elevated levels of transforming growth

factors beta 2 and beta 3 in lambdoid sutures from

children with persistent plagiocephaly. Cleft Palate-

Craniofacial Journal. 34:331-7.

St John D. Mulliken JB. Kaban LB. Padwa BL. 2002.

Anthropometric analysis of mandibular asymmetry in

infants with deformational posterior plagiocephaly.

Journal of Oral & Maxillofacial Surgery. 60: 873-7.

Teichgraeber, JF, Ault, JK, Baumgartner J, Waller, A,

Messersmith M, Gateno J, Bravenec B, Xia, J.

Deformational posterior plagiocephaly: diagnosis and

treatment. Cleft Palate Craniofacial J. 2002: 39: 582-586.

eu eu

tr tr

fz fzex ex ex

tr

in


NUTRIENT DIFFUSION THROUGH ARTICULAR CARTILAGE: DEVELOPMENT AND USE OF A

MODEL SYSTEM.*

Candace Marshall, Ruby Flowers, Neeta Goli, Marianne Vandromme, Douglas Paulsen and Brenda Klement

Dept. of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA 30310

Cartilage is an avascular connective tissue composed of

cells (chondrocytes) suspended in a gel-like matrix that

gives cartilage its ability to bear mechanical stresses

without distortion. The main function of cartilage is to

provide a shock-absorbing and sliding area for joints thus

facilitating bone movements. Components of cartilage

extracellular matrix (ECM) include type II collagen,

glycosaminoglycans (GAGs) and other molecules cross-

linked to give firmness. There is also a substantial

amount of water bound to the negative charges of GAGs

which give the tissue a firm, gel-like consistency allowing

it to act as a biomechanical spring and shock absorber

(Paulsen, 2000).

To maintain structure and function, the chondrocytes

must obtain needed nutrients and molecules via diffusion

through the abundant matrix (Pelletier et al,. 2000). The

composition of the ECM is known to change with age and

with diseases such as arthritis (Bayliss et al., 2000). A

change in composition of the ECM would result in a

change in the arrangement or architecture of the ECM

components, and potentially result in a change in nutrient

diffusion. Disruptions in nutrient flow and availability

have been associated with articular disorders such as

arthritis (Kuettner 1992).

Astronauts experience a dramatic headward fluid shift

in microgravity, which would be expected to decrease the

hydration level of cartilage in the joints of the lower

extremities, and subsequently compromise mobility. To

our knowledge, nutrient flow has not been evaluated in

animal or human articular cartilage during spaceflight.

The goal of this project was to develop a model system

that could be used to assess nutrient diffusion through

articular cartilage matrix.

In this model system, fluorescent labeled glucose (2-(N-

(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-

deoxyglucose), referred to as 2-NBDG, was used. These

initial studies examined only the non-metabolized 2-

NBDG. Future studies will explore the 2-NBDG

metabolized by chondrocytes and the 2-NBDG that

remains associated with the ECM. The model can be

used to assess 1) the 2-NBDG that diffuses into cartilage

within a certain time period or 2) the 2-NBDG that

diffuses out of cartilage after saturation with 2-NBDG.

The ultimate objective is to use this model to determine if

changes in diffusion reflect changes in the composition

and architecture of the ECM occurring with disease, age,

and the fluid shifts associated with spaceflight.

In the original model system, 50µm slices of bovine

articular cartilage were used. The slices were incubated

in 50µM 2-NBDG at room temperature. The longer the

tissue was incubated, the more 2-NBDG diffused into the

tissue, and reached a maximum by 1 hour (figure 1). To

* presented at 2004 meeting

quantitate the amount of 2-NBDG that diffused out of

each cartilage section, the sections were soaked in 2-

NBDG for one-hour then incubated for one-minute

intervals in phosphate buffered saline (PBS). The

quantity of fluorescent 2-NBDG in each PBS aliquot was

measured with a Cytofluor 96-well plate

spectrophotometer. The sum of the fluorescence was

determined for each cartilage section. Cartilage sections

were weighed (wet weight) then dried overnight at 60°C

and re-weighed (dry weight). The amount of non-

metabolized 2-NBDG that diffused out of the cartilage

sections was normalized to section wet and dry weights.

The location of 2-NBDG can be seen in cartilage

sections using confocal microscopy. After soaking in 2-

NBDG the entire tissue fluoresces (Figure 2A). After the

non-bound 2-NBDG has diffused out of the ECM,

metabolized 2-NBDG is seen within the chondrocytes and

some appears to remain associated with the matrix (figure

2B). Quantitation of the metabolized and matrix

associated 2-NBDG has not yet been conducted and is not

part of the current model system. However, this appears

to be an exceptionally interesting aspect and future assays

will be conducted to assess this component as well.

Figure 2. Confocal images of a cartilage section soaked in 2-

NBDG (A) and approximately 1 minute after it had been rinsed

(B). The lighter regions represent areas where the fluorescent

2-NBDG is located.

This model system was used to assess 2-NBDG

diffusion in cartilage obtained from hindlimb unloaded

(HLU) rats. Rat HLU has been used extensively to model

some of the aspects of microgravity such as bone and

muscle loss and headward fluid shift. The tails of HLU

rats were anchored above the cage so that the hindlimbs

were elevated off the cage floor. The tails of the control

rats were also anchored but all four feet remained on the

0 30 60 90 120 150 180 2100

1

2

3

4

5

6

Soak time in 2-NBDG (minutes)

2-N

BD

G (

µµ µµM

)

Figure 1. Amount of 2-

NBDG that diffused into

cartilage sections after

different incubation

times. N≥7, error bars

represent standard error.

A B

C. Marshall et al. – Nutrient Diffusion through Cartilage


cage floor, tethering (TH) them within the cage. The tails

of normally caged rats (NC) were not anchored, and their

movement was unrestricted. Articular cartilage was

collected from NC rats and rats that had been HLU and

TH for 7 or 21 days. Rat tibias were cleaned of soft tissue

and the cartilage from the tibial plateaus were cut in

40µm sections. After incubating in 2-NBDG for 1-hour,

the amount that diffused from the sections was collected

and quantitated as described above (figure 3).

Figure 3. Amount of 2-NBDG that diffused from cartilage

sections obtained from hindlimb unloaded, tethered and

normally caged rats. The amount of 2-NBDG that diffused from

cartilage sections collected from each rat were averaged. The

mean was based on the number of rats within an experiment.

(A) The total amount of 2-NBDG that diffused from the sections.

(B) The amount of 2-NBDG diffusion normalized to wet weight

of the tissue section. N=4 for normally caged rats, N=3 rats for

each group in the 7 day experiment, N=8 rats for each group in

the 21 day experiment. The 21 day data is a combination of

data from 2 experiments. Error bars represent standard error.

There was no significant difference in 2-NBDG

diffusion between cartilage obtained from HLU and TH

rats at 7 days. Cartilage from both 7-day groups showed

less 2-NBDG diffusion than the NC and 21-day

experiment rat cartilage, but was not significant. This

decrease may be due to the initial stress associated with

the experiment, but cannot be determined until additional

parameters are evaluated. The percent of the sections

composed of fluid was the same for the 7-day and NC

groups (figure 4), indicating that the difference in

diffusion was not due to a difference in hydration levels.

In the 21-day experiments the amount of 2-NBDG

diffusion was 42% less in the cartilage sections obtained

from HLU rats than from the TH rats, although this was

not statistically significant (figure 3A). Multiple

experiments were conducted and each showed the same

trend. Combined data from two of the experiments are

shown in figure 3. A significant difference is seen in the

percent of the cartilage sections composed of water in the

HLU and TH rats, indicating that the hydration level of

the cartilage decreased during extended HLU. The

decrease in fluid content may explain the reason for the

reduced 2-NBDG diffusion in the cartilage from the HLU

rats. The amount of 2-NBDG normalized to wet weight

(figure 3B) or dry weight (data not shown), shows the

same pattern as the total 2-NBDG, in all groups.

The headward fluid shift during HLU reduces the fluid

level in the tibial plateau cartilage by 21 days. Diffusion

of 2-NBDG appears to be dependent on the hydration of

the cartilage tissue and may effect the availability of

nutrients within the ECM. This could significantly

impact the structure and function of articular cartilage.

Future studies will determine if the reduction in nutrient

availability has an impact on the health of the

chondrocytes. Expanding the scope of the model system

to include evaluation of metabolized 2-NBDG and 2-

NBDG associated with the ECM will also enable a more

comprehensive evaluation of diffusion in arthritic, and

aged cartilage as well as that from animals subjected to

microgravity, hypergravity and their respective model

systems.

REFERENCES Bayliss, M., Howat, S., Davidson, C., Dudhia, J., 2000.

The organization of aggrecan in human articular cartilage.

Evidence for age-related changes in the rate of

aggregation of newly synthesized molecules. J Biol Chem

275:6321-7.

Kuettner, K.E. 1992. Biochemistry of articular cartilage in

health and disease. Review, Clinical Biochemistry.

25(3):155-63.

Paulsen, D.F. 2000. Histology & Cell Biology:

Examination and Board Review, 4th ed., Lange/McGraw-

Hill, New York, pp.376.

Pelletier, J.P., Jovanovic, D.V., Lascau-Coman, V.,

Fernandes, J.C., Manning, P.T., Connor .JR., Currie

M.G., and Martel-Pelletier, J. 2000. Selective inhibition

of inducible nitric oxide synthase reduces progression of

experimental osteoarthritis in vivo: possible link with the

reduction in chondrocyte apoptosis and caspase 3 level.

Arthritis & Rheumatism. 43(6):1290-9.

ACKNOWLEDGEMENTS We thank Drs. Bayorh and Eatman and their laboratory

assistants for providing the tissue from the unloaded and

control rats. This work was supported by NASA grants

NAG3-2611 and NCC9-112 as well as RR-03034.

0

25

50

75

0 7 21

Unloaded TetheredNormal

*

Hindlimb Unloading (days)

wate

r as %

of

tota

l w

eig

ht Figure 4. Water content

of the cartilage sections

used in figure 3 (wet

weight – dry weight). *

significantly different from

tethered (p=0.0307).

Error bars represent

standard error.

0

1

2

3

4

5

6

7

8

Unloaded

Tethered

NormalA

2-N

BD

G (

µµ µµM

)

0

2

4

6

8

10

12

0 7 21

B

Hindlimb Unloading (days)

2-N

BD

G/w

et

weig

ht

( µµ µµM

/mg

)


HYPERGRAVITY INDUCES DAMAGE TO ROD PHOTORECEPTORS* A.J. Barnstable

1, A.R. Tink

1,2, S. Viviano

1, L. Baer

3, C. Wade

3, C.J. Barnstable

1 and J. Tombran-Tink

1,2.

1Dept. Ophthalmology, Yale University School of Medicine, New Haven, CT,

2Pharmaceutical Sciences, UMKC,

Kansas City, MO and 3Ames Research Center, Moffett Field, CA.

The retina is an outgrowth of the embryonic forebrain

neuroepithelium and provides an excellent model of brain

development and function. The neurons of the retina,

particularly those in the photoreceptor layer, are very

sensitive to environmental perturbations, and is thus a

good sensor for adverse conditions.

The adult retina has a relatively simple laminar

structure containing six neuronal and two glial cell

classes. Light is detected by rod (dim light) or cone

(bright light) photoreceptor neurons in the outer layer of

the retina. The light sensing portion of these cells contain

opsin visual pigment proteins that initiate the visual

transduction cascade that amplifies photon signals into

changes in membrane potential and neurotransmitter

release. The photoreceptor outer segments depend on

intimate contact with both glial cells and the adjacent

retinal pigment epithelial cells for both metabolic support

and regeneration of molecules needed for the visual

transduction cascade.

Retinal rod photoreceptor degeneration can result from

a variety of environmental and genetic factors. Retinitis

Pigmentosa (commonly known as night-blindness) is an

inherited condition in which rod photoreceptors

degenerate. As a secondary effect, cone photoreceptors

are then lost. At least 40 genes have been identified as

causing RP, but no effective therapy has yet been

approved. A number of animal models of RP exist but

there is a strong need for other models that can be

produced without complex genetic or surgical

intervention.

We have shown that the environment encountered in

space shuttle flight can disrupt normal retinal

development and mimic stimuli that induce similar retinal

degenerations (Tombran-Tink and Barnstable, this

volume). We have now examined eyes from adult

animals maintained in a hypergravity environment and

find that these show signs of rod degeneration as well.

Groups of pregnant rats were maintained at 2.0 xg for

approximately 14 days by centrifugation with a normal

lighting schedule. Age- and weight-matched stationary

controls were housed in the centrifuge rotunda, which

allowed them to be exposed to the same environmental

conditions as the hypergravity animals. Eyes from the

adult rats were fixed in formalin, cryoprotected, frozen

and cryosectioned. Sections were labeled with a variety

of cell type-specific antibodies including those

recognizing rod photoreceptor cell bodies, inner segments

and outer segments.

Eyes from rats maintained at 2.0 xg showed loss of rod

photoreceptors with varying degrees of severity. This is

most clearly noted in Figure 1 that shows substantial loss

of rod photoreceptor bodies in the outer nuclear layer. In

* presented at 2004 meeting

the most extreme cases the cell body layer was reduced to

approximately 20% of its normal thickness.

The outer segments were shorter and much more

disorganized although inner segments showed less

change. Stationary control retinas showed normal

photoreceptor morphology, suggesting that the damage

seen was due to the increased g-force.

We measured the thickness of various layers from

cryostat sections of a series of experimental and control

animals. Attempts were made to measure the same areas

in each animal but some variation remained.

Nevertheless, comparisons of relative layer thicknesses

are not affected by this variation. Figure 2 shows the

decrease in ONL thickness in a series of hypergravity

treated and control animals. In all the hypergravity

treated animals the outer nuclear layer is thinner than the

inner nuclear layer, a situation never seen in normal adult

rats. Other cell layers across the retina showed essentially

no change.

The selective loss of rod photoreceptors is similar to

that seen in a number of mutant strains of mice that serve

as models for the human disease of retinitis pigmentosa

(Figure 3). Rd1 mice show an early and rapid loss of rod

photoreceptors such that by 5-6 weeks of age they have

lost essentially all of these cells.

How does hypergravity cause rod photoreceptor

degeneration? One obvious factor to be considered is that

the increase in g-force causes a damaging increase in

Figure 1. After exposure to a 2X g environment for

2 weeks the retinas showed severe damage in the

outer retina. The outer nuclear layer normally

contains about 10 rows of cell bodies (SC215).

After hypergravity exposure this is reduced to

approximately 3 rows of cell bodies (HG203).

ONL, Outer Nuclear Layer; OPL, Outer Plexiform

Layer; INL, Inner Nuclear Layer; IPL, Inner

Plexiform Layer; GCL, Ganglion Cell Layer.

A. Barnstable et al. – Hypergravity damages rod photoreceptors


Figure 2. Summary data for a series of hypergravity

treated (upper histogram) and control (lower histogram)

eyes. Sets of data are for individual animals. All

measurements of layer thicknesses are in microns. Layer

naming is as given in the legend to figure 1.

intraocular pressure. This is unlikely to be the whole

explanation, however, because we observed no loss of

retinal ganglion cells. Increased intraocular pressure is

frequently associated with ganglion cell death in

glaucoma, a disease with little if any effect on rod

photoreceptors. It remains possible, however, that

increased pressure in some way affects the choroidal

blood supply that provides oxygen and nutrients to the rod

photoreceptors but not the retinal blood supply that

provides oxygen and nutrients to the ganglion cells. At

present we have no information about similar

degenerative changes that might occur in other brain

regions.

Further studies are needed to understand the time

course and mechanism of the rod photoreceptor cell death

we have observed. The non-invasive treatment of these

animals provides a valuable model with which to test

novel therapeutic strategies to combat blinding diseases.

One advantage over existing genetic models is that the

degeneration may be induced at different ages, something

that might better mimic a number of human blinding

diseases. Specifically, this model will be valuable for

testing the role of novel neuroprotective factors that can

block rod photoreceptor cell death.

Although short term increases in g-force are

experienced in both jet flight and space flight launches,

there are few studies of the effects of long term increased

g-force on the nervous system in general and the visual

system in particular. Knowledge of how increased g-

force affects cell metabolism and survival in the visual

system, and elsewhere in the nervous system, is important

as we think about exploration of different space

environments.

We thank Louis Ostrach (NASA, Washington, DC) for

advice and help with procuring tissue samples. Supported

by NIH, RPB Inc., and the Connecticut Lions.

wt rd

1

Figure 3. Sections of postnatal day 35 wild-type

(wt) and mutant (rd1) retinas showing the loss of

rod photoreceptors in the mutant. Rd1 mice carry a

mutations in the beta subunit of the rod

photoreceptor-specific cGMP phosphodiesterase.


A GLOBAL TRANSCRIPTIONAL ANALYSIS OF STREPTOCOCCUS PNEUMONIAE IN RESPONSE

TO LOW-SHEAR MODELED MICROGRAVITY

Christopher Allen1, Cristi Galindo

1, Nina Williams

1, Utpal Pandya

1, Ashok Chopra

1, and David Niesel

1

1

University of Texas Medical Branch, Galveston, TX 77555 USA

Long-term habitation in space exposes humans to a

variety of environmental stresses not encountered on earth such as ionizing radiation and microgravity. Decreases in

immune function and increases in microbial growth and

antibiotic resistance during space flight could create an

increased health risk to crewmembers (Sonnenfeld et al.,

1990; Mishra et al., 1992). Streptococcus pneumoniae is

an upper respiratory pathogen which causes pneumonia,

meningitis, and bacteremia. S. pneumoniae has

previously been isolated from crewmembers and could

present a potential health hazard during extended

spaceflight (Cioletti et al., 1991). To facilitate ground-

based microgravity analogue studies, NASA developed

high aspect ratio vessels (HARVs) which model low-

shear microgravity conditions (LSMMG). The LSMMG

environment is generated through the continual rotation of

cell cultures in fluid-filled vessels which minimize

prolonged exposure to gravity through the randomization

of gravity vectors. Recently, these ground-based models

have been used to study the effects of LSMMG on

bacteria to understand the physiological and molecular

changes they undergo in the presence of microgravity as

encountered during spaceflight.

This study utilized S. pneumoniae as a Gram-positive

bacterial model to investigate how LSMMG affects

environmental stress resistance and global transcriptional

activity in opportunistic bacterial pathogens. DNA

microarray analysis was carried out on S. pneumoniae

strain TIGR4 after cultivation in HARVs under LSMMG

and compared to 1 x g and static conditions (Figure 1).

Figure 1. HARV Vessel Orientations for Growth.

Microarrays were acquired from The Institute for

Genomic Research (TIGR) Pathogen Functional

Genomics Resource Center (PFGRC). They were

designed based on the genomic sequence of the TIGR4

strain and contain amplicons representing 2131 open-

reading frames.

Stress analysis was carried out on TIGR4 cultures

grown to mid-logarithmic phase under LSMMG and

controls (1 x g rotating) followed by exposure to thermal

(55°C) and acid (pH 3.5) stress conditions (Figure 2).

Significant decreases (p ≤ 0.05) in both thermal and acid

stress resistance were found in LSMMG-grown cultures

after exposure for 30 minutes using a Student’s t test.

Similar results were found after exposure when stress

conditions were increased up to 1 hour (data not shown).

These results were in contrast to previous reports which

have shown increases in environmental stress resistance

by Salmonella enterica serovar Typhimurium and

Escherichia coli (aWilson et al., 2002; Lynch et al., 2004)

in response to LSMMG. These results may be indicative

of the different responses by Gram-positive and Gram-

negative bacteria to LSMMG. Alternatively, the results

may reflect the different phenotypes exhibited by enteric

and respiratory pathogens which colonize and persist in

distinct niches within the host.

Thermal Stress Resistance

Acid Stress Resistance

Figure 2. Thermal and Acid Stress Resistance in Response to

LSMMG.

In order to understand what genes may be involved in

bacterial LSMMG-mediated responses at the

transcriptional level, global transcriptional analysis was

carried out using mid-logarithmic phase cultures grown

under LSMMG and control cultures (1 x g rotating, 1 x g

static). A stringent analysis of the array data was carried

out using multiple analysis methods (Genepix Pro 6.0,

Spotfire 7.3, SAM, and ANOVA) and a threshold of ≥

1.5-fold was used to identify differentially expressed

genes. Results were confirmed by quantitative real time

C. Allen et al. – A Global Transcriptional Analysis of Streptococcus pneumoniae in Response to Low-Shear

Modeled Microgravity


RT-PCR of six representative genes differentially

expressed under LSMMG. Genes were chosen which

were differentially expressed ≥ 1.5-fold in response to

LSMMG, represented different functional categories, and

located within different regions within the genome.

Pneumococcal genes (81 total) which were found to be

responsive to LSMMG represented different functional

groups located throughout the genome (Table 1). The

statistical analysis program, Significance Analysis of

Microarrays (SAM), confirmed the statistical significance

of these differentially expressed genes based on three

independent experiments.

Functional Category LSMMG-Responsive Genes

Antibiotic Resistance 1

Cell Envelope 11

DNA Repair/Recombination 4

Metabolism 13

Signaling 3

Stress Response 1

Transcriptional Regulation 4

Transporters/Ion Channels 8

Hypothetical/Unknown 36

Table 1. Functional Gene Groups Significantly Altered by

LSMMG.

Interestingly, LSMMG-responsive genes were found to be

predominantly down regulated in response to LSMMG.

Previous studies have shown that global transcriptional

profiles of Gram-negative enteric bacteria contain both

up- and down-regulated genes after growth under

LSMMG (bWilson et al., 2002). This consistant pattern of

down-regulation seen in S. pneumoniae represents a

distinct response by this Gram-positive respiratory

pathogen compared with the more extensively

characterized Gram-negative enteric pathogens.

Hierarchical clustering analysis (Cluster/Treeview,

CLUSFAVOR 6.0, and Spotfire 7.3) revealed several

gene clusters displaying similar expression patterns.

These clusters consisted of different genes from diverse

functional groups. Currently, how these genes function in

response to LSMMG remains unknown. Likewise, a

common mechanism to explain these regulatory changes

in response to LSMMG remains uncharacterized. Further

studies will need to be carried out to identify LSMMG-

responsive regulators.

Controls for the LSMMG experiments included both a

1 x g rotating control and a 1 x g static control (Figure 1)

as a secondary filter to identify LSMMG-responsive

genes. SAM analysis of genes differentially expressed

between static and rotating controls revealed 147

differentially expressed genes. Among these genes, 46

were also altered between 1 x g and LSMMG conditions

while 9 were altered between static and LSMMG

conditions. The results from this analysis clearly indicate

that the choice of control has a significant impact on the

assignment of LSMMG-responsive genes and possibly

physiological properties. Further, the presence of other

external forces such as shear and rotation exert individual

effects on cultured cells independent of LSMMG.

Overall, these results indicate that LSMMG represents

a unique environment that can alter a bacterium’s

transcriptional profile and physiological state.

Investigating how this unique environment can lead to

new properties will allow us to better understand the

impact of LSMMG on microbial virulence and the health

risks these changes may create during long-term space

habitation.

REFERENCES

Sonnenfeld, G., Mandel A.D., Konstantinova I.V., Taylor

G.R., Berry W.D., Wellhausen S.R., Lesnyak A.T., Fuchs

B.B. 1990. Effects of Space Flight on Levels and Activity

of Immune Cells. Aviat. Space Env. Med. 61: 648.

Mishra, S., Pierson, D. 1992. Space Flight, Effects on

Microorgansims. In: Encyclopedia of Microbiology.Vol.

4. Academic Press, Inc. pp. 53-60.

Cioletti, L., Pierson, D., Mishra, S. 1991. Microbial

Growth and Physiology in Space: A Review. SAE

Technical Paper Series No.911512.

aWilson, J., Ott, C.M., Ramamurthy, R., Porwollik, S.,

McClelland, M., Pierson, D.L., Nickerson, C.A. 2002.

Low-Shear Modeled Microgravity Alters the Salmonella

enterica Serovar Typhimurium Stress Response in an

RpoS-Independent Manner. Appl Environ Microbiol

68(11): 5408-5416.

Lynch, S.V., Brodie, E.L., Matin, A. 2004. Role and

Regulation of sigma S in General Resistance Conferred

by Low-Shear Simulated Microgravity in Escherichia

coli. J Bacteriol 186(24): 8207-8212.

bWilson, J.W., Ramamurthy, R., Porwollik, S.,

McClelland, M., Hammond, T., Allen, P., Ott, C.M.,

Pierson, D.L., Nickerson, C.A. 2002. Microarray Analysis

Identifies Salmonella Genes Belonging to the Low-Shear

Modeled Microgravity Regulon. Proc Natl Acad Sci USA

99(21): 13807-13812.


PROTEOMIC RETRIEVAL FROM NUCLEIC ACID DEPLETED SPACE-FLOWN HUMAN

CELLS

D. K. Hammond1, T.F. Elliott

2, K. Holubec

2, T.L. Baker

2, P.L. Allen

3, T.G. Hammond

3 and J.E. Love

4

1Enterprise Advisory Services, Inc., Houston, TX; 2 Wyle Life Sciences, Houston, TX; 3V.A. Medical

Center and Tulane University Health Sciences Center, New Orleans; 4Human Adaptation and

Countermeasures Office, NASA, Johnson Space Center, Houston, TX.

Cells grown in space or in any hostile environment

with a necessity for preservation are subject to

decomposition and loss of valuable scientific

information. To maximize the science return from

flight samples, an optimized method was developed

to recover protein from samples that had been stored

for long periods of time at refrigerated temperatures.

This technique also allows multiple analyses on a

single cellular sample when frozen. Our group

specifically designed this for use with samples from

the International Space Station (ISS) (Love, et al.,

2003) at the same time that a different technique was

established by others (Rodrigo, et al., 2002). This

work extends our initial work, using additional

antibodies and cell lines, as well as corroborating

with histological staining.

Cell cultures were grown in American

Fluoroseal bags (TCM) either on the ISS or in flight

hardware on the ground, and treated at regular

intervals with an RNA stabilizing agent (RNAlater®,

Ambion, Austin, TX) or a formalin solution. The

samples were refrigerated for 3 months. RNA was

purified using an RNAqueous® kit (Ambion) and the

remaining RNA free supernatant was precipitated

with 5% trichloroacetic acid. The precipitate was

dissolved in SDS running buffer and tested for

protein content using a bicinchoninic acid assay

(Sigma, Dallas). Equal loads of protein were placed

on SDS-PAGE gels and transferred using Western

Blotting techniques (Towbin, et al., 1979). Protein

on the blots from preserved cells were analyzed using

horseradish peroxidase antibodies, with an ECL+

fluorescent stain (Amersham, Piscataway, NJ). The

Storm imager and Imagequant software (Amersham)

quantified the pixel volumes for rectangles of equal

size. Human renal cortical epithelial (HRCE) cells

(Cubano, et al., 2004; Cowger,et al., 2002) grown

onboard the ISS during Increment 3 and in ground

control cultures exhibited similar immunoreactivity

profiles for antibodies to the Vitamin D receptor

(VDR) (Fig 1), the beta isoform of protein kinase C

(PKCß) (Fig 2), and glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) (Fig 3). The ground (Grd)

and flight (Flt) samples are presented on the graphs

using an untreated control to normalize the data. A

Vitamin D Receptor

0

20

40

60

80

100

120

140

Grd 3 Grd 6 Grd 9 Flt 3 Flt 6 Flt 9 Control

Perc

ent of C

ontr

ol

Figure 1 - Vitamin D Receptor (VDR) antibody stained

blots, prepared with equal loading of protein (5 µg/lane),

measured a protein with a molecular weight of 60-64kD.

Samples are from ISS Flt samples from days 3, 6 and 9 and

the Grd matched controls from the same days. An example

of one of the blots is shown below the graph, which is an

average of four blots.

PKC-beta

0

20

40

60

80

100

120

140


Perc

ent

of

Contr

ol

Figure 2 - Protein Kinase CβII (PKCβII) antibody stained

blot, prepared with 6 µg/lane of protein, measured a

protein with a molecular weight of 80 kD. The graph is an

average of four blots with an example below the graph.

D. Hammond et al. – Antigenic Protein in LN1 Cells Grown in Space


GAPDH

0

20

40

60

80

100

120


Perc

ent

of

Contr

ol

Figure 3 - Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) antibody stained blot, prepared with 6 µg/lane of

protein per lane, measured a protein with a molecular

weight of 42-44 kD. The graph is the average of three blots

with an example below the graph.

Student's t-test was used to compare the ground and

flight samples at each time point, days 3, 6 and 9.

There was no significant difference between Flt and

Grd samples at any time point (p > 0.05).

Parallel immunohistochemical studies on

formalin-fixed flight and ground cultures also

showed positive immunostaining for VDR (Fig 4) as

well as other biomarkers (not shown here). These

results are consistent with data from antigenic

recovery experiments performed on human mixed

Müllerian tumor cells cultured in microgravity

(Hammond, et al., 2005) as well as data from cells

grown using different culture methods (Cubano,et al.,

2004). Although on a protein per protein basis, the

preserved cells demonstrated slightly less antigenic

protein than untreated cells (control), there was a

high percentage of recovery of antigenic protein

using three different antibodies in both the ground

and flight samples.

These studies demonstrate that quantitative

proteomic information can be acquired from samples

stored in less than optimum circumstances, such as

long term storage at refrigerated temperatures with

different cell lines, using multiple antibodies. Since

space flight experiments are often executed under

resource constraints, this is a valuable addition to our

knowledge of how to best utilize our space samples

and to other work done in extreme environments such

as in undeveloped countries where biospecimens are

kept in suboptimal conditions.

(Supported by NAS9-02078 and NRA NAG8-1362).

Figure 4 - Light photomicrograph (600x) of Vitamin D

receptor immunoreactivity in sectioned formalin fixed

HRCE cells cultured on Cytodex-3 microcarrier beads in

microgravity for 12 days during ISS Increment 4.

Cubano, L., Allen, P.L., Stodieck, L., Genova, J.

Klassen, R.B.B, Love, J., Hammond, T.G. 2004.

Transcription factor translocation during changes in

renal cell mechanical culture. Gravitational and

Space Biology Bulletin 17(2): 75-82

Cowger, N.L., Benes, E., Allen, P., Hammond T.G.

2002. Expression of renal cell protein markers is

dependent on initial mechanical culture conditions. J

Appl Physiol 92:691

Hammond, D.K., Becker, J., Elliott, T.F., Holubeck,

K, Baker, T.L., Love, J.E. 2005. Antigenic protein in

microgravity-grown human mixed Müllerian ovarian

tumor (LN1) cells preserved in RNA stabilizing agent

Gravitational and Space Biology 18(2): 99-100

Love, J.E., Hammond, D.K., Elliott, T.F., Holubec,

K., Baker, T.L., Allen, P.L. and Hammond, T.G.

2003. Antigenic protein from RNALater™ treated

cell cultures grown on the international space station

(ISS), using the cellular biotechnology operations

support system (CBOSS). Gravitational and Space

Biology Bulletin. 17:39

Rodrigo, M.C., Martin D.S.K., Redtzke, R.A., Eyster,

K.M. 2002. A method for the extraction of high-

quality RNA and protein from single small samples

of arteries and veins preserved in RNAlater. J.

Pharm. Tox. Methods. 47:87.

Towbin, H., Staehelin, T., Gordon, J. 1979.

Electrophoretic transfer of proteins from

polyacrylamide gels to nitrocellulose sheets:

procedure and some applications. Proc. Natl. Acad.

Sci. USA. 76:4350.


THE EFFECT OF CHAGES OF GRAVITY ON HUMAN MONOCYTE CELL (TUR) PHAGOCYTOSIS

Cassidy B. Johnson1*

, Lillian S. Waldbeser2

1Dept. of Biochemistry and Cell Biology, PO Box 1892, Rice University, Houston, Texas 77251

2

Dept. of Physical and Life Sciences, Texas A&M University-Corpus Christi, Corpus Christi, TX 78412 USA

The function of the immune system is compromised

during spaceflight (Cogoli, 1996). Many cellular activities

crucial for an effective immune response are diminished

in microgravity conditions. Phagocytosis is a vital,

cytoskeleton-mediated immune process necessary for the

clearance of pathogenic microorganisms; therefore

alterations to this important process may leave astronauts

vulnerable to infection. We sought to determine if

modeled microgravity or hypergravity conditions alter the

phagocytic capacity of a human monocyte-like phagocytic

immune cell (TUR; ATCC, Manassas, V.A.).

Microgravity and hypergravity conditions were

modeled using ground-based systems. A rotary cell-

culture system (RCCS-4D, Synthecon, Houston, TX)

was used to was used to simulate the free-fall conditions

that occur in microgravity, and a novel device called the

Hypergravity 3000 (HG3K), was designed and built for

the analysis of hypergravity. This machine was designed

to fit inside a tissue culture incubator and to concurrently

simulate 4,6, and 8 g.

The TUR cells were cultured under standard tissue

culture conditions until they reached confluence. Cells

were then seeded into the RCCS-4D or the HG3K at a

concentration of 1 x 106. The cells were cultured under

microgravity or hypergravity conditions for 10 days, and

100 µL aliquots of cells were removed from the machines

at 1, 4, 12, 24, 48, 120, and 240 hours, counted and

assayed for phagocytosis. For the phagocytosis assay,

fluorescein-conjugated Escherichia coli bacteria

(BioParticles, Molecular Probes, Eugene, OR) were

added to the experimental cells at a 1:10 cell:particle

ratio. The cells were allowed to incubate with the

BioParticles for 45 minutes, the optimal time allotment

for phagocytosis that minimized re-exposure to 1 g

conditions. After this 45-minute period, the cells were

briefly spun down and washed at 4°C to terminate

phagocytosis and remove the majority of non-

phagocytosed particles. Ethidium bromide was

additionally added to the cell samples to distinguish dead

or apoptotic cells from non-phagocytic cells. An average

of 500 cells were counted and scored as phagocytic or

non-phagocytic at each time point for every level of g and

a linear regression analysis was used to compare the

probability of phagocytosis occurring at each level of g

over time.

* Correspondence to: Cassidy B. Johnson

Dept. of Biochemistry and Cell Biology

Houston, PO Box 1892, TX 77251


Phone: 713-348-2715

Overall, exposure to modeled microgravity increased

the incidence of phagocytosis in TUR cells over the 10-

day exposure period (Fig.1). The most dramatic increase

in phagocytic probability occurred within the first 48

hours of exposure where greater than 60% of the TUR

cells underwent phagocytosis compared to ~42%

observed in the 1 g control cells. This increase diminished

over the next 5 to 10 days, however phagocytosis never

returned completely to 1 g levels. Conversely, 10-day

hypergravity exposure decreased phagocytosis overall in

TUR cells (Fig. 1). Similar to the trend observed in

microgravity, the greatest decrease in phagocytosis

occurred within the first 48 hours, however a ‘peak’ of

phagocytic activity was observed at 24 hours. To further

examine this phenomenon, three additional 5-day

hypergravity experiments were performed to establish if

this peak was due to experimental conditions or sampling

error (Fig.2). Interestingly, after an initial decrease in

activity, phagocytosis increased during all three of these

experiments, the highest proportion of phagocytosis

occurring at 24 hours at all hypergravity levels. The peak

noted at 24 hours during the first 10-day experiment was

also replicated in all three of these concurrent

experiments, suggesting that this observation was not due

to sampling error.

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 40 80 120 160 200 240

Time (h)

0 g 1 g

4.1 g 6 g

8 g

Figure 1. Probability of phagocytosis occurring in TUR

cells during a 10-day exposure to modeled microgravity or

hypergravity conditions. One hour of exposure to modeled

microgravity (0 g) or hypergravity (4.1, 6, and 8 g)

increases phagocytosis compared to control cells (1 g). The

percentage of cells undergoing phagocytosis remains

elevated in microgravity but reduced in hypergravity over a

48-hour period, after which phagocytosis in both conditions

slowly returns to almost 1 g levels at the end of the 10-day

period.

C. Johnson and L. Waldbeser – Effect of Changes of Gravity on Human Monocyte (TUR) Phagocytosis


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60 80 100 120

Time (h)

0 g 1 g

4 g 6 g

8 g

The results of these experiments suggest that that

microgravity and hypergravity conditions modify

phagocytic function in human TUR cells. The

discrepancies between the first 10-day and the concurrent

5-day hypergravity experiments may be explained by the

age of the TUR cells. The cells utilized for the 10-day

experiment were cultured for less than a week at 1 g in

tissue culture flasks before use, while the cells utilized for

the 5-day experiments had been cultured for 10 to 18

days. The synchronously cultured 1 g control cells

exhibited the same phagocytic probability regardless of

their age, indicating that change in gravity coupled with

age may modify phagocytosis. The cells cultured at 8 g

exhibited the lowest phagocytic probability during the

first 10-day trial and the highest during the following 5-

day experiments, whereas the cells exposed to 4 g

remained very close to the “base-line” values of 1 g. This

data suggests that the hypergravitational force acts in a

“dose-dependant” manner. Changes in the levels of

protein kinase C (PKC) may explain these modifications

in phagocytosis, including the ‘peak’ phenomenon that

occurred at 24 hours in all three hypergravity conditions

and the less distinct peak at 24 hours observed in

microgravity. Several PKC isoforms, including PKC α,

PKC β PKC δ and PKC ε are involved in antibody-

receptor mediated phagocytosis (Allen and Aderem,

1996). The translocation and concentration of PKC is

modified in both microgravity and hypergravity

conditions (Schmitt et al., 1996). In microgravity,

translocation of PKC occurs rapidly over a 1-hour period,

then steadily declines over time whereas translocation of

PKC in hypergravity steadily increases over time and with

increased g-level (Hatton et al., 1999). Translocation of

PKC plays an important role in phagocytosis (Larsen et

al., 2000), therefore the additional movement of this

kinase to the cytosol may be causing the enhanced

phagocytic activity of TUR cells, a known PKC deficient

cell (Hass et al., 1993), cultured in altered gravity

conditions. Additional experiments utilizing other cell

lines along with assessment of PKC gene expression are

warranted to fully investigate this phenomenon.

The percent of TUR cells undergoing phagocytosis

started to return to 1 g levels after the 5th

day of

microgravity or hypergravity exposure. The delayed

hypersensitivity reaction of astronauts was additionally

found to normalize after the fifth day of microgravity

exposure (Taylor and Janney, 1992). These observations

suggest that immune cells may have the capacity to adapt

to gravitationally altered environments. This ability to

‘adapt’ may involve the cell’s cytoskeleton, including

actin which is the major cytoskeletal component involved

in phagocytosis. Disorganization and reorganization of

the cytoskeleton in microgravity has been observed in

several cell types, with much of the reorganization

occurring after 24 hours of exposure (Lewis et al., 1998).

This reorganization corresponds to the large phagocytic

peak observed at 24 hours, suggesting a connection

between these two events.

(Supported by: Texas Space Grant Consortium “New Principle

Investigator Grant 2004-2005 ” and Texas Excellence Fund Grant

2005.)

REFERENCES Cogoli, A. 1996. Gravitational physiology of human immune cells: A

review of in vivo, ex vivo, and in vitro studies. J. Grav. Physiology. 3:

1-9.

Allen, L.H., and Aderem, A. 1996. Molecular definition of distinct

cytoskeletal structures involved in compliment and Fc receptor-mediated

phagocytosis in macrophages. J. Exp. Med. 184: 627-637.

Schmitt, D.A., Hatton, J.P., Emond, C., Chaput, D., Paris, H., Levade,

T., Cazenave, J.P., and Schaffar, L. 1996. The distribution of protein

kinase C in human leukocytes is altered n microgravity. FASEB J. 10:

1627-1634.

Hatton, J.P., Gaubert, F., Lewis, M.L., Darsel, Y., Ohlmann, P.,

Cazenave, J.P., and Schmitt, D. 1999. The kinetics of translocation and

cellular quantity of protein kinase C in human leukocytes are modified

during spaceflight. FASEB J. 13: S23-S33.

Larsen, E.C., DiGennaro, J.A., Saito, N., Mehta, S., Loegering, D.J.,

Maxurkiewiez, J.E., and Lennartz, M.R. 2000. Differential requirements

for classic and novel PKC isoforms in respiratory burst and phagocytosis

in RAW 264.7 cells. J. Immuno. 165: 2809-2817.

Hass, R., Hirano, M., Kharbanda, S., Rubin, E., Meinhardt, G., and

Kufe, D. 1993. Resistance to phorbol-ester induced differentiation of a

U937 myeloid leukaemia cell variant with a signalling defect upstream

to Raf-1 kinase. Cell Growth &Diff. 4: 657-663.

Taylor, G.R., and Janney, R.P. 1992. In vivo testing confirms a blunting

of the human cell-mediated immune mechanism during space flight. J.

Leukocyte Biol. 51: 129-132.

Lewis, M.L., Reynolds, J.L., Cubano, L.A., Hatton, J.A., Lawless, B.D.,

and Piepmeier, E.H. 1998. Spaceflight alters microtubules and increases

apoptosis in human lymphocytes (Jurkat). FASEB J. 12: 1007-1018.

Figure 2. Average phagocytic probability for three successive 5-

day hypergravity experiment compared to modeled microgravity

(0 g) and controls (1 g). Hypergravity initially decreases

phagocytosis, however phagocytic activity increases at 12 hours

and the percent of phagocytic cells remains elevated over the 5-

day period compared to1 g controls. Note the peak of phagocytic

activity at 24 hours observed during the 10-day trial at 24 hours


MICROTUBULE DISRUPTIONS AND REPAIR PHENOMENA IN CULTURED GLIAL CELLS

UNDER MICROGRAVITY.

Maria Angela Masini1, Felice Strollo

2, Franco Ricci

3, Martina Pastorino

1, Bianca Maria Uva

1,

1Dipartimento di Biologia, Università di Genova, Italy,

2Unità Operativa Endocrinologia e Malattie del Ricambio

INRCA & Università di Roma La Sapienza Italy, 3ENEA C.R. Casaccia - Roma Italy.

The structure of animal cell cytoplasm is based upon a

scaffolding of proteins called cytoskeleton that is essential

for many processes ensuring mechanical support,

movement, adhesion, polarity and intracellular trafficking.

The cytoskeleton is composed by filamentous proteins

including microfilaments (actin), intermediate filaments

(differing from cell to cell type) and microtubules

(tubulins) all joined by cross-linking proteins.

Microtubules are responsible for mitotic spindle assembly

and chromosome movements as well as vesicular and

cytoplasmic organelles movements inside single cells.

Gravitational vector changes cause severe damages to the

cytoskeleton, as observed in lymphocytes during space

flight (Cogoli-Greuter et al., 1994; Lewis et al., 1998) and

in ground-based experiments (Uva et al., 2002a,b,c).

The aim of the present study was to assess whether

addition of minerals to the culture medium might repair or

prevent microtubules alterations in low g. In this

experiment C6 cells (from rat astroglioma) were submitted

to 3D RPM rotation (56 Deg/sec; 10-6

g) for 1h with

addition to the culture medium (D-MEM) of: 1) mineral

salt integrators (potassium and magnesium citrate,

10mg/ml), 2) calcium (5 µg/ml) and 3) magnesium (0.70

mg/ml). Control cells were cultured in D-MEM without

any added minerals whatsoever and kept rotating for the

same time. Static controls (1xg) were treated in parallel

and positioned on the supporting frame of the RPM in

order to have their cells facing the same vibration stress as

in modeled microgravity samples. At the end of the

rotation, the cultured cells were fixed (4%

paraformaldehyde in PBS) and submitted to

immunohistochemistry using an antibody to α-tubulin.

Apoptosis was visualized by immunostaining of an

apoptosis-related protein (caspase 7 executioner) and

analysis of DNA fragmentation which was visualized by

TUNEL method (Terminal dUTP Nick End Labeling).

Nuclei were stained with 4,6-diammino-2-phenylindole-

dyhydro chloride (DAPI). The cells were observed by

conventional epifluorescence microscope (Olympus).

Percent cells with well organized microtubular array, cells

in mitosis and caspase positive cells, cultured in control

and modified media, were counted from 3 randomly

chosen fields in 5 slide preparations per sample.

Statistical analyses made use of ANOVA with a

confidence of 95%. Data were expressed as means ± SD.

In 1xg control cells the microtubular array was well

organized (Fig. 1a), microtubules radiated from the

organising centre to reach the plasma membrane. After 1h

at RPM rotation in the cells cultured in D-MEM, without

mineral salts or metal addition, microtubules appeared to

be highly disorganized and shortened (Fig. 1b).

Conversely, in cells cultured in D-MEM enriched with

mineral integrators (Fig. 1c), calcium (Figs. 2a, b) or

magnesium (Figs. 2c, d), the microtubular array remained

well organized (Fig. 3).

Figure 1 A, B, C. α-tubulin immunohistochemistry. a)

static 1xg control: microtubules are well organized, b)

after 1h in modeled microgravity: microtubules appear to

be highly disorganized. c) after 1h at modeled

microgravity in the cells cultured in D-MEM enriched

with minerals integrators, the microtubules appear to be

well organized. 1800x.

Figure 2 A, B, C, D. α-tubulin immunohistochemistry. a,

b: D-MEM with addition of calcium: a) static 1xg control,

b) modeled microgravity. c, d: D-MEM with addition of

magnesium: c) static 1xg control, d) 1h modeled

microgravity. The microtubules remain well organized in

both conditions. 800x.

B D

M.A. Masini, et al. - Microtubule Disruptions and Repair Phenomena in Cultured Glial Cells under Microgravity


Figure 3. Number of cells with well organized

microtubules. Means ± SD.

The number of apoptotic cells was very high in the

cultures containing only D-MEM, but got back to control

levels (11±4.2% versus 70.62±11.06%) after magnesium

addition, while the number of cell division increased both

with calcium and magnesium (Fig.4A, B).

Figure 4 A, B. Number of cell divisions (A), and number

of apoptic cells (B) after 1 h at low g in controls and in D-

MEM modified by addition of calcium and magnesium.

Means ± SD

Our data confirm previously reported observations that

severe microtubular changes occur in low g and there is a

lack of microtubular self organization under modeled or

real microgravity conditions (Papaseit et al.; 2000;

Tabony, 1994). However divalent ions, as calcium and

magnesium, are known to play a major role in the

regulation of microtubular polymerization in vivo under

earth gravitational force. In fact a high magnesium

concentration is needed to assemble tubulin dimers into

microtubules in vitro (Gaskin, 1981).

The present results show that the addition of calcium or

magnesium to the culture medium results in protection of

microtubular integrity and chromatin, preventing

apoptosis and enhancing cell divisions, probably due to

the pivotal role of divalent ions in building the mitotic

spindle and the cleavage furrow.

In conclusion these data show that an enriched metal

concencentration in the extracellular compartments may

prevent low g alterations at the cellular level.

REFERENCES

Cogoli-Greuter, M., Pippia, P., Siola, I., and Cogoli, A.

1994. Lymphocytes on sounding rocket flights. J. Gravit.

Physiol. 1: 90-91.

Gaskin, F. 1981. In vitro microtubule assembly regulation

by divalent cations and nucleotides. Biochemistry 20:

1318-1322.

Lewis, M., Reynolds, J.L., Cubano, I.A., Hatton, J.P.,

Lowless, B.D., and Piepmeier, E.H. 1998. Spaceflight

alter microtubules and increases apoptosis in human

lymphocytes (Jurkat). Faseb J. 1007-1018.

Papaseit, C., Pochon, N., and Tabony J. 1998.

Microtubule self-organization is gravity-dependent. Proc.

Natl. Acad. Sci. USA 97: 83634-8368.

Tabony, J. 1994. Morphological bifurcations involving

reaction-diffusion processes during microtubule

formation. Science 264: 245-248.

Uva, B.M., Masini M.A., Sturla, M., Tagliaferro, G., and

Strollo, F. 2002a Microgravity-induced programmed cell

death in astrocites. Proceedings of “Life in Space for Life

on Earth” 23rd

Annual International Gravitational

Physiology Meeting, Stockholm, Sweden.

Uva, B.M., Masini, M.A., Sturla, M., Prato, P.,

Passalacqua, M., Giuliani, M., Tagliaferro, G., and

Strollo, F. 2002b. Clinorotation - induced weightlessness

influences the cytoskeleton of glial cells in culture. Brain

Res. 934: 132-139.

Uva, B.M., Masini M.A., Sturla, M., Buzzone, F.,

Giuliani, M., Tagliaferro, G., and Strollo, F. 2002c.

Microgravity-induced apoptosis in cultured glial cells.

Eur. J. Histochem. 46:209-214.

Control

70.62±1.06

+Ca2+

15±3.6 +Mg2+

11±4.2

A

B


MICROGRAVITY-INDUCED CHANGES IN GENE EXPRESSION IN ACTIVATED T-LYMPHOCYTES INVOLVE MULTIPLE REGULATORY PATHWAYS.

Nancy E. Ward1, Neal R. Pellis

2, Semyon A. Risin

3 and Diana Risin

2

1Wyle Life Sciences, Houston, TX,

2NASA, Johnson Space Center, Houston, TX and

3University of Texas-Houston

Medical School.

We and others have shown that exposure to microgravity

(MG) results in a decline in cellular immune function. To

further investigate this phenomenon, microarray analysis

was employed to identify associated gene expression

changes and possible gravity sensitive genes. CD3- and

IL2-activated human T-cells were seeded at a density of

1 x 106cells/ml and maintained either in static (1g) or

modeled MG conditions for 24 hours at 37°C in an

atmosphere of 95% air and 5% CO2. To model MG

conditions we used the Rotating wall vessel (RWV)

culture system developed at NASA-Johnson Space Center

and commercially available from Synthecon, Inc. This

very low shear culture system randomizes gravitational

vectors and approximates the microgravity environment

by sustaining cells in continuous free fall. RWV

bioreactors were continuously rotated at a speed of

22 rpm.

To decrease biological variation and aid in detecting

microgravity-associated changes, experiments were

performed in triplicate utilizing T-cells obtained from

different blood donors. Total RNA was isolated using the

RNeasy isolation kit (Qiagen, Valencia, CA). RNA was

then analyzed for quality, using the Agilent 2100

Bioanalyzer, and the concentration was determined using

the NanoDrop® ND-1000 Spectrophotometer. Microarray

analysis was performed using the Affymetrix GeneChip®

Human U133A array as described in the Affymetrix

Analysis Technical Manual. The Human U133A array

contains >22,000 probe sets corresponding to 18,400

genes. A cutoff was set at >1.5 fold for analysis and

identification of genes of interest with the criterion for

significance <0.01.

Exposure to modeled microgravity resulted in

alteration of 89 genes, 10 of which were up-regulated and

79 down-regulated. Genes with altered expression were

categorized according to their function and their structural

role as well as association with certain metabolic and

regulatory pathways. A large proportion of them were

found to be involved in fundamental cellular processes:

signal transduction, DNA repair, apoptosis, and multiple

metabolic pathways (Table 1). A group of genes directly

related to immune and inflammatory responses was also

identified.

Up-regulated genes included IL7-receptor which plays

a role in lymphocyte development, and several heat shock

protein family members which function together as

molecular chaperones, stabilize existing proteins from

aggregation and mediate folding of newly translated

proteins.

Down-regulated genes included granzyme B, beta-3-

endonexin, and Apo-2 ligand, all three of which have

various roles in the induction of apoptosis. Among

down-regulated genes directly related to immune and

inflammatory responses we identified granulysin,

proteasome activator subunit 2, peroxiredoxin 4 gene,

HLA-DRA, lymphocyte antigen 75, IL18 receptor gene

and DOCK2 gene. Granulysin is a cytolytic molecule

demonstrating broad antimicrobial activity (Clayberger

and Krensky, 2003). Proteasome activator subunit 2

encodes the beta subunit of the 11S regulator of the

immunoproteasome which is important in the processing

of class I MHC peptides and induced by gamma

interferon (Zhang et al., 1998; Sijts et al., 2002; Kloetzel,

2004). The peroxiredoxin 4 gene encodes a cytosolic

antioxidant enzyme shown to play a regulatory role in the

activation of NF-KB (Kang et al., 1998; Fujii and Ikeda,

2002). HLA-DRA functions in presenting peptides

derived from extracellular proteins (Germain, 1994).

Lymphocyte antigen 75 is involved in the endocytosis of

antigen by dendritic cells (Rajagopal et al., 2004). The

protein encoded by the IL18 receptor gene specifically

binds IL18 and is essential for IL18 mediated signal

transduction (Wu et al., 2003; Subramaniam et al., 2004).

DOCK2 gene product is a protein indispensable for

lymphocyte chemotaxis (Reif and Cyster, 2002).

Also down-regulated were three proteasome beta

family members (10, 9 and 8) making up part of the 20S

proteasome core structure; several genes potentially

affecting the integrity of the cellular cytoskeleton, such as

CD2 associated protein, tubulin, and pericentrin 1; genes

affecting DNA repair, such as nudix and FEN1, and genes

affecting several metabolic pathways.

These data provide base information and a means to

evaluate the possible roles of genes demonstrating altered

expression in lymphocyte response to microgravity at the

molecular level.

Table 1. Functional clustering of genes responding to modeled

microgravity.

Number of genes

down-

regulated

up-

regulated

Tissue /growth regulation 1 1

Protein folding / degradation 3 3

Nucleotide / purine-pyrimidine 19 1

Metabolic pathways / metabolism 5 4

Structure / transport / binding

protein

11

Immune / inflammatory / defense 10 1

Apoptosis 3

Transcription factor 3

Signal transduction 17

Unknown function 7

N.E. Ward et al. – MICROGRAVITY-INDUCED CHANGES


REFERENCES

Clayberger, C. and Krensky, A.M. 2003. Granulysin.

Current Opinion in Immunology 15: 560-565.

Fujii, J. and Ikeda, Y. 2002. Advances in our

understanding of peroxiredoxin, a multifunctional,

mammalian redox protein. Redox Rep. 7(3): 123-30.

Germain, R.N. 1994. MHC-dependent antigen processing

and peptide presentation: providing ligands for T

lymphocyte activation. Cell 76(2): 287-299.

Kang, S.W., Chae, H.Z., Seo, M.S., Kim, K., Baines, I.C.

and Rhee, S.G. 1998. Mammalian peroxoredoxin

isoforms can reduce hydrogen peroxide generated in

response to growth factors and tumor necrosis factor-

alpha. Journal of Biological Chemistry 273(11): 6297-

6302.

Kloetzel, P.M. 2004. The proteasome and MHC class I

antigen processing. Biochim Biophys Acta 1695(1-3):

225-233.

Rajagopal, D., Bal, V., George, A. and Rath, S. 2004.

Diversity and overlap in the mechanisms of processing

protein antigens for presentation to T cells. Indian J Med

Res 120: 75-85.

Reif, K. and Cyster, J. 2002. The CDM protein DOCK2

in lymphocyte migration. Trends Cell Biol 12(8): 368-

373.

Sijts A., Sun, Y., Janek, K., Kral, S., Paschen, A.,

Schadendorf, D. and Kloetzel, P.M. 2002. The role of

proteasome activator PA28 in MHC class I antigen

processing. Molecular Immunology 39(3-4): 165-169.

Subramaniam, S. Stansberg, C. and Cunningham, C.

2004. The interleukin 1 receptor family. Dev Comp

Immunol 28(5): 415-428.

Wu, C., Sakorafas, P., Miller, R., McCarthy, D., Scesney,

S., Dixon, R. and Ghayur, T. 2003. IL-18 receptor

induced changes in the presentation of IL-18 binding sites

affect ligand binding and signal transduction. Journal of

Immunology 170: 5571-5577.

Zhang, Z., Clawson, A. and Rechsteiner, M. 1998. The

proteasome activator 11S regulator or PA28. Journal of

Biological Chemistry 273(46): 30660-30668.


POSSIBLE INVOLVEMENT OF FLOW DETECTION IN THE ACTIVATON OF OSTEOBLASTS*

Muneo Takaoki1

, Hyekeyong Park2

, Naoko Murakami3

, Dai Shiba2

, and Jun-Ichiro Gyotoku2

1

Space Environment Utilization Center, OSFO, JAXA; 2

ISS Science Project Office, ISAS, JAXA, 3Advanced

Engineering Services Co. Ltd.; Tsukuba 305-8505, Japan

One of the most potent mechanical stimulators for bone

cells may be the interstitial fluid flow. Fluid flow induces

various events such as Ca2+

signaling, c-fos expression,

actin fiber reorganization, and appearances of bone

markers in cultured cells (Chen et al., 2000). Fluid shear

stress may distort cellular membrane and other structures,

inducing cellular response. However, the nature of

molecules that convert the mechanical strain into

biological signals remains unclear.

The difficulty in identifying the mechanosensor

molecules seems to lie, at least in part, in the lack of

reliable methods for analysis. In contrast to regular cell

culture experiments, that compare the effects of dilute

substances, cellular responses to mechanical stimulations

are often difficult to interpret. The mechanical stimulation

may alter culture conditions drastically. It is extremely

difficult to apply only mechanical stimulations, leaving all

other conditions uniform. Excluding the effects of such

environmental changes, induced secondarily by the

operational procedure, is almost impossible. Thus, this

study seeks to develop a simple method for applying

mechanical stimulation with minimum secondary

influences, especially from temperature changes during

laboratory procedures.

Figure 1. C-fos expression induced either by rocking (A) or

cooling (B) of the culture flask. MC3T3-E1 cells were pre-

cultured for 3 days at 37°C in T-25 flasks. Culture flasks were

placed in small Styrofoam boxes with heat accumulators (small,

refreezable pack equilibrated to 37°C). This minimized the

cooling during incubator door opening and rocking. Gently

replacing heat accumulators with cold (4°C) packs for 1 min.

achieved cooling with the least possible fluid agitation (B).

After replacing with warm packs again, all flasks were

incubated at 37°C for additional 10 min. Total RNA was

extracted and c-fos gene expression was evaluated with RT-

PCR, and the values were normalized to 18S rRNA as described

previously (Takaoki et al., 2004). Values are means and

standard errors for 3 flasks.

We observed that conventional laboratory handling of

culture flasks could stimulate the murine osteoblastic cell

line MC3T3-E1. Fluid flow, generated either by manual

rocking or by unplanned agitation of culture flasks,

possibly induced c-fos expression to a far greater extent

than that caused by intended stimulation such as

centrifugation (Takaoki et al., 2004). Moreover, cooling

of the culture fluid, due to handling at room temperature,

induced similar response. To distinguish the effect of

rocking from the influence of cooling, we successfully

utilized small Styrofoam boxes and heat accumulators.

With this simple system, it was shown that either fluid

flow or cooling independently induced the responses

(Figure 1). Although flow-cells are normally used for the

purpose (Stevens and Frangos, 2003), this rocking method

was suitable for the parallel analyses of responses in

multiple flasks.

St i l l at 37Åé

10 minafte r 1stRocking

3

2. 5

2

1. 5

1

0. 5

0

Rela

tiv

e

E

xp

ress

ion

c-f

os


10 minafte r 2ndRockingat 60min


Figure 2. C-fos expression response to the second stimulation.

Culture flasks were rocked and then kept undisturbed for one

hour before the second stimulation. Values are means and

standard errors for 3 flasks.

The response reached a peak 10 to 15 min. after the

stimulation and settled to the background level within an

hour. The second stimulation given 60 min. later resulted

in a comparable response (Figure 2). In this study, we kept

all cultures inside the incubator without any disturbance

for at least 2 hours before rocking.

There was some possible mediation of the c-fos

expression response by Ca2+

and the ERK signaling

pathway. The addition of an ERK inhibitor, U0126

suppressed the response (Figure 3). Fluid flow can be more efficiently detected when seen

as a drag force against a projected structure than as a

shear stress to a smooth surface. One such projection,

primary cilium, served as a flow meter in kidney epithelial

cells. A transient receptor potential (TRP) channel family

protein, polycystin-2 (PC2) with polycystin-1, is one of

the key

* correction of article that was published in 2005

(volume 18)

M. Takaoki et al. –Flow detection by Osteoblasts


molecules for this function. PC2 localized at the primary

cilia and functioned as mechanosensitive Ca2+

ion

channel. Ca2+

signalling initiated from primary cilium

distorted by the drag of flowing fluid are sent to

neighbouring cells through gap junctions (Nauli et al.,

2003). Whitfield (2003) discussed the possible

involvement of similar mechanisms in bone cells.

Interstitial fluid flow could be caused by the slightest

distortion of bone tissue by a mechanical load. This flow

could be easily detected by sensitive flow detecting

primary cilia.

Primary cilia were found in the osteoblastic cell line

MC3T3-E1. The cells also expressed PC2. These

observations suggested that bone cells and kidney cells

detected fluid flow through similar mechanisms. However,

the localization of PC2 to the primary cilia was not as

preferential as had been reported in kidney cells (Figure

4). This weak localization of PC2 in the primary cilia and

the difference in reported flow sensitivities (Chen et al.,

2000, Nauli et al., 2003) suggested the involvement of

molecules other than those in kidney cells.

VehicleStill

U0126Still

3

2. 5

2

1. 5

1

0. 5

0

Rel

ativ

e E

xpre

ssio

nc-

fos

VehicleRocked

U0126Rocked

Figure 3. Suppression of c-fos response to fluid flow by U0126

37°C. Values are means and standard errors for 4 flasks.

Figure 4. Immunofluorescent staining of primary cilium

(arrow) of an MC3T3-E1 cell. Immunostainings with anti-

acetylated α-tubulin (AcTub) or anti-PC2 antibodies (right) are

shown.

We observed that MC3T3-E1 cells expressed c-fos in

cooling as well as in rocking of the culture flasks. This

response to the cooling would not seem physiological,

since we could find virtually no literature describing

temperature dependencies in bone metabolism.

Nevertheless, the response could be a factor observable

only under culture conditions with a cell line. Detection

of lowered temperature by PC2 or other TRP channel

superfamily proteins could also be possible. Some TRP

channel superfamily proteins have been shown to sense

either cold, cool or hot temperatures (Clapham, 2003),

while another member was assumed to sense mechanical

stimuli (Corey et al., 2004). Most of these proteins are

expressed in the respective neurons in a highly specific

manner, but not in bone or kidney cells. However, we

have not entirely excluded the possibilities of

mechanosensing and temperature sensing by PC2, or

contributions of other bone-specific TRP channel

superfamily proteins.

The transient c-fos expression response could be

induced by a variety of stimulations. These stimulations

could be applied, either intentionally or unintentionally, to

the cell culture during laboratory procedures. However,

the effects of these stimulations might be considered

trivial in regular experiments, where both reference

controls and experimental groups undergo virtually

identical environmental changes, and are treated side by

side in a similar manner. On the other hand, cultures must

usually be placed in separate locations when applying

different mechanical stimulations. Therefore, it is

extremely important to keep the environmental changes,

such as temperature, humidity, and CO2 concentration

uniform among the groups. This is particularly true in

space experiments, which must be precisely controlled to

enable comparisons between flight and ground specimens.

REFERENCES Chen, N.X., Ryder, K.D., Pavalko, F.M., Turner, C.H., Burr,

D.B., Qui, J., and Duncan, RL. 2000. Ca2+ regulates fluid shear-

induced cytoskeletal reorganization and gene expression in

osteoblasts. Am. J. Physiol. Cell Physiol. 278:C989-C997.

Clapham, D.E. 2003. TRP channel as cellular sensors. Nature

426:517-524

Corey, D.P., Garcia-Anoveros, J.. Holt, J.R., Kwan, K.Y., Shuh-

Yow, L., Vollrath, M.A., Amalfitano, A., Cheung, E.L.-M.,

Derfler, B., Duggan A., Geleoc, G.G., Gray P.A., Hoffman,

M.P., Rehm, H.L., Tamasauskas, D., Zhang D.-S. 2004. TRPA1

is a candidate for a mechanosensitive transduction channel

vertebrate hair cells. Nature 432:723-730.

Nauli, SM, Alenghat, F.J., Luo, Y., Williams, E., Vassilev, P.,

Li, X., Elia A.E.H., Lu, W., Brown, E.M., Quinn, S.J., Ingbar

D.E., and Zhou, J. 2003. Polycystins 1 and 2 mediate

mechanosensation in the primary cililum of kidney cells. Nature

Genetics. 33:129-137.

Stevens H.Y., and Frangos, J.A., 2003. Bone cell responses to

fluid flow. In Helfrich, M.H. and Ralston, S.H. eds. “Bone

Research Protocols”, Humana Press (Totowa, NJ) pp381-398.

Takaoki, M., Murakami, N. Gyotoku, J.-I. 2004. C-fos

expression of osteoblast-like MC3T3-E1 cells induced either by

cooling or by fluid flow Biological Science in Space 18:181-

182

Whitfield, J.F. , 2003. Primary cilium--is it an osteocyte's

strain-sensing flowmeter? J. Cell. Biochem. 89:233-237.


TRANSCRIPTIONAL PROFILING OF THE gps1 MUTANT OF ARABIDOPSIS

Vijayanand Nadella1, 2

, C. Dustin Hildenbrand1 and Sarah E. Wyatt

1*

1

Department of Environmental and Plant Biology, Ohio University, Athens, Ohio, USA; 2Current Address:

Department of Biology, Purdue University, West Lafayette, IN, USA; *corresponding author [email protected]

A plant’s response to gravity is a complex mechanism

involving stimulus perception, signal transduction and a

differential growth response. Disturbance of any of these

steps would affect a plant’s ability to respond to gravity.

The gravity persistence signal (gps) mutants were

identified using a cold treatment to select for mutants

specifically defective in the signal transduction events

prior to auxin redistribution. Plants perceive gravity at

4oC but auxin transport is abolished; transport is restored

upon return to room temperature (RT) (Wyatt et al. 2002).

Although auxin transport is restored in gps1 after return to

RT, auxin is not asymmetrically redistributed, and the

inflorescence stems fail to bend in response to the cold

gravistimulation (Nadella et al., 2006). To identify

additional genes in the pathway, especially those affected

by the gps1 mutation, we analyzed differential transcript

abundance between gps1 and wild-type (WT).

gps1 and wild-type plants were grown to maturity,

gravistimulated at 4oC for 1 h and returned to RT (GPS

treatment) (Wyatt et al. 2002). Inflorescence stems were

sprayed with RNAlater (Ambion) 5 min after return to

RT, and tissue from the region of elongation was

collected and analyzed for relative changes in transcript

levels. As a control for the cold treatment, additional WT

and gps1 plants were placed vertically at 4°C for 1h,

returned to RT and tissue collected as above. Total RNA

was extracted and purified (RNAeasy, Qiagen) from 10-

12 plants for each treatment and sent to the Microarray

Facility at University of California, Irvine for

amplification and hybridization to Arabidopsis ATH1

GeneChips (Affymetrix). Expression values were

normalized across each chip using the Cross Gene Error

Model (GeneSpring, Silicon Genetics, CA) and

significance determined by a Student T-test (p value=0.1).

From the 24,000 transcripts analyzed, 258 genes

showed differential expression: 128 up-regulated and 129

down-regulated in gps1 as compared to wild-type after

cold gravistimulation (Fig. 1). Those genes that were

similarly expressed in response to the cold treatment

alone were eliminated from further consideration.

Ten genes were selected, based on level of differential

expression, for analysis of transcript abundance

throughout the GPS treatment (Table 1). Both WT and

gps1 plants were grown to maturity, cold gravistimulated

for 60 min then returned to vertical at RT (Wyatt et al.

2002). RNA was extracted from the elongation zone of

the inflorescence stems at 0, 30, and 60 min after

gravistimulation and at 70 min after gravistimulation (10

min after return to vertical at RT). Transcript abundance

was determined by real time PCR using an ABI7900HT

sequence detection system according to Kimbrough et al.

(2004). Actin8 expression was measured separately for

Figure 1. Biological function of genes down-regulated (top) and

up-regulated (bottom) in gps1 as compared to wild-type under

the GPS treatment. Numbers indicate the number of genes in

each functional group. Genes selected showed at least a 2-fold

change in expression.

Table 1. Selected genes differentially expressed in gps1

as compared to WT after GPS treatment.

Protein Name Gene

Identifier

Fold

Change

putative 3-methyl crotonyl-

CoA carboxylase

At1g03090 4

putative protein kinase At1g30640 3

Spot 3 protein/vacuolar

sorting receptor homolog

At1g30900 3

hypothetical protein At1g50040 4

F-box protein At1g68050 5

CONSTANS B-box

zinc finger family protein

At1g78600 4

bZIP transcription factor At2g22850 5

cyclobilin like protein At2g39400 6

putative phospholipase At2g39400 6

myb transcription factor At5g12870 5

each time point, and the values used to normalize

transcript abundance of the selected genes.

electron transport/ energy pathways, 3

other cellular processes, 3

transport, 3

signal transduction, 2

cell organization &

biogenesis, 1

other metabolic

processes, 32

biological process

unknown, 31

other physiological

processes, 14 protein metabolism, 9

other biological

processes, 9

transcription,

7

developmental

processes, 5

abiotic / biotic

stimulus response, 5

Stress response , 4

other physiological

processes, 24

biological process

unknown, 7

other cellular

processes, 10

transcription, 9

protein metabolism, 17

other biological

processes, 7

electron transport/

energy pathways, 7

transport, 24

other metabolic

processes, 24

V Nadella et al-Transcriptional profiling of the gps1 mutant of Arabidopsis


Based on expression pattern, most genes fell into two

categories. Transcripts of the Spot 3 vacuolar sorting

protein, 3-methylcrotonyl-CoA carboxylase and

hypothetical protein increased dramatically in WT within

30 min after gravistimulation but were virtually absent in

gps1 (Fig. 2) suggesting that these genes may be regulated

either directly or indirectly by GPS1. Transcript level of

the phospholipase, CONSTANS B box protein, bZIP

transcription factor and cyclophilin were either unaffected

or down-regulated in WT but spiked in gps1 between 30

and 60 min after gravistimulation (Fig. 3). These genes

appear to be negatively regulated by GPS1 or its products.

Figure 2. Time course of transcript intensity of genes up-

regulated in WT (solid line) but absent in gps1 (dashed line).

Time after gravistimulation is indicated on the X-axes and

relative intensity of gene expression (as normalized to Actin8) is

shown on the Y-axes. Two biological replicates, with three

technical replicates each, were performed for each time point.

Bars indicate standard error.

Figure 3. Time course of transcript intensity of genes up-

regulated in gps1 (dashed line) compared to WT (solid line).

Time after gravistimulation is indicated on the X-axes and

relative intensity of gene expression (as normalized to Actin8) is

shown on the Y-axes. Two biological replicates, with three

technical replicates each, were performed for each time point.

Bars indicate standard error.

Although several of these genes are novel to

gravitational biology, potential roles for a vacuolar sorting

protein or a phospholipase could been imaged. Morita et

al. (2002) suggested that vacuole formation and

functioning plays a key role in the early stages of shoot

gravitropism. Phospholipase A2 has also been implicated

in shoot gravitropism of Arabidopsis by auxin mediated

cell elongation (Lee et al., 2003). An increase in the

At2g39400 transcript in gps1 suggests that a

phospholipase, regulated by the GPS1 protein, might be

directly or indirectly involved in lateral auxin

redistribution thereby affecting cell elongation and

contributing to the gps1 no response phenotype. In any

event, we have identified genes that appear to be

regulated early in the gravity signal transduction pathway

and provide interesting new candidates for study.

ACKNOWLEDGEMENTS

Our sincere thanks to the members of the Plant

Gravitational Genomic Center at North Carolina State

University for their assistance with the quantitative RT-

PCR experiments and helpful discussions and to Heather

Sanders for her help in preparing the manuscript. This

project was partially supported by NASA: NAG2-1608 to

SEW and a Summer Undergraduate Research Fellowship

from the American Society of Plant Biologists to CDH.

REFERENCES

Kimbrough, J.M., Salinas-Mondragon, R., Boss, W.F.,

Brown, C.S., Sederoff, H.W. 2004. The fast and transient

transcriptional network of gravity and mechanical

stimulation in the Arabidopsis root apex. Plant

Physiology 136:2790-805.

Lee, H.Y., Bahn, S.C., Kang, Y.M., Lee, K.H., Kim, H.J.,

Noh, E.K., Palt, J.P., Shin, J.S., Ryu, S.B. 2003. Secretory

low molecular weight phospholipase A2 plays important

roles in cell elongation and shoot gravitropism in

Arabidopsis. Plant Cell 15: 1990-2002.

Morita, M.T., Kato, T., Nagafusa, K., Saito, C., Ueda, T.,

Nakano, A., Tasaka, M. 2002. Involvement of the

vacuoles of the endodermis in the early process of shoot

gravitropism in Arabidopsis. Plant Cell 14: 47-56.

Nadella ,V., Shipp, M.J., Muday, G.K., Wyatt, S.E. 2006.

Evidence for altered polar and lateral auxin transport in

the gravity persistent signal (gps) mutants of Arabidopsis.

Plant Cell and Environment 29: 682-690.

Wyatt, S.E., Rashotte, A.M., Shipp, M.J., Robertson, D.,

Muday, G.K. 2002. Mutations in the gravity persistence

signal loci in Arabidopsis disrupt the perception and/ or

signal transduction of gravitropic stimuli. Plant

Physiology 130: 1426-1435.

At1g03090

(putative 3-methylcrotonyl-CoA

carboxylase)

-4

-2

0

2

4

6

8

10

12

14

16

0 min 30 min 60 min 70 min

At1g50040

(hypothetical protein)

-4

-2

0

2

4

6

8

10

12

14

16

18


At1g30900

(Spot3 vacuolar sorting protein)

-5

0

5

10

15

20


At1g78600

(CONSTANS B box Zinc finger family

protein)

-4

-2

0

2

4

6

8

10


At2g39400

(putative phospholipase)

-10

-5

0

5

10

15

20

25

30


At2g22850

(bZIP family transcription factor)

-3

-2

-1

0

1

2

3

4

5


At2g36130 (cyclophilin like protein)

-3

-2

-1

0

1

2

3



NITRIC OXIDE AND CGMP DEPENDENT SIGNALING IN ARABIDOPSIS ROOT GROWTH

Jennifer Jacobi1

, Jacob Elmer1

, Kristin Russell1

, Raj Soundur2

, and D. Marshall Porterfield2,3,4

1Department of Biological Sciences, University of Missouri Rolla, Rolla, MO;

2

Department of Agriculture and

Biological Engineering, 3Department of Horticulture and Landscape Architecture,

4Bindley Bioscience Center,

Purdue University, West Lafayette, IN.

Nitric oxide (NO) and cGMP are established

components of many calcium-dependent signaling

pathways in animal systems. Discovering this has been

fundamental to understanding some of the most basic

physiological processes (Lamattina, et al. 2003;

Wendehenne et al. 2001). In animals NO can act with or

without cGMP to activate protein kinases and cellular

responses. Studies of NO activity in plants have suggested

that the basic NO/cGMP mediated signaling pathway,

which operates in signaling in animals (Figure 1), may

also be active in plant cells. For example, recent work

has shown that NO and cGMP are involved in the Ca++

dependent cellular development in pollen (Prado et al.,

2004)). Here our work focuses on identifying potential

involvement of NO and cGMP in controlling cell

elongation and root extension, with an emphasis on

gravitropism in Arabidopsis roots.

We hypothesized that a calcium-dependent

NO/cGMP dependent signaling pathway may be active in

root gravitropism, either as a primary signal, driving

differential auxin transport in the root cap, or downstream

of an IAA receptor to elicit differential changes in cell

elongation. In these early studies we adopted a

pharmacological approach to investigate the pathway. We

exposed Arabidopsis thaliana seedlings to NO donor

compounds (DEA and Spermine NONOates), dibuturyl

cGMP, L-Name, IBMX, Viagra, and cPTIO. The

seedlings were grown for about a week, then transferred

to agar plates containing varying doses (0–700 uM) of the

drugs, and root growth was monitored for 24 hours.

We tested direct effects of NO on root elongation

(Figure 2) using NO donors (n=6) and found that low

concentrations of DEA (3.0-100 µM) and Spermine (5.0,

10.0 µM) significantly increased root elongation, while

high concentrations of DEA (700 µM) and Spermine

(300-700 µM) inhibited root elongation (t-test: p≤ 0.05).

Two well known phosphodiesterase inhibitors were also

used (n=6). Low concentrations of Viagra™ (1.0-10 µM)

and IBMX (1.0-150 µM) also significantly increased root

elongation (t-test: p≤ 0.05) but this effect was attenuated

as the concentration increased (Figure 2). These two sets

of experiments thereby suggest that there is a connection

between NO and cGMP in A. thaliana root elongation.

A common approach in NO research is to apply

NO using various donor molecules, but to understand the

resulting NO responses we have to account for the

L-Arginine + O2

X L-NamecPTIO –NO

DEA +NO

Spermine +NO

SNP +NO +CN-

NO

GTP

cGMP

PDE

X ODQ

X Viagra, IBMX

Cellular Response

+ Dibutryl cGMP

NO

Synthase

NO/cGMP Signaling Pathway

Guanylate

Cyclase

NO

cGMP

Hydrolysis

Auto-oxidation

L-Arginine + O2

X L-NamecPTIO –NO

DEA +NO

Spermine +NO

SNP +NO +CN-

NO

GTP

cGMP

PDE

X ODQ

X Viagra, IBMX

Cellular Response

+ Dibutryl cGMP

NO

Synthase


Guanylate

Cyclase

NOGuanylate

Cyclase

NO

cGMP

Hydrolysis

Auto-oxidation

L-Arginine + O2

X L-NamecPTIO –NO

PTIO –NO

Spermine +NONO

GTP

cGMP

PDE

X ODQ

X Viagra IBMX

Cellular Response

+ Dibutryl cGMP

NO

Synthase


Guanylate

Cyclase

NOGuanylate

Cyclase

NO

cGMP

Hydrolysis

Auto-oxidation

Calcium/Calmodulin

cPTIO X

Figure 1. NO signaling cascade. Nitric Oxide Synthase (NOS)

is activated by the Ca++ calmodulin complex. NOS converts L-

Arginine and oxygen to citruline and NO. L-Name is an analog

of arginine that inhibits NO production. cPTIO inhibits NOS by

reacting with NO to form carboxy-PTI derivatives. cPTIO also

scavenges NO. DEA, Spermine, and SNP are NO donors. SNP

also produces cyanide. ODQ is a selective inhibitor of the NO

sensitive Guanylyl Cyclase. ViagraTM and IBMX affect the

pathway in similar ways. Both are phospho-diesterase

inhibitors, although Viagra™ is a more specific inhibitor.

Dibutryl cGMP is a cGMP analog that activates protein kinase

G.

NO Donor Concentration (µM)

0 200 400 600

Root E

longation (

rela

tive g

row

th r

ate

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DEA

Spermine

Phosphodiesterase inhibitor Concentration (µM)

0 50 100 150 200 250

Root E

longation (

rela

tive g

row

th r

ate

)

0.8

1.0

1.2

1.4

1.6

1.8

ViagraIBMX

Figure 2. Vertically grown Arabidopsis plants on agar media

that contained experimental concentrations of NO donors (DEA,

Spermine) and phosphodiesterase inhibitors (Viagra™ and

IBMX). The growth after 24 hours was measured and

normalized to the control giving relative growth rate.

Time (minutes)

0 20 40 60 80 100 120

NO

co

nce

ntr

atio

n (

µM

)

0

5

10

15

20

120 140 160 180 200 220 240

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Time (minutes)

0 20 40 60 80 100 120

NO

co

nce

ntr

atio

n (

µM

)

0

5

10

15

20

120 140 160 180 200 220 240

0

1

2

3

4

5

Figure 3. DEA (left) and Spermine (right) drug kinetics. Both

DEA and Spermine modeling is plotted based on NO

concentration vs. time. NO decomposition was modeledfor eack

concentration (700, 500, 300,100 ,50, 30, 20, 10, 7, 5, 3, and 1

µM) as first order and the formation of nitrite was considered to

be second order. The rate constants used were K1-DEA=

1.4x10-3 s-1 (Schmidt et al., 1997), K1-Spermine = 3.2x10-4 s-1

(Ramamurthi et al. 1997) K2 = 9.2x106 M-2 s-1 (Schmidt et al.,

1997) was the same for both drugs.

J. Jacobi et al. – Nitric Oxide and cGMP Dependent Signaling in Arabidopsis Root Growth


balance between NO production, and auto-oxidation in

the system. A key is to understand that NO levels

dynamically change, and the amount of NO available to

the plants at a given time is significantly less than the

actual NO donor drug concentration (Figure 3). Spermine

is a more consistent, sustainable NO source, and as a

result, the pharmacological effects of Spermine are more

dramatic than DEA (Figure 2). Sodium nitroprusside

(SNP) has become a common NO donor in plant

literature, despite being almost completely abandoned by

animal research. We did not use SNP as it is inconsistent,

requiring a reducing agent or light for activation, and is

also produces more cyanide (6:1) than NO (Yamato and

Bing 2000). While SNP has been reported to alter root

physiology (Cassimiro et al. 200l, Correa-Aragunde et al.

2004, Gouvea et al. 1997, Hu et al. 2005, Pagnussat et al.

2003) we found that SNP concentrations ranging from 5

to 300mM completely inhibited growth and killed the A.

thaliana plants.

These experiments suggest that altering NO and

cGMP using NONOate donors (Spermine and DEA),

phospohodiesterase inhibitors (Viagra ™ and IBMX), and

NO synthase inhibitors (L-Name, an arginine/citruline

intermediate) alters root elongation (Figures 2 and 4,) and

gravitropism (Figure 5). In general low doses of NO cause

growth to increase, while high doses of NO cause growth

to slow down and in some instances stop. Both cPTIO and

L-Name (Figure 4) reduce the amount of endogenous NO

production, and enhanced root elongation in a dose

dependent manner (Hilland Plot analysis)

These data are consistent with the effects of

auxin on root growth (Cassimiro et al. 200l, Correa-

Aragunde et al. 2004, Gouvea et al. 1997, Hu et al. 2005,

Pagnussat et al. 2003, Taiz et al. 2002), where high levels

of auxin (10-6

M) reduce/inhibit root growth while low

concentrations (10-10

or 10-9

M) increase elongation. We

believe that these results suggest an auxin-mediated

NO/cGMP signalling cascade may control cell elongation

at the cellular level. Auxin might also play a direct role in

graviperception signalling but further experiments are

required to answer that question.

REFERENCES Correa-Aragunde, N., Graziano, M., Lamattina, L. 2004. Nitric

oxide plays a central role in determining lateral root

development in tomato. Planta. 218: 900-905.

Casimiro, I., Marchant, A., et al. 2001. Auxin transport

promotes lateral rot iitiation. The Plant Cell. 13: 843-852.

Gouvea, C.M.P., Souza, J. F., et al. 1997. NO-releasing

substances that induce growth elongation in maize root

segments. Plant Growth Regulation. 21: 183 – 187.

Hu, X., Neill, S. J., et al. 2005. Nitric oxide mediates gravitropic

bending in soybean roots. Plant Physiology Preview.

Lamattina, L., Garcia-Mata, C., et al. 2003. Nitric Oxide: the

versitality of an extensive signal molecule. Annu. Rev. Plant

Biol. 54: 109-136.

Pagnussat, G.C., Lanteri, M.L., Lamattina, L. 2003. Nitric oxide

and cyclic GMP are messengers in the indole acetic acid –

induced adventitious rooting process. Plant Physiology. July,

132: 1241-1248.

Prado, A. M., Porterfield, D. M., Feijo, J. A. 2004. Nitric oxide

is involved in growth and regulation and reorientation of pollen

tubes. Development. 131: 2707-2714.

Ramamurthi, A., Lewis, R. S. 1997. Measurement and modeling

of nitric oxide release rates for nitric oxide donors. Chemical

Research in Toxicology. 10: 408-413.

Schmidt, K., Desch, W., K, et al. 1997. Release of nitric oxide

from donors with known half life: a mathematical model for

calculating nitric oxide concentrations in aerobic solutions.

Naunyn-Schmiedeberg’s Arch Pharmacol. 355: 457-462.

Taiz, L., Zeiger, E.. 2002. Auxin: the growth hormone. Murphy,

Angus. Plant Physiology,Tthird Addition. 423-460.

Wendehenne, D., Durner, J., Klessig, D. F. 2004. Nitric oxide: a

new player in plant signaling and defense responses. Current

Opinion in Plant Biology. 7:449-455.

Yamato, Tadahiko, Bing, Richard. 2000. P.S.E.B.M. Vol 225:

200-206.

Drug Concentration

0 100 200 300 400

Gra

vitro

pic

Re

ori

en

tatio

n (

Re

lative

to c

ontr

ol)

-1.0

-0.5

0.0

0.5

1.0

1.5

LNAME

Viagra

Figure 5. L-Name and Viagra disrupt the gravitropic response

of horizontally-oriented, gravistimulated roots. These plants

were grown on agar media containing experimental drug

concentrations that were turned so the plants were horizontal.

Reorientation angle was measured after 24 hours of

gravistimulation and compared with controls.

cPTIO Concentration (µM)

0.1 1 10 100 1000

Roo

t E

lon

gation

(R

ea

ltiv

e g

row

th r

ate

)

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

Measured

Hilland Plot

LNAME µM

0 100 200 300

Ro

ot

Elo

ng

ation

(R

ela

tive G

row

th R

ate

)

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

Figure 4: Pharmacological dose response analysis (Hilland

plot) of cPTIO and LNAME. Arabidopsis root growth increased

along with the concentration of cPTIO in the experiment. Low

concentrations of L-Name slowed the growth of Arabidopsis

compared to the control and high concentrations increased

growth. The Arabidopsis plants were grown vertically on agar

plates containing the drugs. Growth rate was measured after 24

hours and compared to the control.


REGULATION OF TRANSCRIPTION IN ROOTS OF ARABIDOPSIS GRAVITY MUTANTS.

Jessie W. Yester1, Jeffery M. Kimbrough

1, Raul Salinas-Mondragón

1

, Patrick H. Masson3, Christopher S.

Brown1,2

, and Heike Winter Sederoff1*

1

Dept. of Botany, and 2

Kenan Institute for Science and Engineering, North Carolina State University, Raleigh

27695; 3Laboratory of Genetics, University of Wisconsin, Madison, WI, USA. * corresponding author:

[email protected]

Plant growth is guided by gravity. The mechanism by

which plants sense gravity is not understood.

Sedimentation of starch-containing amyloplasts (statolith)

in the columella cells is involved in the perception of

changes in the orientation towards the vector of gravity.

Inositol-1,4,5,-triphosphate (InsP3), Ca2+

, and proton

transport, as well as phytohormones (auxin, cytokinin,

ethylene, brassinosteroids), reactive oxygen species, and

nitric oxide have been shown to mediate the gravity

induced signal transduction. Several factors involved in

gravitropism have been identified by mutants defective in

their response to changes in their orientation towards

gravity. In order to elucidate the role of some of these

signal transduction elements in their regulation of gravity

specific transcription, we analyzed temporal changes in

gravity specific up-regulated genes in mutant and their

respective wildtype backgrounds (ecotypes) after

reorientation.

Increases in transcript abundances of some genes occur

within 1 minute after reorientation in the Arabidopsis root

apex, and are regulated specifically by gravity, not

mechanical stimulation (Kimbrough et al. 2004).

Focusing on three of these gravity-specific genes, we

present evidence that gravity-induced transcript

abundance changes of these genes are not strictly

conserved between ecotypes. In the root gravity response

mutants arg1 (defective in a DNAJ protein; Sedbrook et

al. 1999) and arl2 (defective in DNAJ protein similar to

ARG1, Guan et al. 2003), transcript abundance changes in

those selected gravity specific genes were not initiated.

This indicates that the DNAJ protein is required for

gravity-induced transcriptional regulation. In the gravity

response mutant adk1 (defective in adenosine kinase,

Young et al., unpublished) transcript abundance of one

gene was up-regulated, while two other genes did not

show an increase in transcript abundance after

reorientation. This indicates that transcriptional regulation

of those genes requires different signal transduction

elements in response to gravity stimulation.

Methods

We harvested Arabidopsis root apices (7 day old

etiolated seedlings) in order to analyze their relative

changes in transcript level in response to gravistimulation.

For our time course experiment, approximately 150 root

tips were harvested before (0 time point) and 2, 5, 15 min

after 135◦ reorientation by pouring RNAlater (Ambion)

onto the plates and cutting off the root apex (~7.5 mm).

Total RNA was extracted, purified with RNeasy (Qiagen),

and cDNA was synthesized using gene specific primers.

Arabidopsis gravity signaling mutants arg, arl, and adk

along with wild type controls were grown for seven days

in the dark, reoriented (135◦) and time-dependent changes

in transcript abundance measured for the gravity specific

genes (At4g23670; At5g38020; At5g48010).

Results

The gravity-response mutant plants used in this study

were derived from the Arabidopsis ecotypes

Wassilewskja (mutants arg, arl) and Landsberg (mutant

adk). We measured gravity-induced transcript abundance

changes in these ecotypes in comparison to Columbia-O

which was used in the microarray study (Kimbrough et al.

2004). We found that there are differences in the gravity-

induced transcriptional regulation of specific genes

between different Arabidopsis ecotypes in both temporal

profile and magnitude of regulation. (Figures 1 A,B,C).

Figure 1: Transcript abundance changes of At4g23670,

At5g38020, and At5g48010 in response to reorientation

in Arabiodopsis ecotype Columbia (A), Wassilewskija (B)

and Landsberg (C).

0

1

2

3

0 5 10 15

At4g23670

At5g38020

At5g48010

Columbia (Col)

min after reorientation

tra

nsc

rip

t a

bu

nd

an

ce f

old

ch

an

ge

A

0

1

2

3

0 5 10 15

At4g23670

At5g38020

At5g48010

tran

scrip

t ab

un

dan

ce f

old

ch

an

ge


Wassilewskija (WS)B

0

1

2

3

0 5 10 15

At4g23670

At5g38020

At5g48010

tra

nsc

rip

t a

bu

nd

an

ce f

old

ch

an

ge


Landsberg (Ler)C

J.W. Yester et al. – Regulation of Transcription in Roots of Arabidopsis Gravity Mutants.


DNAJ protein is essential for gravity induced

transcription

ARG and ARL code for DNA-J proteins which are

essential for gravity-induced regulation of transcript

abundance changes for the genes we tested. Reorientation

of these gravity mutants did not induce any significant

increases in transcript abundance of the genes tested

compared to their respective ecotype (Figures 2 A,B).

0

1

2

3

0 2 4 6 8 10 12 14min after reorientation

At4g23670

At5g38020

At5g48010

arl(ecotype Ws)

A

tra

nsc

rip

t a

bu

nd

an

ce f

old

ch

an

ge

0

1

2

3

0 5 10 15min after reorientation

At4g23670

At5g38020

At5g48010

arg(ecotype Ws)

B

tra

nsc

rip

t a

bu

nd

an

ce f

old

ch

an

ge



in Arabiodopsis mutants arl (A), and agr (B)

Adenosine kinase (ADK) regulates transcription of some

genes probably involved in hormone synthesis

ADK is involved in modulating root-tip architecture

and gravitropism in the Arabidopsis root tip. Mutation in

this gene alters gravitropic sensitivity (Masson et al.,

unpublished). Transcript levels of At5g38020 (SamT-

functions in Ado-Met pathway as methylating enzyme)

and At5g48010 (AtPEN1 – involved in brassinosteroid

synthesis) do not increase in the root apex after gravity

stimulation, while transcriptional regulation of At4g23670

(Major latex protein-like) is enhanced or at least not

repressed. This indicates that ADK is part of a signal

transduction pathway that activates transcriptional

regulation for At5g38020 and At5g48010. It is therefore

“downstream” of arg and arl in the signal transduction

pathway.

0

1

2

3

4

5

6

0 5 10 15min after reorientation

At4g23670

At5g38020

At5g48010

adk(ecotype Ler)

transc

rip

t abu

ndance

fold

chan

ge



in Arabiodopsis mutant adk.

Conclusions

� We measured variation in the gravity-induced

transcriptional regulation of specific genes between

different Arabidopsis ecotypes.

� ARG and ARL code for proteins which are essential for

gravity-induced regulation of transcript abundance

changes for at least those genes tested.

� ADK expression is required for gravity-induced

transcriptional regulation of some specific genes

(At5g38020, At5g48010) possibly involved in hormonal

regulation.

At4g23670

GS ARG, ARL

ADK

At5g38020

At5g48010

Figure 4: The transcriptional regulation induced by

gravity stimulation (GS) requires the DNAJ proteins ARG

and ARL. The transcription of the Major-latex like gene

(At4g23670) does not require functional expression of

ADK.

(Supported by NASA grant NAG2-1566 to C.S.B. and

North Carolina Space Consortium grant to J.W.Y.)

REFERENCES

Guan, C., Rosen E., Boonsirichai, K., Poff, K., Masson, P. 2003.

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity

signal transduction pathway that is genetically distinct from

the PGM pathway. Plant Physiol. 133: 100-112.

Kimbrough, J., Salinas-Mondragon, R., Boss, W., Brown, C.,

Sederoff H. 2004. The fast and transient transcriptional

network of gravity and mechanical stimulation in the

Arabidopsis root apex. Plant Physiol. 136: 2790-2805.

Sedbrook, J., Chen, R., Masson, P. 1999. Arg1 (altered response

to gravity) encodes a DNAJ-like protein that potentially

interacts with the cytoskeleton. Proc. Natl. Acad. Sci. USA 96:

1140–1145.


MODIFICATION OF RESERVE DEPOSITION IN WHEAT AND BRASSICA SEEDS BY SYNTHETIC

ATMOSPHERES AND MICROGRAVITY

Anxiu Kuang1, John Blasiak2, S. Chen1, Gail Bingham3, Mary E. Musgrave2

1Dept. of Biology, Univ. of Texas Pan American, Edinburg, TX 78541; 2Dept. of Plant Science, Univ. of

Connecticut, Storrs, CT 06269; 3Space Dynamics Lab, Utah State Univ., Logan, UT 84341

Movement of gases in microgravity may be reduced due

to a lack of buoyancy-driven convection (Musgrave, et

al., 1997). We are investigating how seed development in

microgravity could be modulated through changes in

metabolic gas bioavailability (Blasiak et al., 2006). To do

this we have grown wheat (Triticum aestivum L. cv.

Apogee) and Brassica (Brassica rapa L.) in synthetic

atmospheres in which the bulk gas (normally N2 in

ambient air) is replaced by either Ar (slowing diffusion)

or He (speeding diffusion). The seeds produced in the

synthetic atmospheres were then compared with seeds

grown in microgravity on the Mir space station in the Svet

greenhouse. Since starch is the major component of

wheat seeds, while in Brassica protein and lipid are the

major storage reserves, we have compared how these

storage reserves changed in microgravity, ground control,

and synthetic atmosphere environments.

Wheat (Triticum aestivum L. cv. Apogee) and Brassica

(Brassica rapa L.) plants were grown from seed-to-seed

on the Mir space station in the Svet greenhouse. Mature

dry seeds were collected and subsequently examined by

light and electron microscopy. In ground-based studies,

wheat and Brassica plants were grown in synthetic

atmospheres differing in the bulk carrier gas (He, N2 or

Ar), but containing earthnormal concentrations of O2

(21%) and CO2 (400 ppm), to produce treatments in

which the metabolic gases would diffuse at different rates.

The plants were grown under fluorescent lights

(continuous light, 120 mmol/m2/sec, PAR) using a wick-

fed hydroponic system. Gas-flow through the chambers

was approximately 350 ml/min. Dry seeds of wheat and

Brassica were colleted and immature fresh seeds (18 d

post-pollination) of Brassica were fixed and processed for

microscopy using procedures as in Kuang et al. (2000).

Analysis of the wheat seeds produced on the Mir space

station by scanning electron microscopy revealed that

starch grains were deformed, compared to the ground

seeds (Fig. 1). Those starch grains had a rough surface

with microholes that were evenly distributed on the

surface (Fig. 1). In wheat seeds from the synthetic

atmospheres, starch grains revealed comparable

morphology and structures in the He and air (N2)

treatments, however the Ar treatment had smaller,

rounder grains compared to the air (N2) control treatment

(Fig. 2).

Brassica seeds produced on Mir showed retention of

starch grains while lipid and protein were the major

storage reserves in mature ground seeds. Protein bodies

were also a major reserve in Mir seeds but their size was

diminished (Musgrave et al, 2000). Brassica seeds

produced in all of the synthetic atmosphere treatments had

lipid and protein as the major storage reserves, however,

protein bodies in the helium treatment were larger

compared to those in the nitrogen treatment (Fig. 3),

which might have contributed to the greater weight of the

seeds (Table 1). In immature seeds, protein bodies were

smaller and starch grains persisted in embryo cells in the

Ar treatment, while protein and lipid were major reserves

in the seeds produced in the N2 chamber, indicating that

development of Brassica seeds was retarded in the Ar

atmosphere (Fig. 4).

Table 1. Mean seed weight, number of protein bodies per

cell, and mean diameter (µm) of protein bodies in Brassica

dry seeds from the second helium and argon experiments.

Treatment Seed dry Protein bodies Diameter of

weight (mg) (n/cell) protein (µm)

Air 1.84 2.7 6.02

Helium 2.05 2.7 7.54

P-value 0.04 ns 0.002

Air 1.54 2.3 6.80

Argon 1.43 3.0 6.08

P-value ns 0.06 0.09

The results show that small differences in metabolic gas

diffusion rates lead to ultrastructural changes in seed

storage reserves. Starch grains of wheat from the He and

N2 treatments were comparable. While the starch grains

from the Ar treatment did not exhibit the abnormalities

found in the flown material (Fig. 2), the grains were

smaller and rounder than in the N2 treatment. Changes in

starch grain morphology in the spaceflight environment

have been reported by others (Kuznetsov et al., 2001).

Synthetic atmosphere experiments show that diffusion

rate affects development of Brassica seed-storage

components. Development in a He atmosphere enhanced

the size of protein storage bodies and mature seed dry

weight. In contrast, the reduced-diffusion rate of the Ar

atmosphere retarded development: protein bodies were

smaller than in the control treatment, and starch grains

were retained later in seed development. The development

of Brassica seed-storage components under the reduced-

diffusion rates of the argon atmosphere was similar to the

pattern observed under microgravity conditions (Kuang et

al., 2000; Musgrave at al., 2000)

REFERENCES

Blasiak, J, A Kuang, CS Farhangi, and M. E. Musgrave. 2006.

Roles of intra-fruit oxygen and carbon dioxide in controlling

pepper (Capsicum annuum L.) seed development and storage

A. Kuang et al. – Simulation of Changes in Seed Storage Reserves in Microgravity


reserve deposition. J. Amer. Society of Horticultural Science,

131(1):164-173.

Kuang, A, Y Xiao, G. McClure, and ME Musgrave (2000).

Influence of Microgravity on ultrastructure and storage reserves

in seeds of Brassica rapa L. Annals of Botany, 85:851-859.

Kuznetsov, OA, CS Brown, HG Levine, WC Piastuch, MM

Sanwo-Lewandowski, and KH Hasenstein. Composition and

physical properties of starch in microgravity-grown plants.

Advances in Space Research (2001), 28 (4): 651-658

Musgrave, ME, A. Kuang, and SW Matthews (1997). Plant

reproduction during spaceflight: importance of the gaseous

environment. Planta, 203: s177-s184.

Musgrave, ME, A. Kuang, Y Xiao, GE Bingham, L. G. Briarty,

M.A Levinski, VN. Sychev, amd IG Podolski (2000). Gravity

independence of seed-to-seed cycling in Brasicca rapa. Planta,

210(3): 440-406.

Funded by NASA grants NAG2-1375 and NAG10-329 to MEM

and AK.

Figure 1. SEM micrographs showing starch grains in wheat

seeds produced on the Mir space station (A & B) and in the

ground control (C & D). Note that microholes (arrowheads)

are present on the surface of starch grains in the spaceflight

seeds. Bars = 10 µm

Figure 2. SEM micrograph of starch grains in wheat seeds

produced in the synthesized atmosphere experiments. A), air

control for helium treatment; B), helium treatment; C), air

control for argon treatment; D), argon treatment. Bars = 10 µm

Figure 3. Ultrastructure of cotyledon cells of dry Brassica

seeds produced in the air chambers (A & C), helium chamber

(B), and argon chamber (D). Note that both protein bodies and

lipid are major storage reserves but protein bodies (pb) in seeds

produced in the helium chamber (B) are lager in size (see Table

1) compared to seeds produced in air the chamber (A). Bars =

4.2 µm

Figure 4. Ultrastructure of cotyledon cells of Brassica seeds

(18 d old) produced in the argon chamber (A & B) and in the air

chamber (C & D). Starch grains (arrows) were present (A) and

protein bodies (pb) were relatively smaller (B) in seeds from the

argon chamber, compared to the seeds from the air chamber (C

& D). The nucleus (nu) was obvious, whereas the nucleus in

seeds produced in the air chamber became shrunken (D)

indicating that dehydration was in progress. Bars = 3.9 µm.

A

B

C

D

pb

pb

pb

pb

pb pb

pb

pb

pb

A

B

C

D

nu

nu

A B

C D

A B

CD

Gravitational and Space Biology 19(2) August 2006 A - 1

Author Index

Agricola, H-J., 135

Allen, C., 143

Allen, P.L., 145

Ask, M.A., 129

Baer, L., 141

Baker, T.L., 145

Barnstable, A.J., 141

Barnstable, C.J., 141

Benoit, M.R., 31

Bhattacharya, S., 127, 133

Bingham, G., 161

Blasiak, J., 161

Blazevic, E., 133

Boling, J., 91

Boston, P.J., 91

Bourget, C.M., 19

Brown, C.S., 159

Brzezinski, E., 53

Campbell, K.A., 91

Chang, J., 133

Chen, S., 161

Chopra, A., 143

De Carlo, A.R., 123

Duke, J., 137

Echon, C.M., 53

Elliott, T.F., 145

Elmer, J., 157

Emmerich, J.C., 19

English, J., 137

Fahlen, T.F., 133

Ferl, R.J., 3

Flowers, R., 139

Galindo, C., 143

Goli, N., 139

Graham, J.M., 105

Gupta, K.B., 125

Gyotoku, J-I., 153

Hammond, D.K., 145

Hammond, T.G., 145

Hargens, A.R., 53

Hawkins, A., 53

Hawkins, K., 53

Hildenbrand, C.D., 155

Holubec, K., 145

Horn, E.R., 135

Houston, E., 127

Howard, A., 127

Irazoqui, P.P., 123

Jacobi, J., 157

Johnson, C.B., 147

Kakavand, A., 127

Kimbrough, J.M., 159

Kirschnick, U., 135

Klement, B., 139

Kuang, A., 161

Kucik, D.F., 125

Kurk, M.A., 131

Kuznetz, L.H., 85

Lee, R., 137

Lera, M., 133

Leskovsky, D., 127

Levine, H.G., 129

Love, J.E., 145

Lynch, S.V., 31

Maclas, B.R., 53

Maldonado, A., 127

Marshall, C., 139

Masini, M.A., 149

Massa, G.D., 19

Masson, P.H., 159

Matin, A., 31

McLamb, W.T., 123

McSpadden, T., 91

McWilliams, L., 91

Metz, G.W., 131

Meyer, R.S., 53

Mitchell, C.A., 19

Morrow, R.C., 19

Murakami, N., 153

Musgrave, M.E., 161

Nadella, V., 155

Niesel, D., 143

Norikane, J., 129

Author Index

A - 2 Gravitational and Space Biology 19(2) August 2006

Pandya, U., 143

Park, H., 153

Pastorino, M., 149

Paul, A-L., 3

Paulsen, D., 139

Pellis, N.R., 151

Porterfield, D.M., 123, 157

Prenger, J.J., 129

Ricci, F., 149

Risin, D., 151

Risin, S.A., 151

Rokkam, M., 123

Rouleau, R.L., 125

Roux, S.J., 123

Rouzan-Wheeldon, D., 129

Russell, K., 157

Rygalov, V., 129

Salinas-Mondragón, R., 159

Sanchez, M.E., 127, 133

Shenasa, M., 127

Shiba, D., 153

Smith, L.W., 125

Sonnenfeld, G., 45

Soundur, R., 157

Steele, M.K., 127

Stein, T.P., 49

Strollo, F., 149

Takaoki, M., 153

Teichgraeber, J., 137

Thomas, D.J., 91, 131

Thomas, N.A., 131

Tink, A.R., 141

Todd, P., 79, 91, 131

Tombran-Tink, J., 141

ul Haque, A., 123

Uva, B.M., 149

Vandromme, M., 139

Viviano, S., 141

Wade, C.E., 65, 141

Waldbeser, L.S., 147

Ward, N.E., 151

Wells, H.W., 123

Wereley, S.T., 123

Williams, N., 143

Winter Sederoff, H., 159

Wu, X., 125

Wyatt, S.E., 155

Yester, J.W., 159

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