Environmental and Experimental Botany 43 (2000) 33–43

Mycosporine-like amino acids in the marine red algaGracilaria cornea — effects of UV and heat

Rajeshwar P. Sinha, Manfred Klisch, Almut Groniger, Donat-P. Hader *Institut fur Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Uni6ersitat, Staudtstr. 5, D-91058 Erlangen, Germany

Received 29 October 1998; received in revised form 2 September 1999; accepted 2 September 1999

Abstract

Ultraviolet (UV)-absorbing mycosporine-like amino acids (MAAs) were separated from a marine red algaGracilaria cornea using HPLC. The isolated MAAs were identified as porphyra-334 and/or shinorine by comparingthem with various standards. No in vivo induction of MAAs was detected in G. cornea even when the organism wasgrown for 4–5 days either in the presence of UV-A and UV-B only or in combination with photosynthetically activeradiation. In vitro absorption properties of MAAs were unaffected when irradiated with UV-B or subjected to heattreatment (7592°C) for up to 6 h. In comparison to MAAs, other pigments such as chlorophyll a (436 and 665 nm),carotenoids (475 nm) and phycocyanin (618 nm) were severely affected by UV-B irradiation. The results indicate ahighly stable nature of MAAs against the environmental stress factors like UV-B and heat. The SDS-PAGE proteinprofile of G. cornea showed a gradual decrease in the intensity of protein bands at 20 kDa (a and b subunits ofphycocyanin) but at the same time a gradual increase in the intensity of protein bands at 26 kDa (phycoerythrin),showing the phenomenon of chromatic adaptation (changes in pigmentation in photosynthetic organisms in responseto light quality), when the organism was grown in the presence of UV plus PAR. © 2000 Elsevier Science B.V. Allrights reserved.

Keywords: Gracilaria ; Mycosporine-like amino acids (MAAs); UV irradiation; Heat treatment; Pigmentation; Protein profile

www.elsevier.com/locate/envexpbot

1. Introduction

Continued depletion of the stratospheric ozonelayer, mainly due to anthropogenically releasedatmospheric pollutants such as chlorofluorocar-bons (CFCs) is responsible for the increase in

solar ultraviolet-B (UV-B; 280–315 nm) radiationreaching the Earth’s surface (Blumthaler and Am-bach, 1990; Crutzen, 1992; Kerr and McElroy,1993; Lubin and Jensen, 1995). In addition to theAntarctic ozone hole where UV radiation hasbeen detected down to a depth of 70 m (Smith etal., 1992), ozone depletion has also been reportedin the north polar region (Hoffman and Deshler,1991). Biologically effective doses of UV-B radia-tion penetrate deep into the water column (Smithand Baker, 1979) and may thus affect the aquatic

* Corresponding author. Tel.: +49-9131-8528216; fax: +49-9131-8528215.

E-mail address: dphaeder@biologie.uni-erlangen.de (D.-P.Hader)

S0098-8472/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S0098 -8472 (99 )00043 -X

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ecosystems (Hader et al., 1995). The penetrationof UV-B strongly depends on the optical proper-ties of the water column. The depth of waterrequired to remove 90% of the solar radiation at310 nm varies from about 20 m in the clearestoceanic waters to a few centimeters in brownhumic lakes and rivers (Kirk, 1994). All plant,animal and microbial groups appear to be sus-ceptible to UV-B, but to a highly variable ex-tent. UV-B is a small (B1% of total energy) buthighly active component of the solar spectrumwhich has the potential to cause wide rangingeffects, including destruction of proteins, DNAand other biologically relevant molecules,chronic depression of key physiological pro-cesses, acute physiological stress, and conse-quently the productivity of ecosystems (Karentzet al., 1991a; Vincent and Roy, 1993; Bothwell etal., 1994; Williamson, 1995; Sinha and Hader,1996).

Certain organisms have developed mechanismscounteracting the damaging effects of UV-B. Be-sides repair of UV-induced damage of DNA byphotoreactivation and excision repair (Britt,1995; Kim and Sancar, 1995) and accumulationof carotenoids and detoxifying enzymes or radi-cal quenchers and antioxidants that provide pro-tection by scavenging harmful radicals or oxygenspecies (Mittler and Tel-Or, 1991; Middleton andTeramura, 1993), an important mechanism toprevent UV-B-induced photodamage is the syn-thesis of UV-absorbing compounds.

Phenylpropanoids, mainly flavonoid deriva-tives, located in the epidermis have been re-ported to protect higher plants by absorbingharmful UV radiation (Tevini et al., 1991; Koot-stra, 1994). Mycosporine-like amino acids(MAAs) and the cyanobacterial sheath pigment,scytonemin, are thought to accomplish a similarfunction in lower organisms (Garcia-Pichel andCastenholz, 1991; Karentz et al., 1991b; Garcia-Pichel et al., 1993; Ehling-Schulz et al., 1997;Xiong et al., 1997). MAAs are water solublesubstances characterized by a cyclohexenone orcyclohexenimine chromophore conjugated withthe nitrogen substituent of an amino acid or itsimino alcohol, having absorption maxima rang-ing from 310 to 360 nm (Nakamura et al., 1982).

MAAs have been identified in a number of taxo-nomically diverse organisms such as fungi (Fa-vre-Bonvin et al., 1976), marine heterotrophicbacteria (Arai et al., 1992), cyanobacteria (Gar-cia-Pichel et al., 1993; Karsten and Garcia-Pichel, 1996), eukaryotic algae (Carreto et al.,1990; Karentz et al., 1991a; Karsten et al., 1998),marine invertebrates (Karentz et al., 1991b;Shick et al., 1992), fish (Dunlap et al., 1989) anda wide variety of other marine organisms(Karentz et al., 1991a; Dunlap and Yamamoto,1995; McClintock and Karentz, 1997; Carefootet al., 1998; Dunlap and Shick, 1998).

Since macroalgae are sessile, they are restrictedto their growth site and simultaneously exposedto elevated levels of PAR and UV radiation intheir natural habitat. They can not avoid radia-tion stress by migration to less affected areaslike some microalgae. We tested the hypothesisthat macroalgal communities might have devel-oped mechanisms to counteract the damaging ef-fects of radiation stress. Further the hypothesisthat UV-B radiation at doses comparable tothose in natural radiation has a significant im-pact on pigmentation and protein of this organ-ism was tested.

2. Materials and methods

2.1. The organism

The test organism Gracilaria cornea J. Agardh(Rhodophyta) was brought from Gran Canaria,Spain, where it has been routinely cultivated forthe last 6–7 years. In nature, it normally growsfree or attached to small rocks and coral frag-ments at low tide levels. The algae is light red/rust colored, up to 30 cm tall and very rubbery.It does not have leaves and tends to divide byfragmentation. The organism was grown in ar-tificial sea-water (33 ppt; Instant Ocean, Sarre-bourg, France and Mentor, Ohio, USA)supplemented with nitrate (9.8 mM l−1) andphosphate (3.1 mM l−1) and illuminated withfluorescent light (1292 W m−2) at a tempera-ture of 2092°C for a 12 h photoperiod.

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2.2. Radiation source

The organisms were transferred to a transpar-ent container (20×20×8.5 cm) filled up to 3.5cm with artificial sea water and placed on a rotaryshaker to warrant uniform exposure. The algaewere irradiated simultaneously under artificial ra-diation of ultraviolet-B (UV-B; 280–315 nm), ul-traviolet-A (UV-A; 315–400 nm) and fluorescentlight (PAR; 400–700 nm), in the following re-ferred to as UV+PAR (Fig. 1). UV-C irradiationwas eliminated with 295 nm cut-off filters (Ultra-phan, Digefra, Munich, Germany). UV-B irradia-tion was provided by a Philips Ultraviolet-B TL40 W/12 (Holland) tube with its main output at312 nm. The irradiation was adjusted to 1.0 Wm−2. UV-A irradiation was provided by a UV-A-340 tube (Q-Panel, Cleveland, Ohio, USA) withits main output at 340 nm. The irradiation wasfixed at 1.0 W m−2. The source of visible lightwere OSRAM L 36 W/32 Lumilux de luxe warmwhite and Radium NL 36 W/26 Universal white(Germany) tubes, the irradiance of which was

fixed at 12 W m−2. When required, either a 395nm (Ultraphan, UV Opak, Digefra, Munich, Ger-many) or a 320 nm (Montagefolie No. 10155099,Folex, Dreieich, Munich, Germany) UV filter wasused to produce only the PAR and UV-A+PARwaveband, respectively. Alternatively, experimentswere carried out with a transilluminator (Ba-chofer, Reutlingen, Germany) producing exclu-sively UV-B and UV-A (no PAR), with its mainoutput at 312 nm (Fig. 1); the irradiance of thislight source was fixed at 2.0 W m−2 (referred toas UV). The irradiances of the light sources weremeasured with a double monochromator spectro-radiometer (OL 754, Optronic Laboratories, Or-lando, FL, USA).

2.3. Extraction and partial purification ofmycosporine-like amino acids (MAAs)

Aliquots of 0.5 g (fresh weight) of G. corneawere homogenized and extracted in 5 ml of 20%(v/v) aqueous methanol (HPLC grade) by incu-bating at 45°C for 2.5 h. After centrifugation at

Fig. 1. Spectral characteristics of the light source. 1, transilluminator (UV-B plus UV-A; lmax=312 nm, ca. 2.0 W m−2 for bothUV-B and UV-A) with 295 nm cut-off filter; 2, UV-B TL 40 tube (lmax=312 nm, ca. 1.0 W m−2) plus UV-A-340 tube (lmax=340nm, ca. 1.0 W m−2) plus OSRAM L 36 and Radium NL 36 fluorescent tubes (PAR; 400–700 nm, ca. 12 W m−2) with 295 nmcut-off filter; 3, same as in 2 with 320 nm cut-off filter.

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5000 g; the supernatant was lyophilized and redis-solved in 2 ml of 100% methanol, vortexed for2–3 min and centrifuged at 10 000×g for 10 min.Thereafter the supernatant was evaporated to dry-ness at 45°C and the extract redissolved in 2 ml of0.2% acetic acid. The samples were filteredthrough 0.2 mm pore-sized mikro-spin filters inorder to partially purify the MAAs. These par-tially purified MAAs were separated by HPLC.

2.4. Extraction of crude pigments

Fresh G. cornea (0.5 g) was homogenized with amortar and pestle in 5 ml of 100% methanol andkept overnight in a refrigerator at 4°C. The result-ing suspension was centrifuged, and the superna-tant was used for the in vitro UV irradiationexperiments.

2.5. High performance liquid chromatography(HPLC)

HPLC analysis of partially purified MAAs wasperformed with a HPLC (Merck Hitachi; Inter-face D-7000, UV-Detector L-7400, Pump L-7100,Darmstadt, Germany) equipped with a LiCro-spher RP 18 column and guard (5 mm packing;250×4 mm I.D.). The sample was injected with aHamilton syringe into the HPLC column througha Rheodyne injection valve equipped with a sam-ple loop. The wavelength for detection was 330nm at a flow rate of 1.0 ml min−1 and a mobilephase of 0.2% acetic acid. Identification of MAAswas done by comparing the absorption spectraand retention times of several standards such asDe6alearea ramentacea, Porphyra saldanhae,Bostrychia radicans and a supralitoral lichen.

2.6. UV and heat treatment

Purified MAAs or crude pigment extracts of G.cornea were transferred into a quartz cuvette (anoptical path length of 10 mm, 2 mm thickness)and irradiated under UV. Absorption and fluores-cence spectra of the samples were recorded atregular time intervals. For heat treatment, MAAswere transferred in a 2 ml Eppendorf tube, incu-bated in a water bath at 7592°C, and absorptionspectra were recorded at defined time intervals.

2.7. Absorption and fluorescence spectroscopy

Absorption spectra of samples were measuredat regular intervals in a single beam spectrophoto-meter (DU 70, Beckman, Palo Alto, USA). Theraw spectra were transferred to a microcomputerand treated mathematically and statistically usingthe software provided by the manufacturer. Fluo-rescence emission spectra were recorded simulta-neously with a spectrofluorometer (RF-5000,Shimadzu, Kyoto, Japan) at room temperature.

2.8. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE)

SDS-PAGE of G. cornea, irradiated with UV+PAR for up to four days was carried out in avertical system (2001, Pharmacia) with gels of155×130 mm, 1.5 mm thick, using the methoddescribed by Laemmli (1970), with a gradient(5–20% T) in the resolving gel. The electrophore-sis was run initially at 300 V and 30 mA for 1 h.The power was increased to 500 V and 60 mA assoon as the samples had run into the resolving gel.Gels were stained with Coomassie brilliant blue R250 and dried in a gel dryer (Bio-Rad). Sampleswere run along with standard SDS molecularweight (SDS-7; approx. mol. wt. ranging from14.2 to 66 kDa) markers (Sigma Technical Bul-letin, 1996). The protein concentration was deter-mined by the method of Bradford (1976). Bovineserum albumin was used as a standard.

2.9. Statistics

Results are expressed as the mean values ofthree replicates where appropriate and the statisti-cal significance of the means was tested withtwo-way ANOVA with a significance level ofPB0.05.

3. Results

Mycosporine-like amino acids extracted fromG. cornea were purified by HPLC and identifiedaccording to their retention times, absorptionspectra and cochromatography with standards.

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Fig. 2. High performance liquid chromatographic separation of the mycosporine-like amino acids (MAAs) from G. cornea.LiCrospher RP 18 column and guard; mobile phase 0.2% acetic acid; flow rate 1.0 ml min−1; detection by absorbance at 330 nm.1 and 2, mycosporines with a retention time of 2.6 and 4.9, respectively, spectra of which are presented in Fig. 3.

Fig. 3. Absorption spectra of the purified MAAs of G. cornea. 1 and 2, as separated by HPLC presented in Fig. 2.

nm (Fig. 3), respectively. were identified as por-phyra-334 and/or shinorine. There was no in vivo

Two MAAs with retention times of 2.6 and 4.9min (Fig. 2) and absorption peaks at 332 and 334

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induction/retardation of the MAAs in G. corneaeven after 4–5 days of irradiation with any of thelight sources either alone or in combination. Theeffect of heat was assayed by incubating MAAs ina water bath at 7592°C. Absorption spectrawere taken at regular intervals of 1 h. Absorptionproperties of the MAAs were not significantlydifferent (PB0.05) from that of the control evenafter 6 h of heat treatment (Fig. 4).

Absorption spectra of crude extracts of G.cornea showed 6 main peaks at 265 (unknowncompound), 334 (MAAs), 436 (chlorophyll a) 475(carotenoids), 618 (phycocyanin) and 665 nm(chlorophyll a) (Fig. 5). There was a significantdecrease (PB0.05) in the absorbance at all peaksexcept at 334 nm after only 1 h of UV irradiationand a further bleaching with increasing UV irradi-ation time. After 6 h of irradiation all peaks,except at 334 nm had more or less disappeared.There was no effect on the 334 nm (MAAs) peakeven after 6 h of UV irradiation. A significantdecrease (PB0.01) in the absorbance of MAAscould be recorded only after 24 h of continuousUV irradiation, but the complete elimination ofthe MAAs peak did not occur even after 72 h of

UV irradiation (Fig. 5).Fluorescence excitation at the 436 nm peak of

the crude extract of G. cornea resulted in anemission at 670 nm, which first showed an in-crease (PB0.05) in fluorescence and subsequentlya slight shift towards shorter wavelengths after 0.5h of UV irradiation (Fig. 6). A gradual increase(PB0.05) in fluorescence with a further shift to-wards shorter wavelengths continued up to 3 h ofUV irradiation. The fluorescence returned to itsinitial level but with an obvious shift (PB0.01)towards shorter wavelengths after 4 h of UVirradiation. Thereafter, there was a gradual andsteady decline (PB0.01) in fluorescence followedby a further shift towards shorter wavelengthswith increasing duration of UV irradiation (Fig.6). Fluorescence excitation at the 618 nm peak ofthe crude extract of G. cornea resulted in anemission at around 668 nm, which also firstshowed an increase (PB0.05) in fluorescence andsubsequently a slight shift towards shorter wave-lengths after 0.5 h of UV irradiation (Fig. 7).Thereafter, there was a gradual and steady declinein fluorescence (PB0.01) followed by a further

Fig. 4. Absorption spectra of the purified MAAs of G. cornea with increasing time to heat (7592°C) treatment.

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Fig. 5. Absorption spectra of the methanolic extract of G. cornea with increasing UV exposure time. Note the predominantmycosporine-like amino acids peak at 334 nm.

Fig. 6. Fluorescence emission spectra of the G. cornea following increasing exposure time to UV when excited at 436 nm.

shift towards shorter wavelengths with increasingUV irradiation time (Fig. 7).

Changes in the total protein profile of G. corneawere investigated by SDS-PAGE analysis follow

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Fig. 7. Fluorescence emission spectra of the G. cornea following increasing exposure time to UV when excited at 618 nm.

ing increasing irradiation times to UV+PAR(Fig. 8). There was a gradual decrease in theintensity of protein bands at 20 kDa (a and bsubunits of phycocyanin) with increasing irradia-tion of UV+PAR; this band was more or lesscompletely eliminated after 4 days of irradiation,but at the same time there was an increase in theintensity of protein bands at 26 kDa (phycoery-thrin) after the same duration of irradiation. Alsothe protein bands between 36 and 66 kDa initiallyshowed an increase in intensity till 3 days ofirradiation but thereafter remained constant.

4. Discussion

The data obtained in the present study supportthe hypothesis stated in the introduction andshowed the presence of UV-absorbing com-pounds, MAAs, in the test organism G. cornea,which are highly persistent to UV and heat stressand thus may play a vital role as a first line ofdefense against these environmental stress factorsin their natural habitats. Many organisms isolatedfrom marine, freshwater or terrestrial habitats

contain MAAs. A few of them have been iden-tified while most still have to be characterizedchemically. MAAs such as asterina-330, por-phyra-334, shinorine and mycosporine-gly are

Fig. 8. Vertical SDS-PAGE (gradient 5–20% T) protein profileof G. cornea following UV plus PAR irradiation for 4 days.Lanes 1 and 7: marker proteins (66.0, albumin bovine; 45.0,albumin egg; 36.0, glyceraldehyde-3 phosphate; 29.0, carbonicanhydrase; 24.0, trypsinogen; 20.1, trypsin inhibitor; 14.2,a-lactalbumin), lane 2: unirradiated control; lane 3: 1 day; lane4: 2 days; lane 5: 3 days and lane 6: 4 days of UV plus PARirradiation. Equal amount of proteins were loaded in eachwell.

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common in diverse type of organisms (Karentz etal., 1991b). It is evident from the present investi-gation that the organism possesses fairly highamounts of UV-absorbing MAAs even withoutUV induction. While in the present study we didnot attempt to chemically characterize the MAAsisolated from G. cornea, its spectral and othercharacteristics are fully comparable to those ofporphyra-334 and/or shinorine. The occurrence ofhigh concentrations of MAAs in marine organ-isms exposed to high levels of solar radiation hasbeen proposed to provide protection as a UV-ab-sorbing sunscreen (Dunlap and Yamamoto, 1995;Karsten et al., 1998), but there is no conclusiveevidence for the exclusive role of MAAs as sun-screen. It is possible that they play more than onerole in the cellular metabolism of all or someorganisms (Castenholz, 1997).

Studies with cyanobacteria have shown thatMAAs prevent 3 out of 10 photons from hittingcytoplasmic targets. Cells with high concentra-tions of MAAs are :25% more resistant to UVradiation centered at 320 nm than those with noor low concentrations (Garcia-Pichel et al., 1993).Our results indicate that MAAs in G. cornea arehighly stable against UV and heat stress. But theyfailed to provide complete protection to the pho-tosynthetic pigments, chlorophyll and the acces-sory light harvesting pigments such asphycocyanin, from UV induced photobleachingwhen the crude pigment extract containing highconcentrations of MAAs were subjected to UVirradiation. It is pertinent to mention that thebleaching of chlorophyll and carotenoids iscaused by photooxidation processes initiated byUV and visible light absorption of endogenouschromophores. The persistence of MAAs to oxi-dative processes as observed in the present studymay be due to the fact that only oxo-carbonylMAAs have antioxidant properties whereas theimino-MAAs (porphyra-334 and shinorine)present in G. cornea are oxidatively inert. TheMAAs in Nostoc commune have been shown to beextracellular and linked to oligosaccharides in thesheath (Bohm et al., 1995). These glycosylatedMAAs represent perhaps the only known exampleof MAAs that are actively excreted and accumu-lated extracellularly and therefore act as a true

screen (Ehling-Schulz et al., 1997). There may bephysiological limitations to the accumulation ofosmotically active compounds such as MAAswithin the cell, and it seems probable that themaximal specific content of MAAs in the cell isregulated by osmotic mechanisms which isreflected by the fact that field populations ofhalotolerant cyanobacteria contain unusually highconcentration of MAAs (Oren, 1997).

The results on changes in the absorption andfluorescence properties of chlorophyll and phyco-cyanin, following UV irradiation, are in accor-dance with earlier reports (Gerber and Hader,1995; Sinha et al., 1997). The initial increase in thefluorescence of chlorophyll following UV irradia-tion might be due to an accumulation of excita-tion energy in the antenna pigments caused by adecrease of PS II activity. The shift in the fluores-cence emission of chlorophyll seems to be due toa decrease in the fluorescence of chlorophyllmolecules which emit at longer wavelengths andan increase in the fluorescence of the substancessuch as the antenna pigments or pigments fromthe core complex which emit at shorter wave-lengths (Gerber and Hader, 1995). Similarly, UV-induced changes in the fluorescence properties ofphycocyanin followed by a shift towards shorterwavelengths are indicative of an impaired energytransfer from the phycobiliproteins to photosys-tems. The results indicate that pigmented proteinsare one of the main targets of UV (Sinha et al.,1997). The SDS-PAGE protein profile of G.cornea after UV+PAR irradiation shows a lossin the 20 kDa (phycocyanin) and simultaneouslyan increase in the 26 kDa (phycoerythrin)proteins. This could be attributed to a phe-nomenon known as chromatic adaptation(changes in pigmentation in photosynthetic organ-isms in response to light quality) by which anorganism tries to cope with changes in its lightenvironment (Tandeau de Marsac, 1977). Thechange in character of the light harvesting com-plex in response to the different wavelengths oflight allows the cells to use the incident lightefficiently (Grossman et al., 1994). The accessorylight harvesting pigments phycocyanin and phyco-erythrin may operate as a second line of defenseagainst photodamage and help the organism sur-

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vive in highly irradiated environments. Phycobil-isomes are designed to funnel radiant energy spe-cifically to PS II at wavelengths where Chl amolecules do not absorb, hence optimizing theenergy capture and the colonization of environ-ments with different light regimes (Tandeau deMarsac, 1977).

We conclude that the studied macroalga G.cornea has certain properties such as the presenceof UV-absorbing compound, MAAs, and the ca-pacity to vary its phycobiliprotein ratio that mayenable it to survive excessive irradiances in thenatural habitats. Irrespective of the questionwhether the UV-B protective properties of MAAsand phycobiliproteins are a novel function orwhether they are synthesized or accumulated dueto UV-B irradiation, the presence of these com-pounds in an organism may provide protection tothe internal organelles and components from thefull impact of incident UV-B radiation.

Acknowledgements

This work was financially supported by theC.S.I.R. (9/13(795)/96-EMR-I(RK)213108), NewDelhi, India to R.P. Sinha and by the EuropeanUnion (DGXII, Environment programme, ENV4-CT97-0580) to D.-P. Hader. We gratefully ac-knowledge Dr. Garcıa-Reina, Instituto AlgologiaAplicada, Gran Canaria, Spain, who provided theG. cornea strain and Dr. Ulf Karsten, Alfred-We-gener-Institut, Bremerhaven, Germany, forproviding MAAs standards. We thank M. Schus-ter for excellent technical assistance.

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