Life Science Edge

Where professionals become specialists LIFE SCIENCE EDGE

With great pride and pleasure, I take this opportunity to write a

brief “Introductory-note” for the “Life Science Edge” of

2010.

It is with immense interest that I read the entire contents of

this journal put- forth by the Staff and Students of Garden

City College, Bangalore, India.

The journal is well planned and formatted immaculately, with

apt titles for each article, appropriate tables and figures and

finally, relevant references-arranged in a systematic order.

In essence, the journal is divided into several broad sections;

namely, A) Review articles, B) Original research articles on 1)

Plants, 2) Microbes, 3) Insects, 4) Bio-informatics, 5) Data-

mining and 6) on many popular Human Diseases.

I am sure the “Reader”; not necessarily a Scientist; would find it

hard to put down this “PIECE OF ART”, which so beautifully

amalgamates the various forms of Life, that we so often take it

for granted !!!

“HAPPY READING”!!!

Dr. (Mrs) Parvathi CharyDirector R&D - Life Sciences

Garden City College, Bangalore, Karnataka, 560049. INDIA

EDITOR’S DESKPublisher & Patron-in-Chief

Chief Editor

Editorial Board Patrons

Editorial Board

Dr. Joseph V.G.Chairman, Garden City Group ofInstitutions

Dr. (Mrs) Parvathi Chary

Principal Garden City College

Principal Capital College

Vice - PrincipalGarden City College

Prof. Sunil BProf. Sheeja M.SProf. Reena PrathipaProf. V.N. ShettyProf. G.S. PrabhakaraProf. Syed Azhar Ahmed

Vol 1 | No 1 | October 2010

Chitra. G, M. Reena Prathipa, Sunil. BACCELERATED IN-VITRO DEGRADATION OF POLYCAPROLACTONE USING LIPASE ENZYME.

Dr. T. Bhaskar Reddy, Prof. B. Lakshma Reddy & Prof. Meenatchi Sundaram.TEXTUAL DATA MINING

Kshama Prithvi & Pushpa.N EXTRACTION AND ESTIMATION OF PROTEINS FROM DIFFERENT PULSES AND ANALYSIS

Sunil. B, M. Reena Prathipa ANTIMICROBIAL ACTIVITY OF LEAF EXTRACTS OF NYCTANTHES ARBORTRISTIS LINN. (OLEACEAE)

Sreeja BhaskarMICROFLORA OF SALTED DRIED FISH FROM SEA WATER

Mr. Prabhakara G.S, Ms. Proma Chakraborty, Ms. Karishma, Ms. VeenaTHE EFFECT OF HYPERTHYROID DRUG (METHIMAZOLE) ON DROSOPHILA WITH EXTRAPOLATION IN PREGNANT WOMEN ANTIMICROBIAL ACTIVITY OF LEAF EXTRACTS OF NYCTANTHES ARBORTRISTIS LINN. (OLEACEAE)

M. Reena Prathipa, Sunil. B & Chitra. G ANTIMICROBIAL ACTIVITY OF LEAF EXTRACTS OF NYCTANTHES ARBORTRISTIS LINN. (OLEACEAE)

Ms Rupal Gogia , Ms Sayantanee Basu Maitra, Ms Sheeja MSANTIMICROBIAL ACTIVITIES OF VARIOUS PLANT EXTRACTS ON DIFFERENT BACTERIAL SPP

Shruti Awasthi, Ganesh P. Surati, V. N. Shetty & P. CharyIN-VITRO STUDY OF CISSUS QUADRANGULARIS LEAF EXTRACT ON STAPHYLOCOCCUS SPS. ISOLATED FROM OLD BOOKS

Urmimala RayTHE EFFECTS O TIME, TEMPERATURE & IPTG CONCENTRATION ON IPTG INDUCED -GALACTOSIDASE ACTIVITY USING ONPG AS SUBSTRATE

B. Byju & P. George GiriPATENTABILITY OF BIOTECHNOLOGY: A CRITICAL ANALYSIS

Gandhimathi. SWASTEWATER TREATMENT: A BOON FOR WATER CRISIS

Rupa Das, Kirana ShekarTITLE- AYURVASTRA: HERBAL COUTURE FOR IMPROVING HEALTH

Nisha Talukdar“TREASURE TROVE OF SEA”- A KEY TO HUMAN CURSES

Shruti Awasthi , Parvathi Chary, Pooja AwasthiROLE OF NEUROTRANSMITTERSIN MITOCHONDRIAL DISEASE

M. Obulesu, R. Venu, R. SomashekharALZHEIMER’S DISEASE

CONTENTS

LIFE SCIENCE EDGE-03

Chitra.G *, M.Reena Prathipa & Sunil.BDepartment of Biotechnology,

Garden City College, Bangalore – 560049, Karnataka, Indiaemail: * [email protected], [email protected], [email protected]

Accelerated In-vitro Degradation Of Polycaprolactone using Lipase Enzyme.

AbstractBiomaterials are the materials designed for interfacing or interacting with a living system, inducing no adverse reaction at the site of implantation in-vivo or in-vitro and systematically. Biodegradable polymers have found increasing use in controlled release drug formulation, especially in protein and steroid drug delivery. Drug delivery system based on synthetic biodegradable polymers are made from Polycaprolactone (PCL), Poly lactic acid, Poly glycolic acid and their copolymers. These systems can be designed to provide either short term or long term release of bioactive agents to body cavities and capillaries. Lipase is an esterase enzyme having specificity towards carboxyl groups. PCL when reaction with lipase degrades into Caproic acid and unreacted low molecular weight PCL and this resembles in vivo degradation process which is very fast. This criteria is utilised for the correlation between in-vitro and in-vivo degradation of PCL.The liberated Caproic acid is titrated with an alkali of 0.01M using phenolphthalein as indicator. This reaction between Lipase and PCL is used as analytical tool for in-vitro degradation..

INTRODUCTION

Drug delivery systemIn the last few years several novel techniques and methodologies have been devised and evolved in the field of drug delivery. The selection of most appropriate route and delivery system for any bioactive agents depends on many factors such as therapeutic index of the drug molecules and the biological membranes across which the drug should be transported. The choice of the drug delivery system is also influenced by the disease

type of treatment (acute, chronic) and the target organ.

Implantable delivery systems:Implantable delivery systems in the form of hydrogels or pumps facilitate the delivery of drugs directly in to the blood stream without repeated administration or infusion. These system can be designed to provide either short term or long term release of bioactive agents to body cavities and capillaries.

The study prove the use of polymeric implants include poly acrylamide ,silicon rubber , ethylene vinyl acetate copolymers (EVAC) polyvinyl alcohol hydrogels, ethylene vinyl alcohol copolymer ,and poly hydroxyl ethyl methacrylate and poly vinyl pyrollidone, natural micro molecules such as collagen and its graft copolymers for the release of therapeutic agents are also used as Implantable drug delivery devices.

Among the various implantable delivery system being investigated, insulin delivery from implants in the most sorted one and various investigators have used different implantable system for this purpose .Another approach in insulin delivery includes implantable pumps for intravenous or intraperitoneal infusion of insulin solution which can deliver a base line dose of insulin from a reservoir that must be refilled percutaneously once a week.

Polycaprolactone :Polycaprolactone, PCL is a white semi crystalline , thermoplastic, linear aliphatic polyester which is synthesized by the ring opening polymerization of caprolactone and is produced commercially by Union Carbide and Rhonepoulene . This polymer has a melting

opoint of approximately-62 C. PCL is readily degraded


and mineralized by a variety of microorganisms . The degradation mechanism which has been proposed is hydrolysis of polymer to 6-OH Caproic acid an intermediate of omega oxidation and then beta oxidation to acetyl Sco A, which can then undergo further degradation in the TCA cycle .

oPolycaprolactone has ceiling temperature of 22 C . It is a biodegradable polymer obtained from petroleum derived products. It is finely soluble in Acetone, Ethyl acetate, Dichloromethane, Chloroform, DSMO,Toluene DMF .It is insoluble in Methanol, Isopropyl alcohol and in n-Hexane.Polymer Degradation is of increasing importance in the production and use of implantable and other medical devices.

Current research indicates the role of enzymes, peroxides, free radicals phagocytic cells and lipids in the degradation of polymer. But no pattern is clear and consequently there are number of procedures for predicting, measuring, preventing this degradation. Hetrochain polymers, particularly those containing oxygen and/or nitrogen atom in the main chain are generally susceptible to hydrolysis .

Many methods are available for the in-vitro degradation of PCL, the most accurate and reproducible method for in-vitro degradation of PCL by enzymatic degradation using Lipase enzyme. This is an accelerated in-vitro degradation method. The titrimetric method for determining Caproic acid was also proved to be more economical, less time consuming.

Lipase:Lipase enzyme catalysis the degradation of PCL to Caproic acid which resembles the in-vivo degradation process, but very fast. This criteria is utilized for the correlation between in-vitro and in-vitro degradation of PCL.Lipase is an enzyme (or more exactly a group of enzymes) belonging to the esterase. It hydrolyses the fat (present in ester form such as glycerides) yielding fatty acids and glycerol and catalyze digestion.Lipase is an esterase that cleaves large molecular substrate i.e., triglycerides.

Lipase has low molecular weight hence removed from circulation by glomerular filtration but it is completely reabsorbed in proximal convoluted tubule.Lipase are widely distributed in the plant world, also in molds, bacteria, milk and milk products and in animal tissues, especially in the pancreas.

The optimum temperature for activity of lipase is o

between 35-37 C and at pH 5-6 Lipase contain sulfhydryl groups and is inactivated by substrate that keeps SH groups in the reduced state such as glutathione, cysteine and ascorbic acid. The lipase is activated by sulphuric, oxalic, formic, acetic and butyric acids. Caprylic and Caproic acids increase the action of lipase derived from certain mold fungi. Almost all organic solvents decrease lipase activity, petroleum ether being an exception.

MATERIALS AND METHODS:-

Methods of determination of lipase activity:-1. Titrimetric2. Turbidimetric3. Fluorimetric4. Polarographic5. Immunological methodsOf the above methods Titrimetric method of Yamuda et al. is preferred to carry out the study. In this method “one unit of lipase activity is defined as 1micro equivalent of free fatty acid released per minute under standard assay condition”.Lipase is an esterase enzyme having specificity towards carboxyl groups. PCL is a poly ortho ester biodegradable polymers. Upon action with lipase, PCL degrades into Caproic acid and unreacted low molecular weight PCL. Liberated Caproic acid is titrated with sodium hydroxide (0.01 M) using phenolphthalein as indicator. The reaction between Lipase and PCL is used as analytical tool for in-vitro degradation.

Titrimetric procedure:-5ml of PCL/ PVA (homogenized) substrate emulsion was taken and to this 4ml of 0.1 M sodium potassium buffer was added with pH 7.0 and were pre incubated for 1 hr. 1 ml of the enzyme was added and incubated at

o37 C for 20 min. The reaction was terminated by the addition of 20 ml of acetone and titrated against 0.01 M NaOH. A control was carried out similarly except that 1 ml of the heat inactivated enzyme was added to the reaction mixture. The end point is the appearance of pale permanent pink colour.

Table:1 Estimation of Caproic acid.


Sl.No.

Sample Time in hrs

Contents ofconical flask in ml

Blank in ml

Burete Reading in ml

Initial Final

Volume of NaOH

in mlIndicator

Amount of Caproic Acid in

5 ml sample

1 05 ml of PCL in PVA without

enzyme & 20ml of acetone

5 0 5Pheno -

phthalein 0000

2 15 ml of PCL in PVA with 1 ml


0 0 6.2Pheno -

phthalein 0.0004981.2



0 0 7.3Pheno -




0 0 8.5Pheno -




0 0 9.2Pheno -




0 0 10.5Pheno -




0 0 11.5Pheno -




0 0 12.0Pheno -




0 0 13.2Pheno -




0 0 14.3Pheno -




0 0 15.2Pheno -




0 0 16.4Pheno -




0 0 17.2Pheno -



0.006

0.0058

0.0056

0.0054

0.0052

0.005

0.0048

0.0046

0.0044

0.0042

0.004

0.0038

0.0036

0.0034

0.0032

0.003

0.0028

0.0026

0.0024

0.0022

0.002

0.0018

0.0016

0.0014

0.0012

0.001

0.0008

0.0006

0.0004

0.0002

Am

ou

nt o

f C

ap

roic

aci

d in

mo

ls / m

in / 5

ml

0

1 2 3 4 5 6 7 8 9 10 11 12

Amount of Caproic acid

Fig:1 Chart for degradation of PCL.

RESULT AND DISCUSSION:-

Result:-From the above experiment it is evident that the in-vitro degradation of Polycaprolactone under goes first order rate kinetics. It is an useful analytical tool for determining accelerated in-vitro degradation process of materials


containing polyorthoester.

Discussions:-Biomaterials are the materials designed for interfacing or interacting with a living system, which does not give any adverse reaction at the site of implantation in-vivo or in-vitro. A Biopolymer must have High purity, easy fabricability, High stability and Sterilizability. Biodegradable polymers has increasing use in controlled release drug formulation. Numerous polymers have been used in the design of drug delivery systems. Drug delivery system based on syntetic biodegradable polymers are made from Polycaprolactone, Polylactic acid, Polyglycollic acid and their copolymers.

The main constraint in the use of non-degradable polymeric implants is the need for surgical retrieval after their usage. The use of biodegradable or partially degradable polymers as implants obviate the need for surgical removal of the drug depleted device .Further the release rate of drug can be controlled by composition of the polymer matrices, biodegradation of the polymer and the pH of in situ medium.

Poly caprolactone is a white semi crystalline, thermoplastic, linear aliphatic polyester. This polymer

ohas m.pt of approximate 62 C and has a ceiling o

temperature of 220 C. It is a biodegradable polymer obtained from petroleum derived products.

Many methods are available for the in-vitro degradation of PCL ,the most accurate &reproducible method for in-vitro degradation of PCL is by enzymatic degradation using lipase enzyme. Lipase enzyme catalyze the degradation of PCL to Caproic acid. Lipase degrades PCL into Caproic acid & the liberated Caproic acid is titrated with 0.01M NaOH using phenolphthalein as indicator .

From the tabular column it is clear that the enzyme with PCL consume more alkali when the sample time is increased, It means the Caproic acid is released from

PCL because of action of lipase .When the sample time (in hrs.) is increased the liberation of Caproic acid is also increased, Thus it is evident that in-vitro degradation of PCL undergoes I order rate kinetic.

REFERENCES:-1. Polymeric Delivery System for the control release of

biologically active molecules. R. Narayani and K. Panduranga Rao.Trends in Bio materials and artificial organs.Vol. 12 No.1 Mar 1998 (7-18)

2. Shantha K.L and Panduranga Rao .K, J. Polym. Mater. 1994, 1, 157.

3. Davis B. K. Proc. Natl. Acad. Sci, 1974, 71, 3120.

4. Oda. Y. Kamijyo. Y. Okumupura. T. Neurol. Sur. 1985, 13, 1305.

5. Frederic J. L. Shimanuki. T and Dizerega. G. S. Suenie. 1984, 224, 389.

6. Hirai. T., Maniyama. H., Suzuki. T. and Hayashi. S, J. Appl. Polm. Sci. 1992, 46, 49.

7. Miyazaki. S., Yokouchi. C. and Takada. M, Chem Pharm. Bull. 1988. 36 (9), 3689.

8. Kim. S. W., Bae. Y. H. and Okano T. Pharm. Res. 1992, 9 (3), 283.

9. Shalaby. W. S. W and Park. K. Pharm. Res. 1990, 7 (8), 816.

10. Langer. R. Trans. Am. Soc. Artif. Intrn Organs. 1982. 34, 945.

11. Hjorth. S., Dillon. D., Mason. D. and Tice. T. Soc. Neuro. Sci. Abstr. 1988, 14, 764.

12. Hsiue. G. H., Yang. J. M and Wee. R. L, J. Biomed. Mater. Res. 1988, 22, 405.

13. Armoly. M. F. and Rao. K. R. Invest. Ophthal 1973, 12, 491.

14. Powers. K. G. J. Parasitology 1965, 51, 53.


TEXTUAL DATA MINING

Dr. T.BHASKAR REDDYAssociate ProfessorSri Krishna Devaraya UniversityAnantapur-515 003 .A.P .Indiaemail: [email protected]

Prof. B. LAKSHMA REDDYDirector – MCA

Garden City College Bangalore-49, Karnataka .India

email: [email protected]

Prof. MEENATCHI SUNDARAM.HOD - Computer Science

Garden City College, Bangalore-49, Karnataka .Indiaemail: [email protected]

Keywords: Online Analysis and Processing (OLAP), Knowledge Discovery in Databases (KDD), Textual Data Mining (TDM)

1 IntroductionText-mining refers generally to the process of extracting interesting and non-trivial information and knowledge from unstructured text. Text mining encompasses several computer science disciplines with a strong orientation towards artificial intelligence in general, including but not limited to pattern recognition, neural networks, natural language processing, information retrieval and machine learning. An important difference with search is that search requires a user to know what he or she is looking for while text mining attempts to discover information in a pattern that is not known beforehand.

Text mining is particularly interesting in areas where users have to discover new information. This is the case, for example, in criminal investigations, legal discovery and due diligence investigations. Such investigations require 100% recall, i.e., users can not afford to miss any relevant information. In contrast, a user searching the internet for background information using a standard search engine simply requires any information (as oppose to all information) as long as it is reliable. In a due diligence, a lawyer certainly wants to find all possible liabilities and is not interested in finding only the obvious ones.

In addition, caution needs to be taken when these techniques are applied in international cases. Many classic techniques developed in the English language

world; do not always work as well on other languages.

Increasing recall almost certainly will decrease precision implicating that users have to browse large collections of documents that that may or may not be relevant. Standard approaches use language technology to increase precision but when text collections are not in one language, are not domain specific and or contain variable size and type documents either these methods fail or are so sophisticated that the user does not comprehend what is happening and loses control. A different approach is to combine standard relevance ranking with adaptive filtering and interactive visualization that is based on features (i.e. meta-data elements) that have been extracted earlier.

Information retrieval systems that allow users to search large text collections using explicit word-based and phrase-based queries are now widely deployed, and in the hands of a skilled user they can be used to gain access to information from this broad array of sources. But traditional information retrieval systems treat documents as individual entities, placing the burden of recognizing relationships within and across documents on the user. Database systems began in a similar way, but the recent emergence of effective data mining techniques has expanded the range of ways in which users can interact with the data. Data mining essentially provides pattern-based retrieval, in which a pattern in the data is first discovered, and then that pattern is used to present information (the pattern itself or outlier data, perhaps) to the user. Online Analysis and Processing (OLAP) tools that can support interactive visualization of data mining results are becoming more common, and increasingly sophisticated visualization tools are


becoming available as well.

2. Textual Data Mining ArchitectureThe term Data Mining generally refers to a process by which accurate and previously unknown information can be extracted from large volumes of data in a form that can be understood, acted upon, and used for improving decision processes. Data Mining is most often associated with the broader process of Knowledge Discovery in Databases (KDD), “the nontrivial process of identifying valid, novel, potentially useful, and ultimately understandable patterns in data”. By analogy, this paper defines Textual Data Mining as the process of acquiring valid, potentially useful and ultimately understandable knowledge from large text collections.

The distinction between text and data is an important one. Data are numeric or categorical values that acquire their meaning from a fully specified model (or schema) that relates the data to physical reality. Text, by contrast, is expressed in natural language, a format that can (at least in principle) convey any meaning.

The distinction between data and text has important consequences for the formulation of TDM. Fayyad, et al. were interested in a KDD process that was nontrivial, meaning that they wished to find an expression in some language describing some subset of the data rather than simply retrieving that subset of the data as records in their original form. Texts are themselves linguistic expressions, and if the desired information were contained in a single text it would be entirely reasonable to simply find that text. The requirement that the patterns be novel can be omitted from the definition of TDM for the same reason.

Fayyad, et al. described a simplified five-step process KDD model that provided some degree of structure to the analysis of a broad range of KDD approaches. That process model included data selection, data preprocessing, data transformation, data mining, and data interpretation/evaluation. Figure 1 presents a similarly broad model for a six-step TDM process in which three main functions are performed:

• Data Collection, including Source Selection and Text Selection, which corresponds to Fayyad, et al.’s data selection step.

• Data Warehousing, including Metadata Assignment and Data Storage, which corresponds to Fayyad, et al.’s preprocessing step.

• Data Exploitation, including Data Mining and Data Presentation, which corresponds to Fayyad, et al.’s last three steps.

Figure 1. The textual data mining architecture.

• Source Selection is process of selecting sources to exploit. Source selection requires awareness of the available sources, domain knowledge, and an understanding of the goals and objectives of the data mining effort.

• Text Selection is the process of discovering, selecting, and obtaining individual texts from the selected sources. This process might be fully automated or it may be performed interactively by a domain expert.

• Metadata Assignment is the process of associating specific data with individual texts that is formatted in the manner required for data storage and data mining. This corresponds to the data preprocessing step in KDD.

• Data storage is the process of providing storage of and access to data. Data models that specify known relationships in the data can be stored with the data to facilitate subsequent processing.

• Data Mining is the process of fitting models to data. The data transformation function that Fayyad, et al. identify is subsumed in this step for presentation purposes.

• Presentation is the process of explaining and visualizing data mining results to support evaluation of data quality, assessment of whether the selected model is appropriate, and interpretation of the model.

Each step (except, perhaps, data storage) offers significant potential for user interaction, and some (e.g., source selection) might be done entirely manually. The remainder of this section gives an overview of the fundamental issues in each step of the TDM architecture.

2.1. Source SelectionThere are myriad text sources that could be of use for


textual data mining. Source selection has traditionally been performed manually based on a number of criteria, such as:

• Coverage. In order for TDM to be useful, the collection must contain texts that provide the needed information. Many TDM applications are subject-specific, so topical coverage is typically a key source selection criterion. Other criteria that may be important in specific applications are temporal coverage (e.g., earliest date, recency) and geographic coverage (e.g., place of publication).

• Availability. The rapid expansion of the Internet has made it practical to assemble collections that would previously have been impractical. For example, Los Alamos National Laboratories maintains freely accessible pre-print archives in Physics, Nonlinear Sciences, and Computer Science. At the same time, ubiquitous networking is simplifying the delivery of materials from commercially available text collections such as those provided by Dialog and Lexis/ Nexis. Access to legacy documents that exist only in hardcopy form is somewhat more problematic, since the cost of retrospective conversion to an appropriate digital format can be prohibitive in many applications.

• Cost. Although the Internet offers access to a wealth of freely available information, commercial interests often own the copyright on the specialized academic and trade journal texts that can be particularly useful for TDM. Many of these specialized text collections are already available in electronic form at special libraries and information centers. Site licensing arrangements that allow desktop access anywhere in an organization are becoming increasingly common, using either subscription or per-access pricing models.

• Format. Until recently the most widely available text collections included only abstracts, but the availability of full text is now becoming common as documents are captured in electronic form at the time they are generated. The brevity and uniform length of typical abstracts facilitates information extraction by limiting the potential for ambiguity, but full text collections provide far greater scope for the application of advanced information extraction algorithms. Some full text collections also contain potentially useful structural markup that can help guide information extraction by unambiguously

indicating the extent of textual units such as paragraphs, tables, and lists. Common examples of structural markup include Standard Generalized Markup Language (SGML) and derivative formats such as Hypertext Markup Language (HTML) and Extensible Markup Language (XML),

• Metadata. Some text collections, most often those available for a fee from commercial sources, include extensive metadata that can be used directly in TDM applications. For example, the journal articles indexed in Science Citation Index include unambiguous citation links that can be used directly for co-citation analysis, and author fields constructed using name authority control can be used directly for co-authorship analysis. Similarly, use of a controlled vocabulary for subject indexing can facilitate topical clustering.

2.2. Feature ExtractionThe next two steps, text selection and metadata assignment, can include automatic processing that exploits features extracted from each document. There are five fundamental sources of evidence about meaning in texts: orthographic, semantic, statistical, syntactic, usage analysis and multiple sources can be combined to produce stronger evidence than could be obtained from a single source.

• Orthographic Analysis. The most basic form of analysis is the recognition of units of meaning in texts that are made up of character codes. In English, it is common to separate the text at white space (spaces, tabs, line breaks, etc.) and to then treat the resulting tokens as words. For languages that lack reliable orthographic evidence of word boundaries, one common approach is to use a sliding window to form overlapping n-character sequences that are known as "character n-grams" or "n-graphs".

• Semantic Analysis. The goal of semantic analysis is to relate the surface form of a text to a representation of the meaning that it expresses. Different words might be used to represent quite similar concepts, and morphological analysis can help deal with that by conflating several variants into a single root form. In some languages (e.g., English), a similar effect can be achieved through automatic suffix removal, a technique known as stemming. Another common semantic analysis


approach is to exploit relationships among words (e.g., broader and narrower terms) that are encoded in a lexical knowledge structure such as a domain-specific thesaurus or a more general ontology such as WordNet. Simple knowledge structures such as term lists can also be used to recognize multi-word expressions such as idiomatic expressions that are not well handled by compositional techniques.

• Statistical Analysis. Statistical analysis of term usage frequency has proven to be quite useful as a source of evidence about meaning. The simplest approaches are based on counting the number of occurrences of each term (word, word root, word stem, n-gram, phrase, etc.). In collections with broad subject coverage, for example, it is common to find that the same phrase used in different contexts will represent different concepts. Statistical analysis of phrase co-occurrence can help to resolve this word sense ambiguity.

• Syntactic Analysis. Parsers with sufficient coverage and speed to process moderately large text collections are becoming available, and their output can provide several types of evidence about meaning. Part-of-speech analysis, for example, can help to disambiguate word sense, and syntactically identified phrases can provide a basis for further statistical analysis. Syntactic analysis can also be combined with semantic and lexical analysis to identify appropriate word boundaries in un-segmented languages such as Chinese and freely compounding languages such as German.

• Usage Analysis. The way in which a document is used can provide valuable cues about the document itself. For example, if the vast majority of the links to a document on the World Wide Web are from pages that mention known chemical compounds, it would be reasonable to infer that the document might be of interest to chemists. Oard and Kim (1998) identified four categories of potentially observable user behavior that could provide a basis for usage analysis: examination, retention, reference, and assessment.

2.3. Text SelectionManual assignment of metadata is expensive, and automatic techniques for assigning metadata are, at least at present, computationally demanding. It can thus be beneficial to pre-select texts that are likely to be of value

as an efficiency measure when dealing with large text collections. Some automated assistance with this process is typically needed, but integrating human judgment is also important because broad-coverage techniques that are optimized for automated processing of very large collections can not yet approach human performance on this task. Systems designed for this purpose are commonly referred to as information retrieval or, more specifically, text retrieval systems.

Modern text retrieval systems principally rely on orthographic, semantic, and statistical analysis. The usual approach is to use white space to identify word boundaries, followed by stemming to conflate words with similar surface forms into a common term. A weight is then computed for each term in every document using the frequency of the term in the document, the selectivity of the term (the fraction of the collection in which the term is found), and the length of the document. In vector space text retrieval, queries are represented in a manner similar to the documents, and the similarity of each document in the collection to the query is then computed as the normalized inner product of the document and query term weight vectors. In probabilistic text retrieval, a term weight is treated as the probability of relevance of a document to a query, conditioned on the presence of that term in the query. Probabilistic and vector space techniques are often combined with Boolean text retrieval, in which the presence or absence of a term or combination of terms can be explicitly required in the query specification. The principal advantage of vector space and probabilistic text retrieval over a purely Boolean approach is that lists of documents that are ranked in order of decreasing probability of relevance (or decreasing similarity to the query) allow users to interactively decide how many documents are worth examining. Unranked Boolean techniques, on the other hand, might be preferred when no user interaction is possible before the next processing stage. In either case, when the document collection is relatively stable it is common to preprocess the collection to produce an index structure on the feature set that can be searched in sub-linear time.

The utility of a text retrieval system depends strongly on how well the query is constructed, and that depends in turn on how well the user understands the collection and the way in which the indexed features can be used to select documents. It is usually fairly straightforward to find some topically relevant documents, but interactive inspection by the user is generally needed if the relevant


documents must be more carefully separated from the irrelevant ones. An iterative query reformulation process such as Simulated Nucleation [Kostoff, 1997] can be used to speed this process, leveraging inspection of a few documents to produce a query that better separates relevant and irrelevant documents.

2.4. Metadata AssignmentThe association of metadata with individual texts in large collections such as those provided by indexing and abstracting services and libraries has traditionally been a manual process, although interactive machine-aided indexing techniques are becoming more popular. TDM systems that seek to exploit multiple collections that contain manually assigned metadata face three challenges: differing content, differing formats, and differing vocabularies. Metadata content varies widely across collections, with each provider seeking to find a balance between the anticipated needs of their searchers and the expense of providing the metadata. For example, authors’ names are often present in every collection, but organizations with which the authors are associated are less commonly provided. The recent effort to define a standard set of metadata that is known as the Dublin Core should simplify the design of TDM systems that exploit the standardized content, but metadata outside the Dublin Core framework will continue to require collection-specific processing. Some progress has already been made with standardizing metadata formats, but even widely accepted standards such as the Machine Readable Catalog (MARC) records used for library catalogs have national variants. The Resource Description Format (RDF) and the Extensible Markup Language (XML) together provide a standardized metadata format that is compatible with the Dublin Core, and TDM applications will benefit as these standards are more widely deployed.

Even when content and format are standardized, incompatible metadata values pose a challenge for TDM systems. Authority control is used to standardize the representation within a single system for person, place, organization, journal name, subject, and other items for which people might otherwise use different values to represent the same entity. There have been some large-scale efforts to merge the thesauri used to establish subject authority in different systems, and fully automated techniques appear to offer some promise for this task [Gey, et al., 1999].

Many collections of gray literature lack reliable metadata, and the manual creation of metadata in such circumstances might be uneconomical. Automated information extraction algorithms offer a practical alternative in such cases. Information extraction can exploit deeper linguistic processing than the simple term frequency information used in text retrieval because information extraction can be applied to smaller and more coherent document sets. The usual approach is to exploit orthographic and syntactic analysis, typically using extraction rules that fire in two stages. The first step is to recognize the presence of a trigger word that selects a set of candidate syntactic patterns in which that word might occur. The syntactic context of the trigger word is then examined to identify the data to be extracted. These rules can be manually coded or they can be automatically learned from hand-labeled training texts. Recent work on the application of unsupervised learning and statistical analysis to information extraction offers some promise that the requirement for hand-labeled training data can be relaxed somewhat.

Information extraction algorithms can be designed to operate without user interaction, making sharp decisions rather than producing ranked lists of candidates. Results from the Message Understanding Conferences (MUC) suggest that simple tasks such as date extraction and named entity recognition can be done quite accurately, but that more complex tasks in which relationships among data items must also be extracted are still somewhat error-prone. Lawrence, et al. have used information extraction techniques to extract bibliographic citations from gray literature on the World Wide Web [Lawrence et al., 1999].

The raw features exploited by text retrieval and information extraction algorithms offer a third source of metadata for TDM applications. For example, the similarity measure used in vector space text retrieval provides a natural basis for data mining techniques such as clustering.

2.5. Data StorageThe role of the data storage step is to accumulate the metadata, to record and manage information regarding the organization of that data, and to provide access to the metadata that is needed by the data mining step. Data mining applications often exhibit access patterns that differ markedly from those commonly experienced in other database applications. Existing relational and


object oriented database systems may be adequate for relatively small-scale TDM applications, but careful attention to optimization may be required as the collection size grows. When multiple text collections are used in a TDM application, the metadata can be stored on a single machine or federated database architecture in which collection-specific metadata are stored with each collection can be used. Mixing unstructured features such as vector representations of entire documents with more highly structured metadata such as subject terms drawn from a thesaurus is possible, but could pose additional implementation challenges.

2.6. Data MiningThe data mining step includes model selection, transformation of the data into a format appropriate for the selected model, and choice of a method for finding the appropriate parameters for that model. At a very general level, data mining models can be grouped by what they seek to produce (their functions). Functions traditionally associated with data mining include clustering models (producing categories), classification models (producing assignments to predefined categories), and dependency models (producing relationships between data elements). Viewed abstractly, a model is simply a parameterized set of ways in which the data can be used to produce a result, a criterion that describes which results are preferred, and an algorithm for searching the space of possible combinations. The output of the data mining step is the model and the parameters that have been found.

As an illustrative example, consider a TDM system that is designed to identify topically related news stories. A clustering model would be well suited to this task, since clusters could be used to represent topical relationships. If the data storage system contains the frequency of each word appearing in the title, a simple data transformation could reduce this to a Boolean value reflecting the presence or absence of each word. The parameters of a clustering model are the number of categories and the category to which each text is assigned. One simple preference criterion is to assign two texts to the same category if their titles share at least half the words in the shorter title. In this case, a greedy agglomerative clustering algorithm that sequentially makes pair-wise comparisons and either forms or joins clusters will quickly discover the optimal parameters for the model. Better clustering models can (hopefully!) be designed for this task, but the key parts of the data mining step are

illustrated by this example.

2.7. PresentationThe presentation step generally seeks to serve three broad goals: helping the user understand the results, helping the user assess whether the chosen model was appropriate, and helping the user assess whether the quality of the data is adequate to support the desired analysis. It is often useful to provide a range of display capabilities and then allow users to select those that best match their goals and preferences. Presentation involves two sub-processes: summarization and display. Summarization is the process of producing abstract representations that emphasize some features of the data and suppress others. For example, the number of news stories assigned to a topical category might be included in a summary, but the title of each story might be omitted. Display is the process of representing the summary in a form that is appropriate for human perception. Graphical displays are often used because they capitalize on human visual perceptual abilities such as focus and pattern recognition, but other sensory channels (e.g., auditory display) can be useful in some cases. Research on information visualization has produced some tools that can exploit both metadata and text similarity. In TDM applications, those two sources can be augmented with the model parameters produced in the data mining step to produce richer visualizations (Chen et al, 1998).

3. Data Mining TechniquesThe TDM architecture shown in Figure 1 follows the traditional KDD architecture closely, first obtaining a large collection of numeric and/or categorical data and then fitting models to that data. A wide range of data mining techniques has been developed for use with numeric or categorical data. This section surveys some of the most widely used techniques. The classification system used is an augmented compendium of those proposed. Textual Data Mining employs variations of many of these methods, including clustering, classification, and dependency modeling.

3.1. Clustering and Classification MethodsClustering seeks to identify a finite set of abstract categories that describe the data by determining natural affinities in the data set based upon a pre-defined distance or similarity measure. Clustering can employ


categories of different types (e.g., a flat partition, a hierarchy of increasingly fine-grained partitions, or a set of possibly overlapping clusters). Clustering can proceed by agglomeration, where instances are initially merged to form small clusters and small clusters are merged to form larger ones; or by successive division of larger clusters into smaller ones. Some clustering algorithms produce explicit cluster descriptions; others produce only implicit descriptions. Classification methods, by contrast, are used to assign data to predefined categories. A variety of techniques are available (e.g., decision trees, naïve Bayesian classifiers, and nearest neighbor classifiers).

3.1.1. Nearest Neighbor MethodsNearest Neighbor algorithms support clustering and classification by matching cases internally to each other or to an exemplar specified by a domain expert. A simple example of a nearest neighbor method would be as follows: given a set X = {x1 x2 x3… xn} of vectors composed of n features with binary values, for each pair (xi, xj), xi? xj,, create a vector¬ vi of length n by comparing the values of each corresponding feature ni of each pair (xi, xj), entering a 1 for each ni feature with matching values match and 0 otherwise. Then sum the vi values to compute the degree of match. Those pairs (xi, xj) with the largest result are the nearest neighbors. In more complexes nearest neighbor methods, features can be weighted to reflect degree of importance. Domain expertise is needed to select salient features, compute weights for those features, and select a distance or similarity measure. Nearest neighbor approaches have been used for text classification [Cost, S. et al, 1993].

3.1.2. Relational Learning ModelsRelational learning models are inductive logic programming applications. Their foundation is logic programming using Horn clauses, a restricted form of first-order predicate logic. Logic programming describes relations on objects using declarative subject-predicate representations and uses classical deductive logic to draw conclusions. Data mining has been conducted by using inductive logic programming to generate database queries with predicate logic query syntax.

3.1.3. Genetic Algorithms Genetic algorithms can be used both for classification and for discovery of decision rules. Named for their Darwinist methodology, genetic algorithms use

processing that is analogous to DNA recombination. A population of "individuals," each representing a possible solution to a problem, is initially created at random or drawn randomly from a larger population. Pairs of individuals combine to produce “offspring” for the next generation, and mutation processes are used to randomly modify the genetic structure of some members of each new generation.

Genetic algorithms perform categorization using supervised learning, training with a set of data and then using the known correct answers to guide the evolution of the algorithm using techniques akin to natural selection. Genetic methods have advantages over neural networks, because they provide more insight into the decision process. Sheth [1994] describes an example of the use of a genetic algorithm for text processing.

3.2. Dependency ModelsLike clustering and classification, dependency models can be divided into two classes based on the nature of the dependencies being modeled. Sequential dependency modeling seeks to analyze temporal information encoded in the data to detect patterns of variation over time. This can be useful, for example, for performing citation analysis. Static dependency modeling, by contrast, seeks to explore relationships without regard to temporal order.

Dependency models can also be viewed from the perspective of the nature of the models that are considered. In structural dependency modeling, the goal is to discover unknown dependencies that exist among the data. These affinities can be expressed by qualitative rules such as “A causes B,” or by quantitative summaries such as "80% of the records that contain A and B also contain item C.” Quantitative dependency modeling, by contrast, assumes a specific model and seeks only to estimate of the parameters of that dependency relationship. Regression is an example of a quantitative dependency modeling technique.

3.2.1. Graph-theoretic Link Analysis.Many dependency methods employ graph-theoretic approaches, structuring data into representations of causal links or influence diagrams to support sequential or static dependency analysis. The specific formalism adopted and the link and node semantics can vary with the domain, so effective design of the graph can require domain knowledge. Graph-theoretic models are


generally easily visualized as node and link dependency structures.

Bayesian nets are probabilistic graphs that represent causal events as nodes and evidential dependencies as links. The evidence for belief in a leaf node accrues though the system of evidence links, based on the prior probabilities of the root nodes. The most tractable Bayesian networks are those that are instantiated in a Directed Acyclic Graph (DAG). The edges of the graph correspond to the causal or correlation relations that hold between the declarative evidence instantiated in the nodes. Associated with each node is a value corresponding to a belief or a probability whose value lies within the interval [0, 1]. A Bayesian net is evaluated by computing the conditional values of the network nodes based upon inputs applied to the root nodes that have no incoming edges. Formally, for every node n, there is associated evidence e. P (e) is either given by independent prior (root) probabilities, or derived via Bays' Rule. Bayes' Rule provides a tractable method for modeling the strengths of one’s beliefs that an x is a y, given one’s knowledge that x is also a z, but the requirement to estimate prior probabilities could limit the utility of Bayesian networks in TDM applications. To paraphrase Charniak, the numbers have to come from somewhere, and generally that reduces to informed guesses on the part of subject matter experts.

Freeman [1997] describes an interesting case of knowledge discovery within graph-theoretic social network visualization. By rotating the network image around its center with a Virtual Reality Markup Language (VRML) viewer, three-dimensional clustering could be explored. After rotation, a dependency was discovered that would not have been revealed without rotation.

3.2.2. Linear Regression and Decision TreesLinear regression (or correlation) methods are used to determine the relationships between variables to support classification, association and clustering. Variations include univariate and multivariate regression.

One common use of linear regression is to support generation of a decision tree. Decision trees are typically induced using a recursive algorithm that exhaustively partitions the data starting from an initial state in which all training instances are in a single partition, represented by the root node, and progressively creates sub-

partitions that are represented by internal or leaf nodes. At each non-terminal node, the algorithm branches on values of the attribute that best discriminates between the remaining cases. Each node will correspond to a rule characterizing some explicit property of the data, so generation of a decision tree is a restricted form of rule induction in which the resulting rules are mutually exclusive and exhaustive. Decision tree induction is fairly straightforward, but the results will only be useful if the available features provide sufficient basis meaningful categorization. To reduce computational complexity, heuristics are often applied to the selection of linear properties that implicitly omit from consideration the vast majority of potential rules. “This approach may leave valuable rules undiscovered since decisions made early in the process will preclude some good rules from being discovered later”. Rule extraction from decision trees can be used in data mining to support hypothesis validation.

3.2.3. Nonlinear Regression and Neural NetworksNonlinear Regression algorithms are used to support classification, association and clustering. Domains whose properties are not well suited to linear partitioning can sometimes exploit non-linear algorithms such as feed-forward neural nets, adaptive spline methods, and projection pursuit regression.

Neural networks determine implicit rules where the classes invoked are not defined classically (e.g., not conforming to the principle of excluded middle), or are poorly understood and hence are not amenable to linear classification [McCulloch and Pitts, 1988]. A neural network is crafted from layers of neural units with various network typologies (number of units, layers, connections, connection strengths, and information propagation methods). One objection to the use of neural networks is that “the results often depend on the individual who built the model” [Gilmore 1998]. This is because the model, the network topology and initial weights, may differ from one implementation to another for the same data. A "black box syndrome" is also apparent in neural nets because there is rarely semantic insight to be gained from an inspection of the final state of the network. Unsupervised learning methods require no feedback from a domain expert -- instead, the network is used to discover categories based on correlations within the data.


Unsupervised learning using variant of the K-means clustering algorithm has been used with text in self-organizing maps of the type developed by Kaski et al [1995]. The alternative is supervised (or reinforcement) learning, in which expert feedback is given as part of the training set to indicate whether a solution is correct or incorrect.

Conclusion:In this paper we discussed the Text Data Mining as the process of acquiring valid, potentially useful and ultimately understandable knowledge from large text collections. Research and development departments of major companies, including IBM and Microsoft, are researching text mining techniques and developing programs to further automate the mining and analysis processes. Text mining software is also being researched by different companies working in the area of search and indexing in general as a way to improve their results.

Abstract:Text-mining refers generally to the process of extracting interesting and non-trivial information and knowledge from unstructured text. Text mining encompasses several computer science disciplines with a strong orientation towards artificial intelligence in general, including but not limited to pattern recognition, neural networks, natural language processing, information retrieval and machine learning. Text mining is particularly interesting in areas where users have to discover new information. Information retrieval systems that allow users to search large text collections using explicit word-based and phrase-based queries are now widely deployed, and in the hands of a skilled user they can be used to gain access to information from this broad array of sources. But traditional information retrieval systems treat documents as individual entities, placing the burden of recognizing relationships within and across documents on the user. Data mining essentially provides pattern-based retrieval, in which a pattern in the data is first discovered, and then that pattern is used to present information (the pattern itself

or outlier data, perhaps) to the user. Online Analysis and Processing (OLAP) tools that can support interactive visualization of data mining results are becoming more common, and increasingly sophisticated visualization tools are becoming available as well. These papers discuss techniques for text mining using data mining.

Bibliography[1] Chen, H., Houston, A.L., Sewel, R. R., and Schatz,

B.R., Internet Browsing and Searching: User Evaluations of Category Map and Concept Space Techniques, Journal of the American Society for Information Science, 49(7):582-603, 1998.

[2] Cost, S., Salzberg, S. A Weighted Nearest Neighbor Algorithm for Learning with Symbolic Features. Machine Learning, 10, Page 57 1993.

[3] Freeman, L. Using Available Graph Theoretic or Molecular Modeling Programs in Social Network Analysis [http://tarski.ss.uci.edu/new.html] 1997.

[4] Gey, F., Chen, H.-M., Norgard, B., Buckland, M., Kim, Y., Chen, A., Lam, B., Purat, J., Larson, R., Advanced Search Technologies for Unfamiliar Metadata, in Third IEEE Meta-Data conference, Bethesda, MD, April, 1999. Available at http://www.sims.berkeley.edu/research/metadata/papers.html.

[5] Gilman, M. Nuggets™ and Data Mining Data Mining Technologies Inc White Paper, 1988.

[6] Kostoff, R. N., Eberhart, H. J., and Toothman, D. R. (1999a). Hypersonic and Supersonic Flow Roadmaps Using Bibliometrics and Database Tomography. JASIS. 15 April.

[7] Oard, D. W. and Kim, J., Implicit Feedback for Recommender Systems, AAAI Workshop on Recommender Systems, Madison, WI, 1998. A v a i l a b l e a t http://www.glue.umd.edu/~oard/research.html.

[8] Sheth, B., A Learning Approach to Personalized Information Filtering, Master’s Thesis, MIT, 1994.


EXTRACTION AND ESTIMATION OF PROTEINS FROM DIFFERENT

PULSES AND ANALYSIS.-Kshama Prithvi and Pushpa.N

Students Name: Melisa, Shatabdi, Saranya (M.Sc Biotechnology)email-id: [email protected]

email-id:[email protected]

ABSTRACTProteins are composed of the elements carbon, oxygen , hydrogen , and nitrogen. They have a variety of uses in the body, including serving as a source of energy, as substrates (starter materials) for tissue growth and maintenance, and for certain biological functions, such as making structural proteins, transfer proteins, enzyme molecules, and hormone receptors. Proteins are also the major component in bone, muscle, and other tissues and fluids. When used for energy, protein supplies an average of 4 kcal/g.

Proteins are formed by the linking of different combinations of the twenty common amino acids found in food. Of these, ten are essential for the human in the synthesis of body proteins (eight are essential throughout a human's life, whereas two become essential during periods of rapid growth, such as during infancy).

Protein may be found in a variety of food sources. Proteins from animal sources (meat, poultry, milk, and fish) are considered to be of high biological value because they contain all of the essential amino acids. Proteins from plant sources (wheat, corn, rice, and beans) are considered to be of low biological value because an individual plant source does not contain all of the essential amino acids. Therefore, combinations of plant sources must be used to provide these nutrients. When protein intake is inadequate, but total caloric intake is sufficient, a condition known as kwashiorkor may occur .Symptoms of kwashiorkor include an enlarged stomach, loss of hair and hair color, and an enlarged liver. Conversely, if protein and caloric intake are both inadequate, a condition known as marasmus occurs. Marasmus presents with a stoppage of growth, extreme muscle loss, and weakness.

Proteins from different sources like soyabean, rajma, green gram and dal are extracted by salt precipitation method and estimated by Biuret method with reference to ALBUMIN as standard.

INTRODUCTIONThe word protein is derived from Greek word ‘protas’ which means ‘primary’. As the name shows it is of paramount importance for biological systems. It forms the fundamental structural components of the body.

The amino acids are classified into three types

The essential or indispensible amino acids are those in which carbon skeleton cannot be synthesized by human beings and so preformed amino acids are to be taken in food for normal growth. Ex: arginine, histidine, isoleucine, leucine, tryptophan, lysine, methionine, phenylalanine threonine and valine.

The partially essential or semi essential amino acids are histidine and arginine. They required by growing children in their diet but they are not essential for adult individual.

Non-essential or dispensable amino acids are required for the normal protein synthesis. All body proteins do contain all the non essential amino acids. But their carbon skeleton can be synthesized by metabolic pathways and therefore their absence in food will not adversely affect the growth. Ex. Glycine, alanine, serine, cysteine, tyrosine, aspartate, glutamate, proline, hydroxylproline, citruline.

A sound knowledge of the principles of nutrition is of paramount importance for a doctor practicing in developing countries, where more than 60% populations are below the poverty line. Dietetics is the science of food and nutrients, their action, interaction, balance in


health and disease. The main purpose of food is to provide energy for muscular activity and also to supply basic body building materials such as essential amino acids.

Different food stuffs on burning give different amount of energy. How much heat will be obtained by burning a particular foodstuff is expressed by the term ‘Caloric value’. Otherwise it is called as ‘energy density’.

NITROGEN BALANCE:A normal individual said to be in nitrogen balance. Because the dietary intake(I) equals the daily loss through urine(U), faeces(F) and skin(S); or

I=U+F+S

When the excretion exceeds intake, it is negative nitrogen balance.

When the intake exceeds excretion, it is a state of positive nitrogen balance. Nitrogen balance can be actually measured by calculating the dietary intake of protein nitrogen and measuring the daily excretion.

The nitrogen balance is affected by the factors like hormones, growth, pregnancy, convalescence, acute illness, chronic illness and protein deficiency.

NUTRITIVE VALUE OF PROTEINS:Whipple (noble prize, 1934) introduced plasmaphoresis as a means to assess the nutritional value of proteins. Biological value of protein is the ratio between the amount of nitrogen retained and nitrogen absorbed during a specific interval.

BV= Retained Nitrogen ×100

Absorbed nitrogen

NET PROTEIN UTILISATION (NPU):This will assess both the quality and quantity of the proteins in the diet.

NDPV=intake of N X 6.25 X NPU

PROTEIN EFFICIENCY RATIO OR PER:It is the weight gain per gram of protein intake. The essential amino acids content can also be expressed in terms of chemical score (mg of amino acids per gram of protein)

Amino acid score= mg of amino acid /1 gm of test protein

mg of amino acid / 1 gm of reference protein

X 100

Limiting amino acid is that which limits the weight gain when a protein is supplied to animals. This problem may be overcome by taking a mixture of protein in the diet. Mutual supplementation of proteins is thus achieved.

P R O T E I N E N E R G Y MALNUTRITION:It is the most wide spread nutrition problems in developing countries. It is predominantly affecting children. The prevalence rate varies from 20-50% in different areas depending on social economic status and level of education and awareness.

At the end of the spectrum of malnutrition is marasmus (Greek word to waste), which results from a continued severe deficiency of both dietary energy and proteins (primary calorie inadequacy and secondary protein deficiency). At the other end of the spectrum is Kwasiorkor, where isolated deficiency of proteins along with inadequate calorie intake is seen. Kwasiorkor means sickness, older child gets when the next child is born, a term from the local language of Ga tribe of Ghana. A classification by WHO is based on body weight as per % of standard body weight.

TREATMENT OF PEM:Optimal response is observed with the diets providing 150-200 Kcal/ Kg body weight and 3-4gm of protein /Kg body weight. A mixture of three parts of vegetable proteins and one part of milk protein is found to be very effective. The malnourished children will tolerate fat well and may form a major source of calories in the diet upto 30%. It is monitored by disappearance of edema, rise in serum albumin level and gaining weight.

PRINCIPLES

SALT PRECIPITATION METHODThere are hydrophobic amino acids and hydrophilic amino acids in protein molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and


allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water.

When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out.

As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution.

Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known. After removing the precipitate by filtration or centrifugation, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.

BIURET METHODThe biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms a violet-colored complex in an alkaline solution.

The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.

SDS PAGEIt uses an anionic detergent (SDS) to denature proteins. the protein molecules become linearized. One SDS molecule binds to 2 amino acids. Due to this, the charge to mass ratio of all the denatured proteins in the mixture becomes constant. These protein molecules move in the gel (towards the anode) on the basis of their molecular weights only & are separated.

The charge to mass ratio varies for each protein (in its native or partially denatured form). Estimation of molecular weight would then be complex. Hence, SDS denaturizing is used.

The gel matrix is formed of polyacrylamide. The polyacrylamide chains are crosslinked by N,N-methylene bisacrylamide comonomers. Polymerization is initiated by ammonium persulphate (radical source) and catalyzed by TEMED (a free radical donor and acceptor).

The resolution & focus of the protein bands is increased by using discontinuous gels - the stacking gel (pH 6.8, %T=3 to 5 %) & the resolving gel (pH 8.8, %T= 5 to 20 %). %T represents acryl amide percentage. These gels are usually run at constant current. At pH=6.8, most of the glycine in the population exist as zwitterions with no negative charge (pKa 1 =2.45; pKa 2 =9.6; pI=6.025). Only 0.0015% of the glycine is anionic at this pH (refer glycine titration curve & Henderson-Hassel Bach equation). As such, bulk of the current is carried by the denatured, negatively charged, SDS-coated protein molecules. At this stage, the glycine ions lag behind the proteins. The order is as follows- chloride ions, denatured proteins, glycine ions.

Upon entering the resolving gel (pH=8.8), the glycine zwitterions deprotonate to the anionic form. The proportion of these ions increase from 0.0015% to 15.8%. The carrying of the current is now shared by the ions such that protein molecules have a greater freedom to separate on the basis of molecular weights. Due to their small size, the glycine anions also tend to overtake the protein band, thus providing a sandwiching effect & greater resolution in the gel.

COMASSIE BLUE STAININGIn this method ,the gel was fixed stained in the cbb stain solution containing 0.25%cbb in methanol,acetic acid,water in the ratio 5:5:1 and then destained by means of destain solution containing methanol, acetic acid water(5:5:1)gel was stored in 7%acetic acid.

COLOUR REACTIONSThey are of importance in the qualitative detection and quantitative estimation of proteins and for their constituent amino acid.

MATERIALS AND METHODS

Glass apparatusTest tube, conical flask, beaker, pipette, petriplate, glass funnel, measuring cylinder, eppendorf tubes

Instruments

Autoclave, hot air oven, refrigerator, cooling centrifuge,


weighing machine, colorimeter, transilluminator, gel casting apparatus, electrophoresic unit, mortar and pestle.

SamplesGreen gram, dal, rajma, soyabean

Chemical reagents

NH2SO4 SOLUTION,STANDARD PROTEIN SOLUTION,TCA,BIURET REAGENT,SDS KIT.

PROCEDURE: EXTRACTION AND ESTIMATION

Ammonium sulphate method1. 25gms of the sample soaked overnight then

grounded into a fine paste in a blender, volume of paste made upto 100ml with phosphate buffered saline.

2. Extraction of protein should be carried out in ice-cold condition

3. The 5ml of the extract was diluted to 20ml and centrifuged at 3000rpm for 5min at 40C to remove suspended particles

4. Supernatant was collected and 8 to 9gms of Solid ammonium sulphate was carefully added in small amounts on ice. This produced 45%saturation.

5. Beaker was allowed to stand on ice for 45min.

6. The solution was then allowed to be centrifuged at 3000rpm for 10min.The precipitate was collected and dissolved in 5ml of distilled water.

7. To this 5ml, 10%TCA solution was added and again the precipitate was centrifuged at 3000rpm for 10min at 40C.

8. The precipitate was then dissolved in 3ml of distilled water and 1.5ml was taken for estimation of protein.

SDSAssemble plates for casting gel for leakage

Add 500microlitre water in a APS vial and dissolve.

5o microlitre APS mix with 5ml separating gel mix and pour between the plates

Allow the gel to polymerize and wash the top of the gel

20microlitre APS mix with 2ml stacking gel mix, pour above the separating gel, insert comb and allow to

polymerise

Prepare the samples

Fix the plate to the PAGE apparatus with bufferLoad the samplesApply the voltage to start the electrophoresisStop when dye is 0.5cm above the bottom of the gelStain; destain the gel and view for protein bands.

COMMASSIE BRILLIANT BLUE STAININGThe gels are soaked in solution of acetic acid to fix the proteinsThe gels are then put in isopropanol to remove the SDSThe gel is stained with CBB staining solution containing 0.25% CBB,Isopropanol ,acetic acid and water in the ratio 5:5:1After staining the gel is destained with a dilute acetic acid solution so that the protein bands become visible against a clear backgroundThe gel is then stored in 7%acetic acid.

BIURET METHODTake 1ml of the sample and make up the volume with distilled water.5ml of biuret reagent is mixed with the unknown sampleDifferent aliquots of the std. albumin ranging from 0.2 to 1ml is takenAll the standards are made upto 1ml with distilled water.5ml of biuret reagent is mixed with itThe reagent blank is carried out with 1ml of distilled waterAll the tubes are incubated at room temperature for 30minThe readings are taken in the colorimeter at 540nmA graph is plotted with concentration along Xaxis and absorbance along YaxisConcentration of unknown is calculated by extrapolating on the standard graph


Sl. No

Vol of std protein

(ml)

Vol of water (ml)

Amount of protein (mg)

Vol of biuret (ml)

Incubation at room temper-

ature for 30min

O.D at 540nm

OBSERVATION TABLE FOR SOYABEAN

1 0 1 0 5 0

2 0.2 0.8 4 5 0.08

3 0.4 0.6 8 5 0.16

4 0.6 0.4 12 5 0.2

5 0.8 0.2 16 5 0.26

6 1 0 20 5 0.34

7 1 0 ? 5 0.24

Calculation: O.D of test X conc. Of std = 0.24 X 16 = 14.8mg/ml

O.D of std vol of test 0.26 1

OBSERVATION TABLE FOR RAJMA

1 0 1 0 5 0

2 0.2 0.8 4 5 0.08

3 0.4 0.6 8 5 0.14

4 0.6 0.4 12 5 0.2

5 0.8 0.2 16 5 0.26

6 1 0 20 5 0.34

7 1 0 ? 5 0.2

Calculation: O.D of test X conc. Of std = 0.2 X 12 = 12mg/ml


Sl. No

Vol of std protein

(ml)

Vol of water (ml)


Vol of biuret (ml)


ature for 30min

O.D at 540nm

Sl. No

Vol of std protein

(ml)

Vol of water (ml)


Vol of biuret (ml)


ature for 30min

O.D at 540nm

Sl. No

Vol of std protein

(ml)

Vol of water (ml)


Vol of biuret (ml)


ature for 30min

O.D at 540nm


OBSERVATION TABLE FOR GREEN GRAM

1 0 1 0 5 0

2 0.2 0.8 4 5 0.08

3 0.4 0.6 8 5 0.14

4 0.6 0.4 12 5 0.2

5 0.8 0.2 16 5 0.26

6 1 0 20 5 0.34

7 1 0 ? 5 0.16

Calculation: O.D of test X conc. Of std = 0.16X 8 = 9.14mg/ml


OBSERVATION TABLE FOR DAL

1 0 1 0 5 0

2 0.2 0.8 4 5 0.08

3 0.4 0.6 8 5 0.14

4 0.6 0.4 12 5 0.2

5 0.8 0.2 16 5 0.26

6 1 0 20 5 0.34

7 1 0 ? 5 0.08

Calculation: O.D of test X conc. Of std = 0.08X 4 = 4mg/ml


RESULT SOYABEAN-14.8mg/ml

RAJMA-12mg/ml

GREEN GRAM-9.14mg/ml

DAL-4mg/ml


DISCUSSIONProtein is one of the basic building blocks of the body so it is an essential part of your diet and can influence your strength but probably not your energy. Our muscles are built with protein and in fact protein is made up of 20 amino acids. Amino acids are the building blocks of protein and 9 are essential, they cannot be created and must be eaten meaning the other 11 amino acids can be created by our body.

Many times we here that you need to eat steak for energy but in actual fact as we learned earlier carbohydrates are the body's favored energy source. I always think of muscles as being made out of protein but really muscles are mostly water and protein so if you think about how people tend to diet they will cut out things like steak and carbohydrates so the body is not getting as much protein and carbohydrates as it needs to burn for energy so it will tend to burn protein and fat in equal parts to get energy. This is great for losing weight and good for losing fat but it is really bad to lose muscle. One of the ways to stop your body from burning muscle for energy is to do a fair amount of exercise, especially weight training to increase your muscle mass on a consistent basis.

REFERENCES

FOR DIETARY IMPORTANCEK.N. Agarwal,effect of wheat and bengal gram diets on brain 110:2166-2177

A We s t o n , p r i c e , n u t r i t i o n a n d p hy s i c a l degeneration.1939-1948

McCollum,A History of nutrition.1957,p.87

Journal of biology,BRCAI protein products:functional motifs

J-P Borg, NATURE CELL BIOLOGY

R.Legouis ,et al,nature cell biology,415-422

FOR SDS:V.Beisiegal,electriphoresis1986.7:1-18

J.M.Gershoni protein blotting a manual

T.J Nelson analytical biochem

M.R.SURESH et.al,Analysis SDS 83:7989-7993

FOR LITERATUREBiochemistry by d.das,8th edition

Text of biochemistry 3rd edition by D.M. Vasudevan and Sri kumari.S.

Mineral contents in plant food products by M.A. Grusak

Quality of legume seed by T.L.Wang


Homology Modelling of Tumor necrosis factor ligand superfamily

member 6 (Fas antigen ligand) Mus musculus (Mouse).

Sunil.B*, M.Reena Prathipa Department of Biotechnology, Garden City College,

Bangalore – 560049, Karnataka, Indiaemail: *[email protected], [email protected]

AbstractUse homology modelling technique to model Tumor necrosis factor ligand superfamily member 6 (Fas antigen ligand) of Mus musculus (Mouse). Using Deepview v 4.0.1 and SWISS-MODEL which is a fully automated protein structure homology-modeling server, accessible via the ExPASy web server, or from the program DeepView (Swiss Pdb-Viewer). Tumour necrosis factor ligand superfamily member 6 (Fas antigen ligand)(Fas ligand)(CD95L protein)(CD178 antigen) [Contains Tumour necrosis factor ligand superfamily member 6, membrane form;Tumor necrosis factor ligand superfamily member 6, soluble form] [Source:UniProtKB/Swiss-Prot;Acc:P41047] was used for the analysis.

IntroductionWe can observe similarities in topology or even in structural details with protein structures. Comparison of protein structures can reveal distant evolutionary relationships that would not be detected by sequence alignment alone. In inventing measures of similarity and difference between two proteins, it is sometimes more clear-cut to note how to proceed in comparing sequences than to do so in comparing structures. If the amino acid sequences of two proteins can be aligned, then we can either count the number of identical residues or use a similarity index between amino acids.

Fas ligand (FasL or CD95L) belongs to the tumor

necrosis factor (TNF) family and is a type-II transmembrane protein . In the regulation of the immune system and the progression of cancer the Fas ligand/receptor interactions play an important role. Its binding with its receptor induces apoptosis.

Using computer graphics, it is possible to superpose FAS 6 structures in one picture with other similar protein structures or templates available in database, and this can reveal, immediately and obviously, which features are conserved in the FAS 6 proteins or in the conformational changes during allosteric transition/upon complexation. It is this facility in which lies the real advantage of computer graphics.

The methodology used here is - 1. Started from the FAS 6 sequences. 2. Assemble fragments/substructures from different, known homologous structures (1TNRA, and 2TUNA). 3. Carried out limited structural changes from a known neighboring protein (1TNRA, and 2TUNA). 4. Optimize the structure by energy minimization using the Deepview v4.0 .

Materials and method.• Amino acid sequence of FAS 6 Protein was obtained

from UniPortKB Swis-Port.

• Query sequence was subjected to BLAST against the PDB(ProteinData Bank).

• MQQPMNYPCP QIFWVDSSAT SSWAPPGSVF P C P S C G P R G P D Q R R P P P P P P PVSPLPPPSQPLPLPPLTPL KKKDHNTNLW


L P V V F F M V L V A L V G M G L G M Y QLFHLQKELA ELREFTNQSL KVSSFEKQIA NPSTPSEKKE PRSVAHLTGN PHSRSIPLEW EDTYGTALIS GVKYKKGGLVINETGLYFVY S K V Y F R G Q S C N N Q P L N H K V Y M R N S K Y P E D L V L M E E K R L N Y CTTGQIWAHSSYLGAVFNLT SADHLYVNIS QLSLINFEES KTFFGLYKL

Fig1: Raw sequence in Deepview v4.0

• Using DeepView therow sequence was subjected to fend the ExPDb examples and this fetched the result from the database.

• Two top hits ware obtained from the search, that is, 1TNRA, and 2TUNA.

• Rest of the analysis was carried out by using 1TNRA, and 2TUNA as the templates for the model building.

• The protein row sequence was loaded to the workspace of DeepView for further analysis.

• Afetr filling the required details for the swiss model window and selected the checkbox for the program to notify any AA (Amino acids) were exempted from the search.

Fig2: email and other setups for Swiss database access

• Using Deepview then the Find appropriate templates ware done.

• On the result window the top searches which maches even the BLAST search was selected and it was loaded to the workspace.

• After the proteins were centered by the use of keypad and analysed for the further steps.

• Fist made the 1TNRA active and done the analysis by hitting the Magic Fit option.

• This helped us in matching the 1TNRA and 2TNRA at its best match by superimposing.

• Fig3: The magic fit of aligned templates.

1TNRA and 2TUNA has been superimposed

Fig3: 1TNRA and 2TUNA superimposed in Deepview v4.0

• Now further modification done for the bestfitting by the structural window, and realigning the sequence.


• Then using the Iterative Magic Fit tools the superimposing was modified for the bettr accommodation.

• After the superimposing of the template structure we made the raw sequence window active and applied the same Magic Fit for the protein sequence.

• We were able to align the C-terminal domain to the reference sequence, and the first domain was not aligned because of the presence of transmembrane domain counterpart absence.

• After the alignment the threading was done by using Update threading now menu of the Deepview.

• We received the wrapped raw sequence around the first template.

FASL was "wrapped" around 1TNRA.

• Changing the colour of 1TNRA protein to yellow, and the 2TUNA protein to green in the control panel.

• Using the align window up to the first big gap and selectd the region starting from GQSCN up to LNHK. Selection is activated simultaneously in the templates amino acids of 1TNRA and 2TUNA.

Fig5: The alignment window

Fig6: The display window showing the false bond:

Threading correction done by mean force potential• Making the query protein active, the enrgy of the

protein was minimised using the mean force potential energy, computed accordingly to a "Sippl-like"

method.

• By selecting the "smooth" text, and set a smoothing factor of 1. The energy off all the residue averaged of itself plus the energy of 1 flanking residue on each side.

• By selecting the "Auto Color by Threading Energy" item of the "Swiss-Model" menu.

• The recomputed energy obtained was "E= -11.9" for the current layer.

Result and DiscussionThe comparative model of the FAS6 was produced and this tool provides hints and should be used in conjunction with the analysis of the hydrophobicity of residues, the overall structure, and common sense! It works better for displacement of large fragments than for just one or two residues, as it analyses the mean force potential for the whole protein.

The 3-D structure of Tumor necrosis factor ligand superfamily member 6 (Fas antigen ligand) of Mus musculus (Mouse) obtaind by homology modelling shows high similarity to template TUMOR NECROSIS FACTOR RECEPTOR P55 (EXTRACELLULAR DOMAIN) (1TNRA) protein.

Knowledebaseed approached like homology modelling of comaparative modelling can be used as an importtent tool for the rational drug design, mutagenisis analysis, docking analysis and structure based drug designing.

Reference.1. Bengt Fadeel, Christopher J. Thorpe, and Francesca

Chiodi

Mapping of the linear site on the Fas/APO-1 molecule targeted by the prototypic anti-Fas mAb Int. Immunol., December 1995; 7: 1967 - 1975.

2. Richard K. Lee, Julie Spielman, and Eckhard R. Podack

bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis

Int. Immunol., July 1996; 8: 991 - 1000.

3. List of Abstracts from the 15th International Symposium on Olfaction and Taste

Chem Senses, October 2008; 33: S1 - S175.

4. Jui-Han Huang and Mark L. Tykocinski

CTLA-4–Fas ligand functions as a trans signal converter protein in bridging antigen-presenting


cells and T cells

Int. Immunol., Apr 2001; 13: 529 - 539.

5. C Madry, Y Laabi, I Callebaut, J Roussel, A Hatzoglou, M Le Coniat, JP Mornon, R Berger, and A Tsapis

The characterization of murine BCMA gene defines it as a new member of the tumor necrosis factor receptor superfamily

6. B Fadeel, J Lindberg, A Achour, and F Chiodi

A three-dimensional model of the Fas/APO-1 molecule: cross-reactivity of anti-Fas antibodies explained by structural mimicry of antigenic sites

Int. Immunol., Feb 1998; 10: 131 - 140.

7. Tomohiro Takahashi, Masato Tanaka, Johji Inazawa,

Tatsuo Abe, Takashi Suda, and Shigekazu Nagata

Human Fas ligand: gene structure, chromosomal location and species specificity

Int. Immunol., October 1994; 6: 1567 - 1574.

Shuichiro Ito, Tomoko Takayama, Hiroyuki Hanzawa, Kimihisa Ichikawa, Jun Ohsumi, Nobufusa Serizawa, Tadashi Hata, and Hideyuki Haruyama

Crystal Structure of the Antigen-Binding Fragment of Apoptosis-Inducing Mouse Anti-Human Fas Monoclonal Antibody HFE7A

J. Biochem., January 2002; 131: 137 - 143.


MICROFLORA OF SALTED DRIED FISH FROM SEA WATER

Sreeja Bhaskar, Department of Biotechnologyemail : [email protected]

Abstract: This study was aimed to estimate the microbial contamination of salted dried fish. The fish sample used for study was sardine (Sardinella gibbosa).Twenty dried fish samples were examined and a few were found to be contaminated with microbial bioburden.Various microorganisms were isolated from dried fish. The bacterial counts were between 2 x 103 to 10 x 104 CFU/g.the mould count were in the range of 2 x 103 to 5 x 104 CFU/g.The relative humidity and the pH were between 22.7 – 27.6% and 3.0 – 6.0 respectively. In conclusion salted dried fishes were contaminated with fungi and bacteria.

INTRODUCTION Fish supplies a good balance of protein, vitamins and minerals.It has less calorie content of 10% and with good nutritional value. Fish and fish products constitute more than 60% of the total protein intake in adults. They are widely accepted on the menu card and form cherished delicacy that cuts across socio economic, age, religious and educational barriers .The bioburden of the fish is influenced by intrinsic and extrinsic factors. The intrinsic factors include pH,water activity,oxidation reduction potential.The extrinsic factors include temperature, marine ecosystem,fishing devices,catching methods and water used for cleaning.

Fish is most perishable meat product subjecting to autolysis,oxidation, hydrolysis of fat and microbial spoilage. A number of spoilage and pathogenic microorganisms, including lactic acid bacteria, Pseudomonas spp., Staphylococcus spp., Salmonella spp., Clostridium perfringens, Clostridium botulinum, Escherichia coli and Listeria monocytogenes, have been a s s o c i a t e d w i t h f i s h a n d f i s h products.Hence,preservative methods should be initiated from the fishing boat itself. Traditional curing technique based on combinations of salting,drying and

smoking is one of the wide spread preservation methods to increase the shelf life of fish. In the usual process of dry-salting, whole fish are eviscerated, cleaned, washed, dry-salted, stacked in containers with more NaCl in between the pieces, stored for a salting or curing period, and then dried (using sunlight or artificial indoor drying chambers). The salting period depends on several factors including the desired ripened characteristics in fish, the fish species, the amount of salt used, and the storage temperature. The heat of the sun and movement of air remove moisture which causes the fish to dry. In order to prevent spoilage, the moisture content needs to be reduced to 25 per cent or less. The percentage will depend on the oiliness of the fish and whether it has been salted. At certain concentrations, salt was found to prevent growth of many microorganisms by exerting a drying effect on microbial cells and tissue, which concentrates solutes in them, creating an environment unsuitable for microbial proliferation. Because some halotolerant or halophilic (salt-loving) microorganisms are not affected by salt, additional treatments such as drying, heating, curing, or smoking are helpful in controlling them. Salting or brining two aims in removal of water from the fish and movement of salt inside the fish.

Salting requires minimal equipment, but the method used is important. Salt can be applied in many different ways. Traditional methods involve rubbing salt into the flesh of the fish or making alternate layers of fish and salt (recommended levels of salt usage are 30-40 per cent of the prepared weight of the fish). A better technique is brining. This involves immersing the fish into a pre-prepared solution of salt (36 per cent salt). The advantage is that the salt concentration can be more easily controlled, and salt penetration is more uniform. Brining is usually used in conjunction with drying. Effectiveness of salting for preservation depends upon the parameters like uniform salt concentration in the fish flesh, concentration of salt, and time taken for


salting and whether salting is combined with other preservation methods such as drying. Dried fish that contains high levels of lipids—Sardine, herrings, and mackerel—are prone to oxidation and become rancid as microbial spoilage occurs ( Jay). Because of their unsaturated nature, fish body oils are susceptible to oxidation and also easily develop rancid and unacceptable odors and flavors during storage. Once fatty compounds are oxidized, the breakdown products of lipid oxidation that can react with proteins and vitamins, leading to a loss of nutritional value and quality of the fish. Salted sun-dried fish are more prone to oxidation than fish preserved by other methods because of their exposure to light and oxygen (Smith and Hole). Use of crude NaCl (which contains impurities such as chlorides, sulfates, calcium, and heavy metals) accelerates lipid oxidation during fish processing and will adversely affect the overall quality of the finished product.

MATERIALS AND METHODSSample Collection: Dried fishes (Sardine) were randomly sampled and purchased from Adoor market.Twenty fish samples were taken for study and were placed in sterile polyethylene bags,which were used for analyses.

Determination of moisture:

Weighed accurately about 5 gm of the prepared sample in a moisture dish with slip on cover. Dry in an air oven at 100 — 1 ° C for 5 hours. Placed lid on the dish and cooled in a dessicator. Quickly weighed the dish. Returned the dish with the cover to the oven and dried for another 1/2 hour. Cooled in the dessicator and weighed again. Repeated readings were taken for accuracy.

Calculation:

Moisture = M1x100

M2

Where M 1 = Loss in gm in the mass of sample

M 2 = Mass in gm of the sample taken for test

(Ref:- I.S 14950 : 2001 Fish Dry and dry salted

Determination of Sodium Chloride:

Reagents

(1) Standard Silver Nitrate - 0.1 N

(2) Dilute Nitric acid - 1 + 4

(3) Ferric alum indicator - Prepare a saturated solution of Ferric ammonium sulphate

(4) Standard Potassium thiocyanate solution - 0.1 N

Procedure: 2 gm of the dried fish was taken(obtained after determination of moisture)

in a 250 ml beaker and added 50 ml of distilled water free from chloride and heat on a water bath till all the Sod.Chloride goes into solution. Filtered in a 250 ml conical flask and wash with distilled water till the washings are free from chloride. Added 20 ml of dilute nitric acid and a known volume of standard silver nitrate sufficient to precipitate all the chloride. Added 1 ml of ferric alum indicator and titrate with standard Potassium thiocyanate solution until a permanent light brown colour appeared.

Calculation

Sodium Chloride(on dry basis) m / m = 5.85 x (V1N1-V2N2)

M

Where

VI = Vol of standard solution of silver nitrate

Nl = Normality of standard silver nitrate solution

V2 = Vol of standard Pot. Thiocyanate solution

N2 = Normality of standard Pot. Thiocyanate sol

M = Mass of dried material taken for test

(Ref:- I.S 14950 : 2001 Fish Dry and dry salted)

Isolation of Microflora: Attempts to isolate fungi and bacteria from the dried fish samples were made aseptically on Saboraud dextrose agar, Martins Rose Bengal Agar and Nutrient Agar respectively. Ten grams of the fishes samples were weighed aseptically and macerated in 90 ml sterile distilled water using a blender. From this, subsequent tenfold dilution was made up to 10-5 [11]. One milliliter of each dilution was dispensed in triplicate in sterile MRBA, SDAand NA plates. Petri dishes, which were gently rotated to ensure even dispersion. The plates were allowed to solidify.MRBA, SDA plates were incubated at 28±1°C for 3-5 days and NA plates at 37°C for 24-48 hrs. All observed colonies were subcultured to obtain pure cultures which were subsequently isolated and identified.

Enumeration of Microflora: The pure culture plates were screened thoroughly for the total count of bacteria and fungi per dilution using qubec colony counter.

Identification of Microflora:The isolated


microorganisms were identified using morphological characteristics(colony characterization,staining) spore formation, the production of fruiting bodies and biochemical reactions

Determination of pH: Two grams each of the macerated fish samples were weighed in triplicates. Water was added and mixed thoroughly to make a fish slurry. The pH readings were taken using digital pH meter equipped with a glass electrode (digital thermo pH meter mod B-E105) slurry. The pH reading were then recorded. Determination were done in triplicates and the mean value were obtained.

RESULTS AND DISCUSSIONResults obtained from this study showed that

Aspergillus flavus, , A.fumigatus, Aspergillus niger, Rhizopus sp., , Mucor sp.

., Penicillium sp, Candida sp and Fusarium sp were found from SDA and MRBA plates.Where as the NA plates showed the presence of Escherichia coli, Serratia sp, Micrococcus, Pseudomonas.

The moisture content present in the collected sample were between 22.7 – 27.6%.

The salt concentration was estimated to be around 15-18%.

From the pure culture technique, the bacterial count was with in a range of 2 x 103 to 10 x 104 CFU/g. and the mould count were in the range of 2 x 103 to 5 x 104 CFU/g.

The pH of the sample were with in the range of 3-6.

Fig. 1: The rate of occurrence (%) of fungi

Fig. 2: The rate of occurrence (%) of bacteria

The presence of these microorganisms in the samples might probably make its consumption hazardous to health. During the drying period, the overloading of the fishes on the trays leads to improper processing which in turn encourages bacterial and fungal attack [5]. The water used for the brine preparation can be a source of impurity.Storage of dried fish in non ventilated areas aids in microbial attack. The environment in which fishes are displayed in the market is not always hygienic and this is another avenue for microbial contamination.The study revealed that the average bacterial count ranged from2 x 103 to 10 x 104 CFU/g. and the mould count were in the range of 2 x 103 to 5 x 104 CFU/g. The microbial levels obtained in this report could be considered hazardous to consumers because of the possibility of the presence of enterotoxigenic strains.

CONCLUSIONThe dried fish sample of sardine studied from the Adoor market,Pathanamthitta Dist. Kerala was contaminated with different types of bacteria and aflatoxin producing moulds.The consumption of the same with improper cooking can be hazardous for human health. The studies have revealed that all these microorganisms are contaminants and not of the fish origin.Hence better preservation techniques like using food additives,low temperature preservation,combination of preservative methods can eliminate microbial contamination.

REFERENCES1. ALMAS, A.K. Chemistry and microbiology of fish

processing. Dept. of and Application. Agro Botanica. Bikaner, (India), Pp 650 –652. (1981).

2. APHA. Compendium of methods for the Microbiological Examination of Foods. (Ed.) M.L. Speck, Published by America Public Health Association Inc., New York. 701 p. (1971).


3. APHA. Compendium of methods for the mic rob io log i ca l examina t ion o f foods 3rd(VANDERZANT. C & SPLITTSOESSER. D, Eds,), APHA, Washington, DC. (1992).

4. CIFT. Annual Report 1993-94, Central Institute of Fisheries Technology, Cochin, India. (1994).

5. FOOD AND DRUG ADMINISTRATION. Reference Manual to codes of practices for fish and fishery products. pp 152, FAO, Rome. (1982).

6. GUPTA, R, & SAMUEL, C, T. A. niger in dried fish samples in rainy season in Cochin coast, Fish. Technol. 22, 132. (1985).

7. IYER, T.S.G. & SHRIVASTAVA, K.P. Fish.Technol. 25:39. (1989).

8. Claus, J. R., C. Jhung-Won, and G. J. Flick. "Processed Meats/Poultry/Seafood." In Muscle Foods: Meat, Poultry, and Seafood Technology, edited by D. M. Kinsman, A. W. Kotula, and B. C. Breidenstein. New York: Chapman and Hall, 1994.

9. Jay, J. M. Modern Food Microbiology. 4th ed. New York: Van Nostrand Reinhold, 1992

10. Pszczola, D. E. "Salty Developments in Food." Food Technology 51 (1997): 79–90.

11. Salt Institute. Facts about Salt. Alexandria, Va.: Salt Institute, 2000.

12. Smith, G., and M. Hole. "Browning of Salted Sun-Dried Fish." Journal of the Science of Food and Agriculture 51 (1991): 193–205.

13. Zakhia, N., Coulibaly,Ab., Coulibaly, Az., Traore Cisse, O., 1998.Use of some additives for improving preservation and quality of traditionally dried fish in Mali. In : FAO Fisheries Report, n° 574, Sixth FAO Expert Consultation on Fish Technology in Africa, 27-30 August 1996, Kisumu, Kenya, pp. 117-122, FAO, Rome, Italy.

14. Zakhia,N., Themelin,A.,1990. Quality assessment and process controlling of the traditionally dried fish in Mali. In : International Agricultural Engineering Conference and Exhibition, 3-6 December 1990, Bangkok, Thailand, vol. 2, pp. 561-569, Asian Institute of Technology, Bangkok, Thailand.


The Effect Of Hyperthyroid Drug (methimazole) On

Drosophila With Extrapolation In Pregnant Women

Mr. Prabhakara G.S, Ms. Proma Chakraborty,

Ms. Karishma, Ms. Veena

Department of Genetics, Garden City College Bangalore-560049email : [email protected]

AbstractHyperthyroidism is the term for overactive tissue within the thyroid gland which causes an overproduction of thyroid hormones. This hormone controls the metabolism in the body. When there is excess production of thyroid hormone, all functions of the body tend to speed up. Therefore, in order to control this condition, several anti thyroid drugs are being used. But these drugs have several side effects especially in pregnant women. The present toxicological study on Drosophila involved methimazole which is known to delay its life cycle, due to reduced activity of ecdysone. Administration of high levels of this drug can caused an increase in fetus size with major structural abnormalities like extension and curving of abdomen, wing and limb deformities. This is because the drug interferes with the expression of zygotic gene (bicoid, hunchback and pair-rule) patterning genes, pax family and colt genes. In the case of humans, prenatal exposure of the drug leads to stillbirth at 32 wk of gestation and exhibited cardiac defects, esophageal atresia with tracheoesophageal fistula, omphalocele, and hydrops. Thus it has been concluded that methimazole is a teratogen.

Key words: Hyperthyroidism, methimazole, thyroid hormone, teratogen

INTRODUCTIONThyroid hormones are lipophilic substances that are able to traverse cell membranes even in a passive manner. At least 10 different active, energy dependent and genetic regulated iodothyronine transporters have been identified in humans. They guarantee that intracellular levels of thyroid hormones are higher than in blood

plasma or interstitial fluids. In adult mammals thyroid activity is required for control of metabolic rate. The thyronines act on nearly every cell in the body. They act to increase the basal metabolic rate, affect protein synthesis, help regulate long bone growth (synergy with growth hormone), neuronal maturation and increase the body's sensitivity to catecholamines (such as adrenaline) by permissiveness. The thyroid hormones are essential to proper development and differentiation of all cells of the human body. These hormones also regulate protein, fat, and carbohydrate metabolism, affecting how human cells use energetic compounds. They also stimulate vitamin metabolism. Numerous physiological and pathological stimuli influence thyroid hormone synthesis. Thyroid hormone leads to heat generation in humans. However, the thyronamines function via some unknown mechanism to inhibit neuronal activity; this plays an important role in the hibernation cycles of mammals and the moulting behaviour of birds. Additionally, the importance of the thyroid gland in development has long been recognized, as iodine deficiency during pregnancy causes mental and physical retardation in the infant.

In amphibian development the thyroid axis plays another critical role in regulating metamorphosis. The prohormone T4 (thyroxine) is generated in the thyroid from thyroglobulin and tyrosine by TPO (thyroid peroxidase). T4 is converted into the active form T3 (tri-iodothyronine) in peripheral tissue by 5-DII (type II iodothyronine 5-deiodinase) and to a lesser extent type I deiodinase. The hormonal signal is terminated by 5DIII (type III iodothyronine 5-deiodinase), which converts T3 into the inactive form, reverse T3.In amphibians the relative levels of 5-DII and the inhibitor 5DIII control


the timing at which different tissues undergo metamorphosis. Thyroid hormones play an important role in early embryonic development prior to the development of a functional thyroid. Studies in Xenopus laevis have shown that the TRa (TH receptor a) is expressed in developing oocytes and at all stages of embryonic development. The TRß protein has been detected in early neurula stage embryos. Hormones are deposited in the eggs during oogenesis and are retained during early development.A number of studies have shown that TH is required for normal development of the nervous system.TH is capable of upregulating neurogenesis in the spinal cord of larval stage X. laevis.

The thyroid hormone is specifically synthesized by the thyroid follicular cells of thyroid gland. The differentiative program of thyroid follicular cells (TFCs) is completed only when the gland reaches its final location. TFCs express a series of proteins that are essential for thyroid hormone biosynthesis. Because there will be no further differentiation event in the life of TFCs, this last differentiation could be called terminal or functional differentiation. Genes typical of this stage appear according to a given temporal pattern: Tg, TPO, and TSH receptor (Tshr) genes are expressed by embryonic gestation period (E14.5); sodium/iodide symporter (NIS) is detected by E16. T4 is first detected at E16.5. The other genes involved in thyroid gland development are Titf1/Nkx2-1 which is a thyroid transcription factor required for early morphogeneis. Pax8 (paired box gene 8) is maintained in the thyroid follicular cells during all stages of development and also in adult hood. Foxe1 (formerly called TTF-2 for thyroid transcription factor-2) was originally identified as a thyroid-specific nuclear protein that recognizes a DNA sequence present on both Tg and TPO promoters under hormone stimulation. Hhex (hematopoietically expressed homeobox) is a homeodomain-containing transcription factor which is expressed in adult thyroid at highest level.

Any mutation in the above genes can lead to severe human pathology. The most common form of thyroid hormone disorders are hypothyroidism and hyperthyroidism. Hypothyroidism is the disease state in humans and in vertebrates caused by insufficient production of thyroid hormones by the thyroid gland. Cretinism is a form of hypothyroidism found in infants. The candidate genes associated with this genetically heterogeneous disorder form two main groups: those

causing thyroid gland dysgenesis and those causing dyshormonogenesis. Genes associated with thyroid gland dysgenesis include the TSH receptor in non-syndromic congenital hypothyroidism, and the thyroid transcription factors (TTF-1, TTF-2, and Pax-8), associated with different complex syndromes that include congenital hypothyroidism. Among those causing dyshormonogenesis, the thyroid peroxidase and thyroglobulin genes were initially described, and more recently PDS (Pendred syndrome), NIS (sodium iodide symporter), and THOX2 (thyroid oxidase 2) gene defects.

Common symptoms include fatigue and lethargy, cold sensitivity, dry skin and lifeless hair, impaired concentration and memory, increased weight with poor appetite and constipation. Patients may also fairly often experience a hoarse voice, tingling of the hands (carpal tunnel syndrome), heavy and, later, absent periods, deafness and joint aches. In childhood there may be delayed development and in the adolescent precocious puberty. The elderly may develop memory disturbance, an impaired mental state or depression, and in rare cases coma can occur, resulting in death if left untreated. ‘Myxoedema facies’ in which the face looks puffy due to the accumulation of subcutaneous fluid, slow pulse rate, slow tendon reflex relaxation time, slow pulse rate and hoarse voice. The thyroid may be enlarged (causing a goitre) in some patients due to accumulation of lymphocytes (Hashimoto’s thyroiditis).

Hyperthyroidism is the term for overactive tissue within the thyroid gland causing an overproduction of thyroid hormones in blood. Familial non-autoimmune hyperthyroidism is a rare autosomal dominant genetic disease resulting from mutations in the thyroid-stimulating hormone receptor (TSHR) gene. Common symptoms of hyperthyroidism include fatigue, heat intolerance, sweating, weight loss despite good appetite, shakiness, inappropriate anxiety, palpitations of the heart, shortness of breath, tetchiness and agitation, poor sleep, thirst, nausea and increased frequency of defecation. The elderly may complain predominantly of heart problems with a fast or irregular heartbeat, breathlessness and ankle swelling, fast tendon reflexes, an enlarged thyroid gland and prominent bulging eyes.

Drosophila being a model organism is extensively used for drug toxicological studies. Since the organism shares 50% homology with human genome most of the studies conducted in the fly are extrapolated in humans. The


pertinent literature deals with the effect of hyperthyroid drug methimazole in drosophila with co-relation in pregnant women.

Methimazole is a drug used to treat hyperthyroidism. Methimazole inhibits the addition of iodine to thyroglobulin by the enzyme thyroperoxidase, required for the synthesis of triiodothyronine (T3) and thyroxine (T4). Thus lowering the level of the hormone in the blood. It does not inhibit the action of the sodium-dependent iodide transporter located on follicular cells basolateral membranes.

MATERIALS AND METHODThe experiment was conducted on the wild type strain Oregon-R of Drosophila melanogaster. The optimum temperature required for the survival, growth and breeding is 20-25? C. temperatures above 31? C, makes the fly sterile and reduce the oviposition. It may also result in death. The food media used for maintenance of the fly was “ Cream of Wheat Agar Medium” . The ingredients used for the media preparation were:

• 1000ml Distilled water

• 100gm Wheat flour (Suji)

• 100gm of jaggery

• 10gm of Agar-agar

• 7.5ml of propionic acid

• Yeast granules(crushed)

1000 ml distilled water was boiled and 100 gm jaggery was added to it followed by further boiling until jaggery melts to form thin syrup. To that 100 gm suji was added slowly and ingredients were cooked properly to form a thick paste. In order to solidify the media 100gm Agar- agar was added, prior to the removal of the media from the flame, followed by addition of 7.5ml propionic acid to give a fruity smell which attracts the flies.

The media prepared was then transferred to heat sterilized culture bottle s(sterilized in hot air oven at

120○C for 1 hour). Media was allowed to solidify overnight and yeast granules were added next day

followed by plugging the bottles with cotton plugs.The wild stock of Drosophila was cultured for three

generations in the medium at 23○C. The flies obtained were

of wild type without any variants or cross contaminations. The virgin flies were isolated immediately after emergences

and sexes were separated. Two sets of culture bottles, each containing 12 bottles were provided with 15ml of the

standard media. Two bottles from each set are marked as

control with no drug added to them. Remaining 10 bottles of

each set were marked between (0.01mg- 0.1mg) in which we

have added the drug in ascending concentration. The first

set of 12 bottles was named as “Master copy” and the second

set as “Replica copy”. The Methimazole tablets were

crushed into fine powder using mortar and pistle. The powdered drugs were weighed in amounts ranging from

0.1mg-1.0mg and were mixed with yeast granules and were

added to above marked bottles. The virgin flies from pure

culture were then transferred into each bottle, containing

five males and five female flies. The flies were closely

monitored during the mating period. Once the eggs were

laid, the each developmental stage starting from egg, first,

second, third instar larva, pupa and adult morphological

changes due to acute drug exposure were recorded. The

time required for larval ecdysis, pupal conversion followed by emergence of the adult were compared with the control

flies.

RESULTSAfter closely monitoring the drug treated flies following

observation were recorded. Life cycle was delayed with

increasing drug concentration (table: 1.1) followed by increase in larval size but with a thinner morphology

compared to wild type. Reduced puffing was seen in drug

treated third instar larvae. Adult flies had emerged with morphological changes including thin and elongated

abdomen, abdomen curving (Fig: 1.2), deformed wings

andl imbs (Fig:1 .3)a longw ithd epigmentation.

Days control 0.01mg 0.02mg 0.03mg 0.04mg 0.05mg 0.06mg 0.07mg 0.08mg 0.09mg 0.1mg1 Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs2 Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs3 Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs4 Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs Eggs

5 First First First First First First First First First First First instar instar instar instar instar instar instar instar instar instar instar

6 First First First First First First First First First First Firstinstar instar instar instar instar instar instar instar instar instar instar

7 Second Second Second Second First First First First First First Firstinstar instar instar instar instar instar instar instar instar instar instar

8 Third Third Third Third Second Second Second Second Second Second Second instar instar instar instar instar instar instar instar instar instar instar

9 Pupa Pupa Pupa Pupa Third Third Third Second Second Second Secondlarvae larvae larvae larvae instar instar instar instar instar instar instar

10 Flies Pupa Pupa Pupa Third Third Third Third Third Third Thirdlarvae larvae larvae instar instar instar instar instar instar instar

11 Flies Pupa Pupa Pupa Third Third Third Third Third Third Thirdlarvae larvae larvae instar instar instar instar instar instar instar

12 Flies Flies Flies Flies Third Third Third Third Third Third Thirdinstar instar instar instar instar instar instar

13 Flies Flies Flies Flies Pupa Pupa Pupa Pupa Pupa Pupa Pupalarvae larvae larvae larvae larvae larvae larvae

14 Flies Flies Flies Flies Pupa Pupa Pupa Pupa Pupa Pupa Pupa15 Flies Flies Flies Flies Pupa Pupa Pupa Pupa Pupa Pupa Pupa

16 Flies Flies Flies Flies Flies Flies Flies Pupa Pupa Pupa Pupa17 Flies Flies Flies Flies Flies Flies Flies Flies Flies Flies Flies

Table: 1.1 showing the life cycle of Drosophila melanogaster at mg weight of drug with methimazole.


Figure: 1.2 Thin and elongated abdomen of drug treated flies

Fig: 1.3 leg and wing deformities of drug treated flies

DISCUSSIONMolting in insects are regulated by the steroidal moulting hormones ecdysteroids. In the immature stages of insects, the prothoracic glands are the primary source of ecdysteroids.. Ecdysone undergoes 20-hydroxylation in certain peripheral tissues, yielding 20-hydroxyecdysone, which is considered to be the major active moulting hormone in most insect species. At the onset of Drosophila metamorphosis, the steroid hormone 20-OH ecdysone directly induces a small number of early puffs in the polytene chromosomes of the larval salivary gland. Proteins encoded by the early genes corresponding to these transcriptional puffs then regulate the activity of both the early puffs themselves and a much larger set of late puffs. Three of these early genes encode transcription factors that play critical regulatory roles during metamorphosis. In Drosophila, changes in the concentration of the steroid hormone 20-OH ecdysone regulate specific developmental events, metamorphosis from the larval to the adult form. The early stages of metamorphosis are coordinated by three


discrete ecdysone pulses. The late-larval ecdysone pulse triggers metamorphosis and leads to puparium formation some 6 hours later, transforming the feeding larva into an immobile white prepupa. Immediately after pupariation, the whole-body ecdysone concentration rapidly declines to a low level by the mid-prepupal stage, followed by a second, prepupal ecdysone pulse that peaks 10 hours after pupariation. Head eversion occurs 2 hours later, giving rise to a fully formed pupa. Pupation then is followed by a broad pupal ecdysone pulse that is necessary for tissue differentiation and adult development.

The hormonal signal is transduced primarily through the action of an ecdysone receptor that binds both DNA and hormone as a heterodimer of two nuclear receptor proteins encoded by the ecdysone receptor (EcR) and ultraspiracle (usp, the Drosophila RXR gene) genes . The EcR gene encodes three protein isoforms (EcR-A, EcR-B1, and EcR-B2), that 44are differentially expressed in tissues with different hormone responses . Although all three isoforms contain the same DNA-binding domain, functional differences between the isoforms may contribute to the cellular and tissue specificity of the hormone response . Formation of a functional ecdysone receptor also requires the activity of a complex of molecular chaperones.

Ecdysone hormone cannot bind directly to DNA. It requires ecdysone hormone receptor for its expression in the tissue. This ecdysone receptor is similar to thyroid hormone receptor. When the flies are treated with methimazole (hyperthyroid drug) they go and bind to these receptors thus ecdysone cannot bind effectively to this receptor resulting in delay in life cycle by affecting metamorphosis.

In some polyploid insect cells, the sister chromatids do not separate during replication and they aggregate to form polytene chromosomes. With a thousand copies all aligned, the more tightly condensed regions appear as dark bands while the relaxed regions appear as light bands. The result is a distinct banding pattern for each chromosome. The banding patterns also reveal the transcriptional activity of the chromosomes. In regions of high transcriptional activity, the polytene chromosomes decondense at the sites of the induced genes. This decondensed chromatin appears as “puffs” or looped regions in the chromosome. Puffing is induced by ecydsone hormone. Ecdyscone binds to

DNA via ecdysone receptor receptor and brings about puffing in the polytene chromosome. Methimazole interferes with the binding of ecydsone to its receptor resulting in reduced puffing in polytene chromosome.

Zygotic genes like Bicod. Hunchback and caudal transcriptionally regulate gap genes. After expression of gap gene product pair-rule gene is expressed followed by segment polarity genes. They all are expressed in zygotic condition. After their expression the syncytial blastoderm is converted into a cellular blastoderm. The cellular blastoderm undergoes gastrulation movement morphogenesis and differentiation to form first instar larvae which undergoes a series of molting to form the imago. Methimazole interferes with the above genes resulting in thin long larvae and the adults with elongated curved abdomen.

The colt gene consists of a single exon and encodes a protein of 306 amino acids made of three tandem repeats, each characterized by two predicted transmembrane segments and a loop domain. The colt protein shares extensive homology with proteins in the mitochondrial carrier family protein, which has been shown to be maternally required for embryonic tissue differentiation. The zygotic colt gene is involved in normal embryonic morphogenesis, gas-filling of the tracheal system, at hatching time of the embryo and for normal epithelial morphogenesis of the wings. Methimazole may interfere with the normal functioning of colt gene and thus leading to wing deformities.

The development of limbs depend directly on pax family gene, wingless gene, decapentaplegic, homeothorax and distal-less gene. Genetic analysis have shown that these genes functions as a developmental switch that is required to promote the development of limb structures above the evolutionary ground-state of body wall. Reduction of this activity has a global effect on pattern formation in the limb. Methimazole may interfere with the expression level of these gene products leading to limb deformities.

The Abdominal-B Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. Methimazole may interfere with the expression of this pigmentation gene and thus leading to de-pigmentation of drug treated flies.

In human it has been reported that methimazole crosses the placenta and has been possibly linked to a number of birth defects. Case reports have suggested that severe


anomalies can occur in infants exposed to methimazole during pregnancy. Stillborn at 32 wk of gestation and exhibited cardiac defects, esophageal atresia with tracheoesophageal fistula, omphalocele, and hydrops. Methimazole readily crosses the placental membranes and can induce goiter and even cretinism in the developing fetus. Thus methimazole is a teratogen and not suggested during pregnancy.

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genome sequence of Drosophila melanogaster”. Science 287(5461);2185-95.

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• Barnes, G.L., Mariani, B.D. and Tuan, R.S., Teratology, 1996, 54, 93-102.

• Berglund J, Christensen SB, Dymling JF, Hallengren B (May 1991). "The incidence of recurrence and hypothyroidism following treatment with antithyroid drugs, surgery or radioiodine in all patients with thyrotoxicosis in Malmo during the period 1970-1974". Journal of Internal Medicine 229 (5): 435–42.

• Casares, F. and Mann, R.S., Nature, 1998, 392,723-726.

• Crescioli C, Cosmi L, Borgogni E, et al. (October 2007). "Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes". J. Endocrinol. 195 (1): 145–55. doi:10.1677/JOE-07-0240.PMID 1 7 9 1 1 4 0 6 . h t t p : / / j o e . e n d o c r i n o l o g y -j o u r n a l s . o r g / c g i / p m i d l o o k u p ? view=long&pmid=17911406.

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• Nakamura H, Noh JY, Itoh K, Fukata S, Miyauchi A, Hamada N (June 2007). "Comparison of methimazole and propylthiouracil in patients with hyperthyroidism caused by Graves' disease". The Journal of clinical endocrinology and metabolism 92 (6): 2157–62. doi:10.1210/jc.2006-2135. PMID 17389704


Antimicrobial Activity of leaf Extracts of Nyctanthes arbortristis linn. (Oleaceae)

M. Reena Prathipa*, Sunil.B & Chitra.G

Department of Biotechnology, Garden City College, Bangalore – 560049, Karnataka, India

email: * [email protected], [email protected], [email protected]

AbstractThe extract of the leaves of Nyctanthes arbor-tristis Linn. belonging to family of Oleaceae widely used medicinal plant in Ayurvedic system of medicine for the treatment of sciatica, arthritis, fevers, various painful conditions and diuretics as laxative. The aim of the present study was to evaluate the antimicrobial activity of the leaf and seed extracts. The test organisms were Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Salmonella Typhi. The zone of inhibition and Minimum Inhibitory Concentration (MIC) of the extracts were determined and compared with the standard antibiotics Tetracycline, Ampicillin, Gentamycin. The methanol extract was found to have good antibacterial whereas the acetone shows active only in Escherichia coli.

Key words: Nyctanthes arbortristis, Oleaceae, Antimicrobial, Ayurvedic, medicinal plant.

IntroductionThe medicinal plants are the plants whose parts (leaves, seeds, stem, roots, fruits, foliage etc.), extracts, infusions, decoctions, powders are used in the treatment of different diseases of humans, plants and animals (Nostro et al., 2000). Extracts from many plant parts are highly efficient against parasitic as well as microbial infections. The frequency of life-threatening infections caused by pathogenic microorganisms has increased worldwide and is becoming an important cause of morbidity and mortality in immune compromised patients in developing countr ies and many infect ious microorganisms are resistant to synthetic drugs; hence

an alternative therapy is very much needed. It is estimated that around 70,000 plant species, from lichens to tall trees, have been used at one time to other for medicinal purposes (Purohit & Vyas, 2004). The use of different parts of several medicinal plants to cure specific ailments has been in vogue from ancient times. The indigenous system of medicine namely Ayurvedic, Siddha and Unani have been in existence for several centuries. This system of medicine caters to the needs of nearly 70% of the population residing in villages. Besides the demands made by these systems as their raw material, the demands for medicinal plants made by themodern pharmaceutical industries has also increased manifold (Gupta et al., 1999; Ashraf & Orooj, 2006; Ashraf et al., 2006).

The medicinal plants occupy a significant place in modern medicine as a raw material for some important drugs, although synthetic drugs and antibiotics brought about a revolution in controlling different diseases. But these synthetic drugs are out of reach to millions of people. Those who live in remote places depend on traditional healers, whom they know and trust. The judicious use of medicinal herbs can even cure deadly diseases that have long defied synthetic drugs (Bhattacharjee, 2001). The plants used, as drugs are mostly unobjectionable and relatively free from toxic effects or were so toxic that lethal effects were well known. The nature has provided the storehouse of therapeutics to cure all ailments of humankind. They are effective in the treatment of infectious diseases while at the same time mitigating many of the side effects that are often associated with synthetic antimicrobials . There is no doubt that plants are a reservoir of potentially useful


compounds which serve as drugs, are provided fresher leads and clues for modern drug design by synthesis . Antimicrobials of plant origin have enormous therapeutic potential. Nyctanthes arbortristis Linn. commonly known as Harsinghar or Night Jasmine or Parijatah or Harsinghar or Pavizhamulla is a popular plant with fragrant flowers described in Ayurveda is being sporadically used for its anti-microbial activity by several Ayurvedic physicians. Different parts of N. arbortristis are known to possess various complaints by rural mainly tribal people of India (Kerala, Orissa and Bihar) along with its use in Ayurveda, Sidha and Unani systems of medicines. Traditionally the powdered stem bark is given in rheumatic joint pain, in treatment of malaria and also used as an expectorant. Plant is used as anthelmintic, laxative, dysentery, sores, ulcers, leafs for fever, rheumatism, chronic fever, intestinal worms.

MATERIAL AND METHODSPlant material collection and identification:

Leafs and seeds of N. arbortristis Linn. was collected from Thiruvannamalai hills of Tamil Nadu, India in October, 2009. The plant was authenticated at Department of Botany, NSS College, Pandalam, kerala.

Preparation of crude extract.

The leaves and seeds were freed from dirt by washing with distilled water. The moisture content was removed by air drying. After excising, the leaves were mixed with 10 mM sodium acetate buffer (Terras et al., 1993). The seed were powdered and the extracts were prepared in a solution mixture of 10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 2 mM EDTA, 1.5% polyvinylpolypyrrolidone (PVPP), 1mM phenyl methyl sulfonylfloride (PMSF) and 2 mM thiourea. The leaf and seed mixtures were centrifuged at 12,000 xg at 4oC for 20 min. The supernatant was filtered with whatmann filter paper no-1 to remove debris present in the supernatant. The filtrates were stored at -20oC in 100 mL sterilized bottles (Terras et al., 1992).

SEPARATION:Spot the sample of plant extract at one side of the slide at a distance of 0.5cm from the edge . Place the slide obliquely in the solvent containing chamber in such a way that the spotted edge just dips above the solvent and allow the solvent to run. Different combination of solvents (mobile phase) in 1:1were used for various extracts.

DEVELOPMENT AND ANALYSIS:After the run is over, the slides are dried and observed under the UV trans illuminator to

observe the different bands of different chemical compounds. The bands in extract which

were good were subjected to column chromatography to purify that extract.

Column Chromatography of plant extractsThe method as described in MSC Part 1 journal of Institute of science was used to carry

out the separation of compounds present in the plant extract.

Silica gel slurry:-Slurry of silica gel and eluting solvent is prepared.

Experimental procedure:-The extracts are added directly (or carefully down the side of the column) to the column.Then the mobile phase is drained continuously to the top of the column. The bottomoutlet of the column is opened. The eluent flows down through the column. The column,with the adsorbent and the sample, is 'developed': As the eluent passes down the column,the components of the mixture begin to move down the column. The separated zones 'flow out' of the column, where the eluates are collected in tubes. The tubes are changed as the eluate changes colour. A bright beam of light is directed at the leaf extract and at the samples eluted from chromatography column. Leaf extract and the samples containing chlorophyll or pheophytin or any other compounds produce a reddish glow. This phenomenon is known as fluorescence, through which the study was carried out.

Salmonella typhiiMethanol Extract 1.9 2.0 0.6 0.65Diethyl ether Extract 1.7 1.5 0.7 0.55

Klebsiella pneumoniaeMethanol Extract 1.5 1.5 0.8 0.65

Staphylococcus aureusMethanol Extract 1.8 1.8 0.7 0.9

E. coliMethanol Extract 1.4 0.6 00 1.0Ethanol Extract 1.5 2.5 7.0 4.5

PseudomonasHexane Extract 1.7 1.0 00 00

GENTAMYCIN TETRACYCLIN AMPICILLIN EXTRACT

Salmonella typhii

Ethyl acetateExtract 1.7 1.6 0.8 1.1

Klebsiella pneumoniae

Acetone Extract 1.2 1.6 0.8 0.5

Staphylococcus aureusAcetone Extract 1.6 1.6 00 00

GENTAMYCIN TETRACYCLIN AMPICILLIN EXTRACT

TABLE II: Showing comparative antibiotic analysis of seed extract


RESULTS

COMPARATIVE ANTIBIOTIC ANALYSIS

TABLE I: Showing comparative antibiotic analysis of leaf extract


GRAPHICAL COMPARATIVE DATAFig 1. Graph for Antibacterial activity of leaf extract.

Fig 2. Graph for antibacterial activity of seed extract.

DISCUSSIONFrom the following results, it can be ascribed that the antibiotic property of leaf and seed extract of Nyctanthes arbortristis is very potent when compared to standard antibiotics present and thus it is highly effective against preventing the growth of microorganisms. The concentration of compounds present in the extract is almost 10- 15 % of the total sample taken. From this observation, the concentration of extracts is nearly 20 microgram. While the concentration of standard antibiotics taken for the comparative study was 400 microgram. Thus, it can be ascribed that at equal concentration, pure extract from Nyctanthes arbortristis will have much better activity when compared to the standard antibiotics. This property of the extracts relies

on the compounds present in the extract. Further study on this aspect has the potential to lead towards a few more astonishing facts towards the antibiotic property of its extracts.

REFERENCE1. Al-Bari MAA, Sayeed MA, Rahman MS.

Characterization and antimicrobial activities of extracts in a phenolic acid derivative produced by Streptomyces bangladeshiensis, a novel species collected in Bangladesh. Res. J. Med. and Med. Sci., 2006, 1: 77-81.

2. Kiew R, Baas P. Nyctanthes is a member of Oleaceae. Proc. Indian Acad. Sc. (Plant Sc.). 1984, 93(3): 349-358.


3. Iwu MW, Duncan AR, Okunji CO. New antimicrobials of plant origin. In: J. Janick (ed.), Perspectives on new crops and new uses. ASHS Press, Alexandria, VA. 1999, 457-462.

4. Nadkarni AK. Indian Materia Medica, Vol.I, 3rd ed. (Popular Prakashan Pvt. Ltd.,) 1982, 857-858.

5. Kirtikar KR, Basu BD. Indian Medicinal Plants, Vol.VII, (Sri Satguru Publications, New Delhi,) 2000, 2110-2113.

6. Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, Vol.VII, (National Institute of Science Communication, CSIR, New Delhi), 1997, 69-70.

7. Sasmal D, Das Sanjita, Basu S P. Phytoconstituents and therapeutic potential of Nyctanthes arbortristis Linn., Pharmacognosy Reviews, 2007; 1 (2): 344-349

8. Khandelwal KR, Kadam SS, Singhama. Antibacterial acivity of the leaves of Nyctanthes arbortristis Linn. Indian J. Nat. Prod., 1999; 15: 18-20.

9. Cowan M, Steel L. Preparation of the test organisms. Niger. J. Microbiol. 1965; 22: 56-60.

10. Osadebe PO, Ukwueze SE. A comparative study of the phytochemical and antimicrobial properties of the Eastern Nigerian species of African Mistle foe (Loranthus nucranthus) sourced from different host trees. J. Biol. Res. Biotechnol., 2004; 2(1): 18-23.

11. Iwaki K, S Koya-Miyata, Kohno K, Ushio S, Fukuda S. Antimicrobial activity of Polygonum tinctorium Lour: extract against oral pathogenic bacteria. Nat. Med., 2006; 53: 72-79.

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antifungal and cytotoxic activities of tuberous roots of Amorphophallus campanulatus. Turk. J. Biol., 2007; 31: 167-172.

13. Reddy, P.B., Reddy, C.K., Rambhau, D., Venkateshvaralu, V. and Murthy, V.N., Indian J. Pharm. Sci., 1993, 55, 134. Back to cited text no. 1

14. Handa, S.S., Pharma Times., 1991, 23, 13. Back to cited text no. 2

15. Kirtikar, K.R. and Basu, B.D., In; Indian Medicinal Plants, 3rd Edn., Vol. II, International Book Distributors., Dehradun, 1995, 1526. Back to cited text no. 3

16. Yoganarasimhan, S.N., In; Medicinal plants of India - Karnataka, Vol. I; Interline Publishing Pvt. Limited., Bangalore, 1996, 333. Back to cited text no. 4

17. The wealth of India, A Dictionary of Indian raw materials and Industrial products, Vol. VII, New Delhi publication and Information Director., CSIR, New Delhi, 69. Back to cited text no. 5

18. Ghosh, M.N., In; Fundamentals of Experimental Pharmacology, Scientific Book Agency, Kolkata, 1984, 156. Back to cited text no. 6

19. De, S., Sheila, V.J., Ravishankar, B. and Bhavasar, G.C., Fitoterapia., 1996, LXVII, 106. Back to cited text no. 7

20. Chandrashekhar, V.M., Abdul Haseeb, T.S., Habbu, P.V. and Nagappa, A.N., Indian Drugs., 2004, 41, 366. Back to cited text no. 8


Ms Rupal Gogia Ms Sayantanee Basu Maitra

Ms Sheeja MS*Department of Biotechnology, Capital College, Bangalore – 36

*Authors Correspondence: [email protected]

Antimicrobial Activities of Various Plant Extracts on Different Bacterial SPP

ABSTRACTNature is the abode to various forms of life that show different relations with the other life forms. This study aims at attempting to understand the antimicrobial actions of Betel leaf (Piper betle), Capsicum (Capsicum annum), Clove (Eugenia caryophyllus), Garlic (Allium sativum) and Hibiscus (Hibiscus rosasinensis) on organisms like E.coli, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella and Klebsiella. The method used to study was the antimicrobial activity by zone of inhibition process. Each of these sources was tested on the different organisms mentioned above. The zones of inhibition were tabulated for each and every source. The antimicrobial activity shown by the different sources could be due to the various phytochemical or biochemical components present in the sources. These antimicrobial properties can be further exploited to study their effects and their uses medically.

Key Words: Antimicrobial activity, Zone of inhibition, sources, organisms.

INTRODUCTION

The nature is an abode of various forms of life living in harmony or antagonistic to each other. Apart from this, there are various chemicals in abundance in the nature in the form of spices, fruits, vegetables which inhibit the growth of other forms of life. This present paper aims to study the antimicrobial activity of certain nature procured forms such as spices, vegetables, flowers etc.

The following is a study of antimicrobial activity of Betel leaf (Piper betle), Capsicum (Capsicum annum), Clove (Eugenia caryophyllus), Garlic (Allium sativum) and Hibiscus (Hibiscus rosasinensis) on organisms like E.coli, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella and Klebsiella.

Betel (Piper betle) is a creeper, mostly cultivated in South and South East Asia(3). The potent chemicals found in betel leaf are a class known as allylbenzene which include particular chemicals like chavibetol, estragole, eugenol, methyl eugenol, hydroxycatechol. Apart from this it also contains terpenes and terpenoids(3). It also has many medicinal properties like it has analgesic which gives relief in headache. It is useful in the treatment of nervous pains and beneficial in pulmonary affection in childhood and old age. It is also effective in treating sore throat, inflammation such as arthritis and orchitis (inflammation of the testes) (5).

Capsicum (Capsicum annum) a native spice of America and is cultivate all over the world (3). The ethanolic extract of the fruit shows antimicrobial activity against both Gram positive and Gram negative bacteria and some reports show Capsicum to be antifungal(4).

Clove is a dried flower bud of Eugenia caryophyllus, family Mytraceae(1). Literature sites that the essential oil of the spice has a greater antibacterial activity. Eugenol has the major role in the antimicrobial activity upon food borne pathogens (2).

Aqueous and ethanolic extracts of Garlic (Allium sativum) shows a wide range of antimicrobial activity (6). It is not only known as a bactericidal agent but it is also known for its effect against cold, cough, asthma. The agent also has the ability to boost up the immune system lower blood cholesterol, fight against cancer and virus (7). The key to its antimicrobial activity is thiosulfinates(8) which at times referred to as allicin (Cavallito and Bailey,1944)(7).

Malvaceae family member Hibiscus rosasinensis has considerable amount of antimicrobial activity against Gram negative bacteria. Chemicals such as cyaniding, quercetin, hentriacontane, calcium oxalate, thiamine,


riboflavin, niacin, ascorbic acid and flavonoids are present in the flower extract (9). The family shows its usage as a cardioprotective, hypocholesterolemic, antioxidative agent.

MATERIAL AND METHODS

1. Preparation of Test organisms: The test organisms used were E.coli, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella and Klebsiella.

The primary cultures of the organisms were produced by inoculating the test organisms in nutrient broth and growing them for 18hours at 370C .

The test organisms were then streaked aseptically on to nutrient agar plates and incubated at 370C for 24 hours.

A loopful of the cultures were transferred into 4% peptone broth and incubated at 370C for 18 hours.

The test organisms were then used to swab on Muller Hilton agar medium for study of antimicrobial activity.

2. Preparation of the extracts

1. Betel LeafThe leaf was surface sterilized using 70% ethanol and then dried aseptically to evaporate the excess ethanol. The leaf was then placed in a 600C preheated hot air oven for 4 hours. The leaf was then crushed and 1g of this powder was taken and suspended in 10 ml of chloroform. The set up was left for 24 hours. Next day the suspension was homogenized in mortar and pestle under aseptic condition and filtered through Whatmanns filter paper.

2. CapsicumThe fruit was surface sterilized using 70% ethanol and then cut into small pieces and left for drying in a preheated (60 0C) oven for 72 hours. It was then powdered using a sterile grinder. 1g of this powder was suspended in 10 ml of ethanol and left over night. The suspension was filtered through Whatmanns filter paper.

3. Clove1g of surface sterilized clove was suspended in10 ml sterile distilled water for 24 hours. The suspension was then homogenized using sterile mortar and pestle. The extract was then passed through Whatmanns filter paper.

4. Garlic100g of garlic was peeled and then surface sterilized using 70% ethanol and then left in sterile condition for the ethanol to evaporate. The cloves of garlic were then crushed using sterile grinder and passed through doubled muslin cloth to extract the 100% garlic juice. It was further passed through Whatmanns filter paper.

5. Hibiscus1g of Hibiscus flower was surface sterilized (using 70% ethanol) .The flower was dried in a 600C preheated hot air oven for 24 hours. It was then made into a fine powder. 1g of this powder was suspended in 10ml of ethanol and left over night. This suspension was further filtered using Whatmanns filter paper.

All the extracts can be stored at 40C when not in use.

ASSAY OF ANTIMICROBIAL ACTIVITYMuller Hilton agar plates were prepared and swabbed with18hours culture of the test organisms and left aside for 5 min in the upright position.

Wells were bored in each plate such that each well could hold up to 100µl of the extract.

The extracts were then introduced into the well as per designated on the labels. The plates were incubated at 370C for 24 hours in the upright position.

All organisms except Staphylococcus aureus showed result within 24hours which in case of Staphylococcus aureus was 48 hours and the zone of inhibition was checked.


RESULTSThe zones of inhibition obtained are tabulated below.

Test Organisms/Agents Betel leaf Capsicum Clove Garlic Hibiscus

E.coli 20 mm No zone 18 mm 36 mm 20 mm

Staphylococcus aureus 46 mm 14 mm 25 mm 20 mm No zone

Pseudomonas fluoresens 25 mm No zone 10 mm 40 mm 25 mm

Salmonella 25 mm 30 mm 40 mm 34 mm No zone

Klebsiella 22 mm No zone 15 mm 56 mm No zone

The above mention study infers garlic as a potent antimicrobial agent.

The antibacterial activity of all the spices is shown in the following graphs.

Fig:1 Antimicrobial activity of Betel leaf on test organisms

Fig:2 Antimicrobial activity of Capsicum on test organisms

Fig:3 Antimicrobial activity of Clove on test organisms


Fig:4 Antimicrobial activity of Garlic on test organisms

Fig:5 Antimicrobial activity of Hibiscus on test organisms

DISCUSSIONBetel leaf shows a wide range of antimicrobial activity against both Gram positive and Gram negative organisms.

Capsicum does not show a wide range of result but is again effective against both classes of Gram positive and Gram negative organisms.

The growth of Salmonella has been highly prevented by the use of clove extract where as the minimum is seen in case of Pseudomonas.

Garlic is also effective against all the microorganisms used as the test organisms.

Hibiscus shows a considerable amount of antibacterial activity against Gram negative organisms.

CONCLUSIONThe study sites the screening of other common agents for its antimicrobial activity which not only inhibits growth of microorganisms and gives strength to our body to fight against disease but also provides minimum or no side effects which in turn will be a mile stone in the

long journey towards newer and newer drug discoveries.

REFERENCES1. Antibacterial activity of clove extracts against

phagogenic strains including clinically resistant isolates of Shigella and Vibrio cholerae Sk. Nazrul Islam* Abu J. Ferdous,** Monira AhsaN** and A.B.M. Faroque** *Institute of Nutrition and Food Science, University of Dhaka, Dhaka 1000, Bangladesh. **Department of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh

2. Md. Mahfuzul Hoque, M. L. Bari, Vijay K. Juneja, and S. Kawamoto, Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh. National Food Research Institute, 2112 Kannondai, Tsukuba, Ibaraki 305864 2, Japan Food Safety Intervention Technologies Research Unit, Eastern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA

3. www.wikipedia.org


4. S.Soetarno, Sukrasno, E. Yulinah and Sylvia, Department of pharmacy, faculty of Mathematics and Natural Science, Jl. Ganesa 10, Bandung, 40132 – Indonesia, September 27th, 1997

5. www.kiva.org

6. African Journal of Biotechnology Vol. 3 (10), pp. 552-554, October 2004, Studies on the antimicrobial effects of garlic (Allium sativum Linn), ginger (Zingiber officinale Roscoe) and lime (Citrus aurantifolia Linn) Onyeagba R.A.1*, Ugbogu O.C. 2, O k e k e C . U . a n d I r o a k a s i . O . 1 1 Department of Microbiology, Abia State University, PMB 2000 Uturu, Nigeria

2 Department of Botany, Abia state University,PMB 2000 Uturu, Nigeria

7. Current Research Journal of Biological Sciences 1(3): 123-126, 2009) Antibacterial Activity of Garlic Varieties (Ophioscordon and Sativum) on Enteric Pathogens 1S. Shobana,2V.G. Vidhya and 3M. Ramya 1Department of Bioinformatics, School of Bioengineering, SRM University, Kattankulathur-

603203, 2 Department of Biotechnology, Science and Humanities, SRM University, Kattankulathur-603203, Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur-603203

8. Electronic Journal of Biology, 2009, Vol. 5(1): 5-10, In vitro Antibacterial Activity and Stability of Garlic Extract at Different pH and Temperature Srinivasan Dura i r a j 1 , * , S ang ee tha S r i n iva s an2 , P. Lakshmanaperumalsamy3, 1 Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing MI-48824, USA, 2 Department of Crop and Soil sciences, Michigan State University, East Lansing MI-48824, USA, 3 Department of Environmental Sciences, Bharathiar University, Coimbatore-614046, India

9. Antibacterial Activity of Some Selected Indian Medicinal Flora R. NAIR, T. KALARIYA, Sumitra CHANDA Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat – INDIA


In-vitro Study of Cissus QuadrangularisLeaf Extract on Staphylococcus Sps.

Isolated from Old Books*Shruti Awasthi,Ganesh P. Surati,

V.N. Shetty & @ P.chary

Garden City College, BANGALORE-49email ID: *[email protected] | [email protected]

ABSTRACTMicro – organisms are omnipresent. Thus, the pages on which we pen down our imagination are also the habitat for these microbes, with this in mind ,we isolated micro – organisms from a very old book of 25 years from our College library. By gram – staining and observation under the microscope, Cocci were observed. On performing various biochemical tests such as catalase , oxidase, starch hydrolysis, gelatin, we identified the organisms to be a species staphylococcus sps . Antibacterial test were performed in – vitro using Cissus quadrangularis leaf extracts to study its role on the inhibition of this pathogen. A standard borewell method was employed.

Keyword : Cissus Quadrangularis, Staphyloccocus ,Cataalase test, Indole test

INTRODUCTIONMicroorganisms like bacteria are omnipresent ,which exist either as Cocci or rods. Among the Cocci, Staphylococcus sps., E.coli sps., Bacillus sps. And Diplobacillus sps. are some common examples

Staphylococcus sps. is a gram-positive bacterium that appears as a grape like clusters. The Staphylococcus genus has about thirty-three species. Most of them are harmless and reside normally on the skin and mucous membranes of humans and other organisms. Also found worldwide, they are a small component of soil microbial flora. Staphylococcus can cause a wide variety of diseases in humans and other animals through either toxin production or invasion. Toxins from this organism is a common cause of food poisoning, When food is improperly stored.

Cissus Quadrangularis linn. is an ancient medicinal plant native to Sri Lanka and India.Common folk in India uses this plant for promoting the fracture healing process of bones. Thu, Cissus Quadrangularis is an indigenous medical plant. It has been prescribed in Ayurveda as an alterative, anthelmintic, dyspeptic, digestive, tonic, analgesic in eye and ear diseases, and in the treatment of irregular menstruation and asthma. In Cameroon, the whole plant is used in oral re-hydration, while the leaf, stem, and root extracts of this plant are important in the management of various ailments.

MATERIALS AND METHOD

MATERIALS :Petridish, cotton swabs, boiling tube, NA media , gelatin, iodine, inoculation, alcohol, spirit lamp,etc.

METHOD :Using basics concepts of microbiology.We prepared agar media, blood agar media as a nutrient media (S. S. FINE CHEM. LIMITED, MUMBAI.)bacteria needs agar media i.e. Na media for its growth .Now we isolated the bacteria by swabbing with the cotton swabs from middle of the book and applied it on NA media plate .Now we incubated it for 18 hour at 37 deg.C and we observed white coloured colonies on NA media plates .We sub cultured the already prepared bacteria and thus we obtain NA media plate containing a pure culture. GRAM STAINING we did gram staining using the gram’s iodine , crystal violet stain , saffranin , alcohol and bacterial pure culture and we performes biochemical test such as starch hydolysis , gelatin test , indole test , methyl red test , catalase test and oxidase test.


ObservationThe results for various biochemical and staining tests are given as below in the table:.

RESULTS:The bacteria which were isolated from old books were found to be Cocci. These were confirmed to be Staphylococcus sps. Based on biochemical tests such as catalase , oxidase , methyl red and gelatine test which were positive, whereas the starch hydrolysis and indole test showed a negative results , this confirms the presence of Staphylococcus sps .

The antimicrobial effect using the crude extract of Cissus quadrangularis was conducted. Cissus quadrangularis which was extracted from organic solvent showed a antibacterial property against pathogenic bacteria.

Conclusion and DiscussionWe concluded that that the organism isolated from the old books was found to be Staphylococcus in-vitro antibacterial tests using a crude juice of Cissus qudrangularis on Staphyloccus showed a clear zone of inhibition.. Pathogenic bacteria like Staphylococcus can cause a wide variety of diseases in humans and other animals through either toxin production or invasion. Staphylococcal toxins are a common cause of food poisoning, as it can grow in improperly-stored food. In year 1964 two scientist namely Evenleigh and Brewer observed bacteria isolated from old papers from Canadian paper Mill. Research shows that Cissus has been "discovered" by sports and nutritional supplement manufacturers and, mostly based on anecdotal evidence, many claims for "weight loss" and "muscle protective" effects are being made. Cissus is known to be an ancient medical plant, with optimal healing in white tissue areas of the body ( tendons & ligaments).Our study concludes that this ‘medicinal plant’ as well as ‘ bone healer’ also showed a ‘antibacterial property against pathogenic bacteria .

ACKNOWLEDGEMENTWe are greatful to our beloved chairman Dr. Joseph V. G. and Principal Mr. K.C. JOHN for providing us with an opportunity to work on this project.

REFERENCE

1. ANATHANARAYAN AND PANIKAR’S TEXTBOOK OF MICROBIOLOGY, BY: C.K.J. PANIKAR.,(7th edition)

2. ADVANCES IN GENERAL MICROBIOLOGY SHARAD SHRIVASTAVA and VINEETA SINGHAL (VOLUME-5)

3. LABORATORY MANUAL IN GENERAL MICROBIOLOGY N.KANNAN (H.O.D OF MICROBIOLOGY P.S.G COLLEGE OF S C I E N C E A N D A RT S C O I M B T O R E ,TAMILNADU

4. T E X T B O O K O F M I C R O B I O L O G Y CHANDRAKANT KELMANIEDITED BY : PROFESSOR S.B.SULIA.PUBLISHED BY : UNITED PUBLISHERS

5. RESEARCH PAPER :U.S. PATENT 2007-10020719 APPLICATION PUBLICATION DURAN VILA et. al.

Tests Inference

STARCH HYDROLYSIS -ve

GELATINE HYDROLYSIS +ve

INDOLE TEST -ve

CATALASE TEST -ve

OXIDASE TEST +ve

METHYL RED TEST +ve

TABLE 1: Note Gram – staining and biochemical tests indicated that the Micro -organism was Staphyloccous sps.

LEGENDS:Figure 1 : BACTERIAL CULTURE

Figure 2 : PURE BACTERIAL CULTURE

Figure 3 : Oxidase test

Figure 4 : Catalase test

Figure 5 : Starch hydrolysis

Figure 6 : M.R. TEST

Figure 7 : GELATIN TEST

Figure 8 : INDOLE TEST

Figure 9: GRAM STAINING STAPHYLOCOCC GRAM POSITIVE

F i g u r e 1 0 : C R U D E J U I C E O f


C.QUADRANGULARIS

Figure 11: ANTIBACTERIAL TEST

Figures:

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8


Figure 9

Figure 10

Figure 11


The Effects of Time, Temperature & IPTG

Concentration on IPTG Induced ?-galactosidase Activity using Onpg as Substrate

Urmimala Ray, Department Of Biotechnology,Garden City College

email : [email protected]

ABSTRACT

?-Galactosidase is one of many enzymes that are involved in breaking down lactose to glucose and galactose. IPTG, isopropylthiogalactoside, is a lac inducer that either upregulates or down regulates the

lacZ gene for transcription of ?-galactosidase. The

substrate o-nitrophenyl-?-D-galactopyraniside

(ONPG) can be a substitute for lactose. When the ?-galactosidase cleaves ONPG, o-nitrophenol is released,

which has a yellow color, and absorbs 420 nm light. ?-galactosidase activity can be measured by monitoring the accumulation of yellow color (increase 420 nm absorbance)/minute. Varying the physical conditions, it was observed that with increasing time of reaction and temperature, the activity also increased. Also with different IPTG concentration, it was concluded that a concentration of 5µm produces a significantly greater amount of protein expression and is the optimal amount

for maximal ?-galactosidase activity.

INTRODUCTIONß-galactosidase, (ß-gal) is a hydrolase enzyme that catalyzes the hydrolysis of ß-galactosides into monosaccharides. The LacZ gene is derived from the E.Coli chromosome. And when the gene is induced, its product is the protein, ß –Galactosidase, which is able to hydrolyze (cleave) ß -D-galactosides. This enzyme facilitates growth on carbon sources like lactose by cleaving it into a molecule of glucose and a molecule of galactose which the cells can catabolize and grow on. IPTG can have dual roles. It is used as a promoter, but also acts as an inhibitor. Lactose is a type of sugar found in milk. Like lactose ONPG is a molecule composed of two rings held together by an oxygen bridge. This bridge can be broken when the enzyme ß-galactosidase binds to lactose and a water molecule reacts with the oxygen atom in the bridge by hydrolysis reacyion. ß-galactosidase also

forms a complex with ONPG molecules and is able to hydrolyze them, facilitating the hydrolysis of ONPG. When ONPG is hydrolyzed a yellow product ONP is produced. The intensity of color produced can be used to measure the rate at which â-galactosidase can hydrolyze ONPG (or lactose).

MATERIALS /METHOD: Z buffer, per 50 mL:

O. 8 0 g N a 2 H P O 4 . 7 H 2 O ( 0 . 0 6 M ) , 0 . 2 8 g NaH2PO4.H2O (0.04M) , 0.5 mL 1M KCl (0.01M), 0.05 mL 1M MgSO4 (0.001M) , 0.135 mL â -mercaptoethanol (BME) (0.05M) , Brought to approximately 40 mL with H2O, all the salts were dissolved , pH was adjucted to 7.0, Graduated cylinder was used to bring the buffer to 50 mL , Stored at 4 C.

Preparation of CellsE.coli cultures were incubated for 20' on ice to stop growth and was washed. At least 2 mL of cells at 4 C were pelleted by centrifuging for 10' at 6,000 rpm. Supernatant was poured out and the cell pellet was resuspended in the same volume of chilled Z buffer. OD600 of the resuspended cells was measured (blank against Z buffer). Cells were diluted in Z buffer to 1 ml. The diluted cells were permeabilized by adding 100 µl chloroform and 50 µl 0.1% SDS (sodium dodecyl sulfate). Vortexed the tubes for 5' in a 28 C water bath.


ONPG test. 100 µl of each concentration of lysed cell culture was transferred into each well of a microplate. 20 µl of ONPG, o-nitrophenyl-beta-d-galactopyranoside, was added to the microwells. The ONPG interacted with the amount of b-galactosidase; in turn, measured activity, by turning yellow. At first sight of color change, the reaction was stopped by adding 50µl of sodium bicarbonate. Various conditions of time, temperature & IPTG conditions were used. The absorbance was measured.

RESULTS

Figure ONPG & Beta-galactosidase Activity

The twelve induced samples are arranged in increasing concentrations from 0µm to 2000µm in order of 1-12 on a microwell plate. ONPG turned from light yellow and darker in accordance with increased beta-galactosidase activity. Columns A,B,C consist of the same samples replicated. Column D has alternately arranged positive and negative controls

Effect of varying time on Beta-galactosidase activity

Concentration vs. B-Galactosidase Activity

Figure 2- The numbers on the X-axis represent the tube numbers who’s

concentrations are 1=0µm, 2=0.1µm, 3=0.5µm, 4=1µm, 5=5µm,

6=10µm, 7=50µm, 8=100µm, 9=500µm, 10=1000µm, 11=1500µm12=2000µ and

the Y-axis has the activity value ranging for 1-100.

DISCUSSION/CONCLUSION

During the procedure, the cells that were to be induced actually grew from the expected experimental absorbance level of 0.4nm to 0.59nm. Therefore, the cell were still very much actively growing. The ONPG interaction with b-galactosidase reacted in varying shades of yellow. The range was from a light yellow color , from the result of low activity level of b-galactosidase; to dark yellow reacting to high activity level of b-galactosidase. Time also played a role. With increasing temperature, the activity increased but the optimum


temperature was 37 after which the activity remained constant. The color change evolved more rapidly due to increased enzyme activity than that of low activity level- which took more time to develop its color reaction.

The amount of b-galactosidase activity was calculated using the following equation:

1000 x A(420)-(1.75 x A(550) )

t x 0.1 x A (600)

It is critical to not store the lysate below 4°C because it would substantially decrease enzyme activity yielding inaccurate or no results.

IPTG, indeed, initiated transcription of the lacZ gene. Results indicated that transcription was inhibited with 1.0µm IPTG. B-Galactosidase activity showed maximal increased activity/expression at a concentration of 5µm. Activity showed a constant decrease with 50µm, 100µm, and 500µm and so on till a 2000µm concentrations. IPTG starts to display its inhibition to almost the extent of the .5µm and .1µm concentrations. It is possible that those concentrations are too little for the lacZ gene to recognize. The 1000µm and greater concentrations may saturate the gene’s ability to initiate transcription.

REFERENCES1. Dorland's Illustrated Medical Dictionary.

2. Matthews B (2005). "The structure of E. coli beta-galactosidase". C R Biol 328 (6): 549–56. PMID 15950161.

3. J.H. Miller in "Experiments in Molecular Genetics" 1972 Cold Spring Harbor Laboratories pages 352-355

4 .http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed& pubmedid=7909660.

5. Beta-Galactosidase Assay (A better Miller) - OpenWetWare

6. “Monitoring Expression by B-Galactosidase Activity” Expression of Cloned Genes in Escherichia coli. 17.35.

7. Quantitative â-galactosidase assay suitable forhigh-throughput applications in the yeast two-hybrid system Natalie Mockli and Daniel Auerbach Dualsystems Biotech AG, Zurich, Switzerland BioTechniques 36:872-876 (May 2004)

8. Journal of bacteriology, mar., 1966 copyright © 1966 american society for microbiology effect of isopropylthiogalactoside on induction of the galactose operon by d- fucose in a lactose deletion mutant of E.coli D. C. H. mcbrien and V. moses lawrence radiation laboratory, University of California, Berkeley, California

9. Jeffrey H. Miller. A short course in bacterial genetics. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1992. isbn:0879693495. [Miller-1992]

10. Zhang X and Bremer H. Control of the Escherichia coli rrnB P1 promoter strength by ppGpp. J Biol Chem 1995 May 12; 270(19) 11181-9. pmid:7538113. PubMed HubMed [Zhang-JBC-1995]

11. Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. isbn:0879691069. [Miller-1972]


Patentability of Biotechnology: A Critical Analysis

B. BYJU &P. GEORGE GIRI*

Patents are the lifeblood of biotechnology . The origins of patents for invention are obscure and no one country can claim to have been the first in the field to have to come up with a patent system. However, Britain does have the longest continuous patent tradition in the world. Its origins can be traced back to the 15th century, when the Crown started making specific grants of privilege to manufacturers and traders. Such grants were signified by Letters Patent, open letters marked with the King's Great Seal. The basis of the patent system is a “social contract” between the Crown on one hand and an inventor on the other hand. The contract is based on the proviso that if an inventor makes his or her invention available to the public, the Crown will grant to the inventor a monopoly in that invention for a fixed period An important milestone in the further development of the British patent system was a limited investigation into the novelty of the invention before a patent was granted

The world is fast moving towards an era where life forms, including plants and animals, are heading for a remote control by a few private conglomerates. The existing problem is patent ability of new life forms and processes of the same are still not accepted in majority of the countries. However there are international conventions and treaties like the Paris Convention, the Patent Cooperation Treaty and the Budapest Treaty and the agreement on Trade Related Aspects of Intellectual Property Rights (TRIPS) a part of World Trade Organization (WTO) providing norms and standards to be abided by member countries in respect of seven categories of intellectual property right.

The use of IPRs (particularly patents) in the context of biological resources and living organisms is a relatively new phenomenon. The patent system evolved and widely used in mechanical and non-living inventions. Now significant investments are made in the research and development of biotechnology. A patent can cover the method for creating the new life form, the life form itself and the production of substances using the new

life form. The issue of whether genes and life forms should be excluded from the patent system is to be considered.

The current controversy in biotechnology is to find an answer for the vexed question what is patentable and what is not. Generally, an invention is patentable while a discovery is not. While this rule in other areas of specifications and application, may appear well defined, in biotechnology, ands the line is blurred it is often the cause of differences in regulations as obtained in different countries. Discovery is merely making available what already exists in nature. A substance freely occurring in nature, if merely found or discovered, is not patentable. However, if the substance found in nature has first to be isolated from its surroundings, and a process for obtaining it is developed, that process is patentable.

The rules and regulations governing biotechnology patents are country-specific. Each country has set its own standards for granting biotechnological patents and they are not able to give a clear-cut idea about patentability of life forms. The rapid advances in biotechnology have generated questions about the protection of intellectual property rights and the potential conflict that may result over those seeking to make broad use of the new developments even then, strict IPR regimes, especially patents, were not legislated in many developed countries until the 1970s. It was in the mid-1990s that many developed countries began to move beyond the original tenets of patent law by allowing patents on living organisms, although there were numerous differences in the extent of patentability in each country.

This paper focuses on current national and international legislative efforts to regulate and protect indigenous genetic resources and the products that local people have developed from them., and it explores the development of effective policy to arrive at comprehensive understanding of patentability or otherwise of living


organism.

Special conditions that a patent should fulfill;-

To be patentable your invention must:

• Be new

The invention must never have been made public in any way, anywhere in the world, before the date on which an application for a patent is filed.

• Involve an inventive step

An invention involves an inventive step if, when compared with what is already known, it would not be obvious to someone with a good knowledge and experience of the subject.

• Be capable of industrial application

An invention must be capable of being made or used in some kind of industry. This means that the invention must take the practical form of an apparatus or device, a product such as some new material or substance or an industrial process or method of operation.

“Industry" is meant in its broadest sense as anything distinct from purely intellectual or aesthetic activity. It does not necessarily imply the use of a machine or the manufacture of an article. Agriculture is included.

Articles or processes alleged to operate in a manner clearly contrary to well-established physical laws, such as perpetual motion machines, are regarded as not having industrial application.

• Not be "excluded"

An invention is not patentable if it is:

• a discovery;

• a scientific theory or mathematical method;

• an aesthetic creation such as a literary, dramatic or artistic work;

• a scheme or method for performing a mental act, playing a game or doing business;

• the presentation of information, or a computer program.

If the invention involves more than these abstract aspects so that it has physical features (such as a special apparatus to play a new game) then it may be patentable.

Government grants a patent for an invention to the inventor, giving the inventor the right for a limited period

to stop others from making, using or selling the invention without the permission of the inventor. When a patent is granted, the invention becomes the property of the inventor, which - like any other form of property or business asset - can be bought, sold, rented or hired. Patents are territorial rights.

Patent law was framed in an industrial context, and typically applied to objects, chemicals, designs and processes. But, with the rise of biotechnology, a shift has occurred, partly in technical sense, and partly in our perceptions.

What is patentable in biotechnology?

This is the question that India must decide in regards to new biotechnology (read genomics, proteomics, bioinformatics, genetic engineering). As per the Indian Patent Act, the following are patentable:

?Micro organisms: under the Indian Patent Act, microbiological processes can be patented. Also patentable are processes for producing new-micro organisms through genetic engineering and the products that result out of this process, such as micro organisms including plasmids and viruses if they are non-living.

?Cell lines-A cell line is patentable if artificially produced. R-DNA, RNA, AMINO ACID: if the end result is non-living, it is patentable.

?Hybridoma technology: patents are also allowed on hybridoma technology, but not on protoplast fusion.

?Expressed sequence tag’s, or ESTs, are small fragments of genetic material obtained by reverse transcriptions of messenger RNA (mRNA) from expressed genes. The gene sequence, or expressed sequence tags (ESTs), can be patented if it has a use, such as if it works as a probe.

What is not patentable in biotechnology? The Indian Patent Act defines what is not patentable biotechnology: invent ability does not apply to plant or animals. Accordingly, a method of producing a new form of a known plant or tissue culture method for production of plant variety is not patentable, nor is a method of treatment of a human body by surgery or operation for diagnosis. Nor is a method of improving or changing the appearance of the human body or parts of it patentable.

Still, these categories are not as clear-cut as they appear.


Skilful wording may decide whether a finding is an “invention” or a “discovery.” "However, it is and always has been a principle of patent law that mere discoveries or ideas are not patentable, but those discoveries and ideas which have a technical aspect or technical contribution are. Thus the concept that what is needed to make an excluded thing patentable is a technical contribution is not surprising.

Patents on Micro Organisms:- The term "micro-organisms" is used very widely and it includes cell lines and monoclonal antibodies. The GATT negotiation ended with all member states accepting that they would provide patent protection for microorganisms. Patents on microorganisms like bacteria, algae, fungus and virus will have far reaching consequences for developing societies India could have tried to get out of the patent on microorganisms by invoking the clauses of public and offence to prevailing norms of morality. Unfortunately it did not. . Patents on microorganisms have been introduced in the draft Patent Amendment Act that is awaiting approval by Parliament.

Primarily, the strategy is to keep the definition of microorganisms conservative and not agree to the inclusion of unconventional categories like genes and cells. The details are as follows.

1.Microorganisms for the purpose of Indian Act include:

?Bacteria,

?Virus,

?Viroid

?Protozoa

?Algae

?Lichen

?Actinomyces

?Fungi except edible fungi

2. Microorganisms do not include:

Variant forms

?DNA

?plasmids

?prions

?hybridomas

?cells

?cell lines

3. Naturally occurring microorganisms are not patentable.

4. Patentable microorganisms are those, which have been produced by adequate human intervention and fulfill the criterion of novelty, non-obviousness and industrial utility. Mere discovery and isolation will not be considered sufficient human intervention.

5. Patents will not be granted on materials obtained from national and international collections and depositories.

6. When material is taken from a country, Article 15 of the Convention on Biological Diversity will have to be respected. No patents will be granted without prior informed consent and material transfer agreements.

7. When a patent is granted, the patent holder will be obliged to share the economic benefits with the communities of the country from where the material was obtained.

8. In view of the critical nature of the subject matter, the patented microorganism will remain free for scientific research and experimentation.

9. Patents will not be granted on a broad basis (overarching patents with a very wide scope).

Patents will be granted for the organism only with respect to that particular function / property that constitutes the invention. The organism will remain free for others to create other inventions.

10. A Compulsory licensing provision will be made available to safeguard Food, Health, and Defense & Environmental security.

To review Article 27 3(b) of the TRIPS Agreement

TRIPS does not provide a definite code regarding new life forms. The present version of TRIPS allows members to exclude from patent protection:

• plants and animals other than micro-organisms;

• and essentially biological processes for the production of plants or animals other than microbiological processes.

• The TRIPS should ensure sustainable use of genetic resources. To avoid conflicts, Art 27.3(b) should be amended to include the requirements of

(a) Identification of sources of the genetic material,

(b) Related traditional knowledge


(c) (TK) used to obtain that material

(d) Evidence of fair and equitable benefit sharing, and

(e) Evidence of prior informed consent from the government or the indigenous community for the exploitation of the subject matter of the patent.

Art. 27.3 (b) should consider the possibility of providing flexibility for the member to limit or deny IPRs over such technologies.

“Members may exclude from patentability, plants and animals other micro-organisms and essentially biological processes for the production of plants or animals other than non-biological and micro-biological processes. However, Members shall provide for the protection of plant varieties either by patents or by an effective sui generis system or by any combination thereof...”

The TRIPs Council put forward a check list of issues on the review: the link between Art. 27.3(b) and development; the technical issues relating to patent protection under Art.27.3(b); technical issues relating to sui generis protection of plant varieties; ethical issues relating to patentability of life forms; relationship to the conservation and sustainable use of genetic material; and the relationship with the concepts of traditional knowledge and farmers rights.

The scope of ‘micro-organisms’ would be relevant to ensure greater consistency in application of TRIPS.

TRIPS has also an inbuilt agenda for review of why IPR protection of biological innovations is so controversial.

Microorganisms’ and that the ‘dictionary meaning’ (meaning anything that could not be seen with a naked eye but needs a microscope) would do. Transgenic micro-organisms as organisms which, except for all or part of plants or animals, express, by means of direct human intervention in their genetic composition, a characteristic normally not attainable by the species under natural conditions.”

When a micro-organism cannot be described in the specification sufficiently to allow a person with the relevant skills to use the invention, then an alternative means of "disclosing" it must be found.

Providing the micro-organism is not available to the public at the date of filing the patent application, the disclosure may be satisfied by depositing a culture of the micro-organism in a depository institution. This process is therefore only an acceptable way of disclosing an invention if the nature of micro-organism cannot be

sufficiently disclosed in writing.

Budapest Convention: The Convention enables inventors applying for patent protection in several countries to satisfy the provisions of Schedule 2 of the Patents Rules 1995 - and the equivalent rules in other signatory countries. This is done by depositing just one sample of a microorganism in an "International Depositary Authority" recognized by the Convention - without needing to make separate deposits in every country.

When making a deposit, the scientific description and/or proposed taxonomic description of the micro-organism must be supplied. Ideally, the deposit should be made about ten weeks before the patent application is filed, because viability and contamination testing may take some time. Deposited micro-organisms should not be made public until the Certificate of Viability and Certificate of Deposition have been obtained. Evidence to prove monoclonal antibodies exist

What exactly has to be deposited tends to vary. However, for monoclonal antibodies, it is usually a culture of the hybridoma, which produces the antibodies. There have been cases (in 1992 and 1993) in which patents for monoclonal antibodies were found to be invalid, on the grounds that the invention was insufficiently disclosed, because of the lack of a deposit. In both these cases, a hybridoma was deposited, but turned out to produce antibodies that did not conform to the patent claims.

Written descriptions had also been provided, but it was held that these did not disclose the exact nature of the monoclonal antibodies produced by the hybridomas in the absence of the deposit of the hybridoma itself. No other deposit had been made, and so the patents were held to be invalid.

Gene ModificationEthical, cultural and religious beliefs on patenting of life forms vary among countries, and often within a country, and hence should be taken into account in the review process. While property rights over life forms are recognized in most cultures, patents over life-forms such as micro-organisms do not limit control over individual living beings but over whole group of specimens and this is considered morally wrong for many cultures.

The new technologies usually called 'genetic engineering' or 'genetic modification' (GM) promise to revolutionize medicine, animal husbandry and agriculture. The development of GM plant technology raises two kinds


of issues: the scientific and the ethical.

Science is concerned with understanding the world in which we live and in particular the causal relationships that shape that world: for example, the association between genes as a molecular sequence and the characteristics, such as resistance to frost, that the genes express. Understanding such causal patterns is necessary if we are to alter or change the characteristics of plants in an informed way.

Ethics, by contrast, is concerned with what we ought or ought not to do. Ethical principles provide standards for the evaluation of policies or practices, for example, indicating that it would be wrong to carry out a certain genetic modification because to do so would threaten human health or harm the environment. Although it may be scientifically possible to undertake a certain experiment or introduce a new type of crop for commercial planting, it does not follow that it would be ethically right to do so. Working out what it is right or permissible to do involves, therefore, bringing together our scientific understanding with our ethical principles to decide what we should do given the capacities for genetic modification that have been developed.

There are three main types of principle that are relevant to the evaluation of policies or practices. The first principle is a principle of general welfare, which enjoins governments (and other powerful institutions) to promote and protect the interests of citizens. The second is the maintenance of people's rights, for example their rights to freedom of choice as consumers. The third is the principle of justice, and it requires the burdens and benefits of policies and practices to be fairly shared among those who are affected by them. The moral and ethical consequences of inventions are best dealt with directly - by inhibiting the development of the technologies themselves.

The rapid advances in biotechnology have generated questions about the protection of intellectual property rights and the potential conflict that may result over those seeking to make broad use of the new developments. " At the same time, patent law must be structured in a way that not only encourages the original inventor but also provides access to others so that the new product can be improved upon. And it must protect against commercial concerns that obtain patents in the area of biotechnology without carrying through with a concrete product, thus depriving society of a direct benefit.

The government needs to make a concerted effort to amend its Patent Laws, strengthen its IPR regime, so as to protect the economic interests of those who innovate. Additionally, India needs to sign the “Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure” to assist in the standardization process of biotechnology patenting.

The establishment of minimum standards for patent and related intellectual property protection, including those for products resulting from genetic engineering is purely need of the hour. Such standards are vital not only for protecting products from industrial countries but also for encouraging the creation of and protecting developing country inventions.

The amendment in the Patents Law should aim to:

?Widen the definition of invention in view of Article 27 of the TRIPS Agreement. The definition proposed is "...a new product or process involving an inventive step and capable of an industrial application";

?Widen the term "chemical process" to include biochemical, bio-technological and micro-biological processes.

?Provide for publication of applications after eighteen months. At present an application is substantively published only after examination and acceptance of the complete specification;

?Provide for examination of an application only upon request. A period of four years is provided for making the request. Upon failure to request examination, the application shall be treated as withdrawn;

?Shorten the time for putting an application in order for acceptance subsequent to its first examination to 12 months. The present law provides for a maximum of 18 months;

?Provide for importation of patented article to constitute a valid defence in revocation proceedings initiated for not working the patent.

Patents on life forms do not protect inventors' rights to their creations; instead, they award monopolies on organisms or their components. This distortion of the patent system hamstrings scientific and medical research by restricting access to information and materials, and by preventing competition.


The patents applications should specify and identify the sources of the genetic material used in the invention

The NGOs have demanded that the Article 27.3(b) review should result in banning patenting of all life forms (and not merely an enabling provision for national jurisdictions to exclude from patents), and also protection of indigenous knowledge, and ensuring that TRIPS sub serve the CBD.

It should be ensured that patenting of genetic resources does not run counter to the basic principles of the CBD -sovereignty of countries over their genetic resources, principles of benefit sharing and of prior informed consent, the protection at national level of traditional knowledge. The patents over micro-organisms, and such other patents to a member’s genetic resources granted outside raises issues of potential conflict with the principle of sovereignty of contracting parties in the CBD.

It would be less cost-effective to establish an internationally accepted solution to prevent biopiracy than to divert national resources to expensive judicial processes for revocation of patents that including illegal genetic resources. Developing countries do not have the resources to follow each and every patent outside their territories on the use of their resources.” The dominant welfare-based concern is that genetic modification, like some other technological advances, will not be an unqualified success and that we risk damaging the economic and amenity resources of the environment

India's resistance to patenting life is the culmination of many years of activism by Indian farmers against the corporate control of agriculture. A balance needs to be struck between the legitimate desire of farmers to provide a livelihood for themselves and their families, the reasonable demands of the food producers, retailers and the consumers, and the continuing need to maintain a sustainable environment for future generations. . Self -reliant , sustainable agriculture will be adversely affected if our ability to develop biofertilisers and biopesticides, both based on micro organisms, will be hindered by foreign patents. The role of micro organisms in other areas like pharmaceuticals, bio-mining, energy etc. is well known. Patents will also affect self-reliance in these sectors.

The Government needs to incorporate explicit and robust measures into national law to restrict the patenting of new products that are made with traditional knowledge or that utilize material derived from living

organisms originating in India. In this process the role of indigenous and local communities must be recognized by the equitable sharing of the benefits arising from the utilization of their knowledge, innovations and practices.

The Acts which are to be enacted should ensure that the patent system is well suited to the needs of modern society, sufficiently flexible to accommodate future changes in technology and adapted to operate in an international context.

Globalization of intellectual propertyThe second circumstance supposedly enabling the North to conduct its cultural piracy and biopiracy is related to the implementation of the TRIPS. The TRIPS was concluded in 1994, in the course of the establishment of the World Trade Organization (WTO). Ratification of TRIPS is a prerequisite for membership of the WTO. Of course, developing countries practically had no choice but to adhere to TRIPS. Their economic development made it absolutely necessary to join the WTO, which allows them to freely trade their products around the world. Hence, they were forced to implement TRIPS in their national legislations. The North insisted on the connection between TRIPS and the WTO as it would enable it to effectively enforce the intellectual property rights pertaining to some of its most important export products, i.e. technology and artistic creations such as medicines and films. At the same time, it is clear that developing countries do not have the means to participate in the 'race to innovation'. The state of their technological and economic development does not allow them to compete with equal arms. Therefore, the South feels that it is not only confronted with cultural piracy and biopiracy, but that it is even forced to collaborate. It not only finds that its genetic and knowledge resources are appropriated, but that it must even legitimize the 'theft' through granting and enforcing intellectual property rights. In view of this, some of these countries have made national legislation providing measures against the appropriation of those resources through intellectual property law.

National legislation to prevent piracyIn the past few years, many developing countries have implemented sui generis regimes for both the offensive and the defensive protection of their genetic resources and the traditional knowledge of their indigenous communities. For example, Costa Rica, Brazil, Peru and India have implemented legislation for that purpose.


These statutes have in common that they condition access to genetic resources and traditional knowledge upon the fulfilment of certain requirements, such as prior informed consent of a national office governing the country's biological resources and the indigenous communities involved. Furthermore, biological samples and knowledge can only validly be transferred if proper benefit-sharing agreements are concluded, allowing the source country and the indigenous communities involved to share in the proceeds deriving from the commercial exploitation of the material or knowledge concerned. The statutes presuppose the existence of intellectual property rights in the materials and knowledge concerned, and often negate the existing public/private domain paradigm. Even the transfer of biological samples or of traditional knowledge does not exhaust the proprietary rights pertaining thereto - the provider usually remains entitled to exploit the object of transfer regardless of any subsequent intellectual property rights of the acquirer. Most importantly, these statutes generally indicate that within the countries concerned no intellectual property rights can be obtained if the aforementioned requirements, such as benefit sharing, are not fulfilled. Sometimes, violating the requirements of these statutes amounts to a criminal offence and may be prosecuted accordingly.

Obviously, these statutes violate TRIPS in various manners, most importantly by negating the distinction between the public and the private domain, adding additional requirements to the patentability of inventions and apparently providing for continuous licenses to the transferors by operation of law. However, these statutes seem to correspond more or less with another treaty which existed before the conclusion of TRIPS - the Convention on Biodiversity.

Intellectual Property Rights Protection.Modern biotechnology has accentuated the differences in intellectual property rights (IPRs) protection (example, patents, breeders= rights) between developed and developing countries.

IPRs were introduced by the industrialized countries. They are explicit on being designed to protect only individual interests of members of the industrial society. One condition for patentability is that the technology be industrially applicable. By denying patentability for non-industrial applications the system discriminates against collectively produced and communally used indigenous and local community technologies. Initially, it treated

such communal knowledge and technologies as unpatentable. But this condition has been relaxed and community knowledge and technologies are being taken as fair game for the industrial sector to privatize, e.g. the old Indian technology of parboiling rice has been patented.

One condition of patentability within the industrial system itself has been that what is to be patented should be an invention, not a discovery. With the development of biotechnology, discovery is being subsumed in invention so that the mere identification of a DNA sequence which determines a trait is being taken to be an "inventive step", the same as if describing the sequence creating it from scratch were the same thing. Even if that DNA sequence were made in the laboratory, it would merely be synthesizing a natural product, which is a chemical achievement but not an invention of the natural trait determined by that product. This has lead to the patenting of living things by merely describing a DNA sequence in them. Such patenting will lead to complex legal barriers that will stand in the way of the use and conservation of biodiversity. For example, by merely decoding the genes responsible for gluten in wheat and patenting it, one could control all the research and development in wheat. It is said that a comparable actual patent taken out on cotton is causing problems in cotton research and development.

In the United States, it has now become possible to patent traits without even decoding their genetic causation. For example, male sterility in quinoa has been patented. It should be added that this trait in quinoa was developed by Andean farming communities and an American patent on it is thus unjust.

If adherence to the criterion of invention were adhered to, active ingredients of herbal medicines, even when synthesized, would not be patentable.

It is contrary to the letter and the spirit of industrial society IPRs that all these patents are allowed. It is contrary even when farmers' varieties of crops from farming local communities are taken and, with little or no further breeding, given Breeders' Rights protection as the intellectual property of individuals. Unfortunately, this is happening extensively in industrialized countries, and increasingly so in developing countries.

The Convention on Biological Diversity recognizes that it is indigenous and local communities who have generated and given us our knowledge and technologies on, and who continue to conserve and use, biodiversity


sustainably. It stipulates that the knowledge, technologies and biodiversity of indigenous and local communities should be accessed and used with their prior informed consent, and with their involvement. It also stipulates that IPRs should be supportive of, and should not run, counter to the its objectives, which are the conservation and sustainable use of biodiversity and the fair and equitable sharing of its benefits.

It is obvious, therefore, that the predatory and disruptive IPR systems related to biodiversity and the knowledge and technologies on it go contrary not only to our sense of justice, but also to international law.

In contrast to this, the Trade Related Intellectual Property (TRIPs) component of the Uruguay Round of negotiations which created the World Trade Organization insists that microbiological applications and plant varieties shall be protected by IPRs.

ConclusionEthically, we can apply the principle of love or beneficence. Does allowing patenting of biotechnology give more benefits than a ban to the most people? Does it work for love of life and the environment, compared to alternatives? The benefits should be in terms of general medical or agricultural development, rather than economic prosperity of one company or country over another. At the same time, love says that no one should be stripped of the respect that others owe to them, and we should work for the benefit of all without ignoring the weak.

Biotechnology companies must consider the novel aspects of any new technology or process they use, beyond simply human health concerns. The first is the impact on the environment. Now every industrial sector tackles with the environmental problem. In fact, the application of biotechnology to clean polluted environments such as bioremediation is one emerging area. This is beyond the development of cleaner procedures and products. Since biotechnology companies use technology with living organisms, they need to especially take account of biosafety, biodiversity and the complexities of intricately tuned ecosystems.

The second is the impact on society. Biotechnology is often said to be a technology that may alter people’s view of “life”. Biotechnology companies must face the issues concerned with human rights immediately in their operations and bioethical procedure are inevitable, such as keeping customer’s privacy and informed consent. It

is necessary for them to consider the values and concerns of users and community, and to convey the right information through labeling, education etc.

The third one is economic impact. New alternative products can substitute for old, and newer methods to produce refined products can reduce the need to import the raw materials. As artificial sweeteners caused damage to the sugar industry in tropical countries, this is collapsing some industry in developing countries. Thus, epoch-making technology may cause direct and indirect influences on regional and international economy, which may differ depending on the country. One big multinational company can change the structure of agricultural trading. Sharing benefit from biotechnology is another issue, if we think its resources as common property.

The progress of modern biotechnology has raised a variety of bioethical issues. As seen in medical ethics, where doctors and patients need to be on similar levels and let patients express the results of their decision making while facing new medical care, companies, as producers, and consumers, need to both participate in making decisions.

We should not merely criticize companies’ profit making, since we use their products or services and the ethical principle of beneficence supports our community including companies if they do no do harm. However, we cannot overlook the influence of commercial biotechnology upon global economics and trade, and we must not sit complacently ignoring our part in choosing the goals of global society with growing economic disparity.

In conclusion we should reflect further on how to reward intellectual property, and be more open to diverse approaches that are appropriate in different societies. In Europe a conference was held in Oviedo in May 1999 to discuss whether the Council of Europe should develop a further Bioethics Convention to meet the challenges that ethical patenting faces from biotechnology. Despite the acceptance by the European Patent Office of the European Parliament’s 1998 directive on patenting inventions produced through biotechnology, there are ethical issues that require further resolution. The patent situation will also depend upon the relationship between TRIPS, UPOV Convention and the Convention on Biological Diversity, which oversees the international situation.

Globalization has many implications; however it is not


clear that patenting will be the best solution to the questions of sharing the benefits gained by human curiosity with all of society. In existing law there is room for interpretation of the public morality clause in different ways, but whatever decision is made at least the law should be predictable within a given society otherwise industry cannot make long-term policy to pursue research and brig new products to market. Public opinion is heated as seen in the December 1999 World Trade Organization meeting in Seattle, where protestors against global free trade and economic paradigms

gathered. We can expect further questioning of the economic imperative as the prime motivator for social systems, and a debate between the United Nations and the WTO could be expected, despite the recent avoidance of discussion of the morality of patents in the United Nations agencies. This is ethically justified, as every society should seek deeper ethical goals of beneficence, justice and rights beyond economic development itself.


Wastewater Treatment: A Boon for Water Crisis

Review Article

Gandhimathi.S* Lecturer, Department of Microbiology

Garden City College, BANGALORE-49.email ID:*[email protected]

ABSTRACT:Water crisis poses a serious threat due to pollution, global warming and urbanization which directly affects environment and ecosystem. The reduction in fresh water level gives us a wakeup call to minimize the maximum wastage of water and implies the recycling of wastewater. This includes both domestic sewage and industrial effluent. Thus the composition of wastewater varies widely. The presence of different organic and inorganic constituents in the wastewater gives an unpleasant color and odour. The unamiable color is due to the presence of recalcitrant polymers, such as melanoidin, caramel, polyphenols and alkaline degradation products. Unfortunately with the complicated color compounds decolorisation is a difficult and challenging task . The public demand for color free water discharge to receiving waters and tougher color standards have made decolorisation of various industrial wastes a top priority. Different techniques including physical, chemical and biological methods are used.A combination of these process chemical-biological, bio-physical and physico-chemical ones are used is used. Microbial decomposition is an eco friendly technique to remove color from sewage and effluents which makes water amicable for recycling.

Key Words: Decolorisation, sewage, effluent, Lactobacillus plantarum, Coriolus versicolor

INTRODUCTION:Wastewater is any water that has been adversely affected in quality by anthropogenic influence. It comprises liquid waste discharged by domestic residences, commercial properties, industry, and/or agriculture and can encompass a wide range of potential contaminants and concentrations.

Scarcity of fresh water, reduction in water table level.pollution, urbanization, global warming, and lack of rain gives naturalist and aware public an alarming signal. The possible solution to the growing water crisis will be the recycling use of waste water after appropriate treatment. There are different types of waste water each requiring different treatments and disposal systems. The effluent disposal of industries is a difficult and costly problem. Most petroleum refineries, chemical and petrochemical plants have onset facilities to treat their wastewater, so that the pollutant concentrations in the treated water comply with the National regulation. But the treated waste water of paper and pulp industry, textile industry, food industry and distillery are rich in nitrogen, phenols, flavanoids and dyes which gives it a crude color.Highly colored water is not preferred even though they are non-toxic. Conventional methods are ineffective for decolorizing, and hence novel methods are tried and tested.

Origin Wastewater or sewage can come from several sources.

vHuman waste (fæces, used toilet paper or wipes, urine, or other bodily fluids), also known as black water, usually from lavatories;

vCesspit leakage;

vSeptic tank discharge;

vSewage treatment plant discharge;

vWashing water (personal, clothes, floors, dishes, etc.), also known as greywater or sullage;

vGroundwater infiltrated into sewage;

vSurplus manufactured liquids from domestic sources (drinks, cooking oil, pesticides, lubricating oil, paint, cleaning liquids, etc.);


vUrban rainfall runoff from roads, car parks, roofs, sidewalks, or pavements (contains oils, animal fæces, litter, fuel or rubber residues, metals from vehicle exhausts, etc.);

vIndustrial site drainage (silt, sand, alkali, oil, chemical residues);

vIndustrial cooling waters (biocides, heat, slimes, silt);

vIndustrial process waters;

vOrganic or bio-degradable waste, including waste from abattoirs, creameries, and ice cream manufacture;

vOrganic or non bio-degradable/difficult-to-treat waste (pharmaceutical or pesticidal manufacturing);

vextreme pH waste (from acid/alkali manufacturing, metal plating);

vToxic waste (metal plating, cyanide production, pesticide manufacturing, etc.);

vSolids and Emulsions (paper manufacturing, foodstuffs, lubricating and hydraulic oil manufacturing, etc.);

v Agricultural drainage, direct and diffuse.

Wastewater constituentsThe composition of wastewater varies widely. This is a partial list of what it may contain:

vWater ( > 95%) which is often added during flushing to carry waste down a drain;

vPathogens such as bacteria, viruses, prions and parasitic worms;

vNon-pathogenic bacteria (> 100,000 / ml for sewage);

vOrganic particles such as faeces, hairs, food, vomit, paper fibers, plant material, humus, etc.;

vSoluble organic material such as urea, fruit sugars, soluble proteins, drugs, pharmaceuticals, etc.;

vInorganic particles such as sand, grit, metal particles, ceramics, etc.;

vSoluble inorganic material such as ammonia, road-salt, sea-salt, cyanide, hydrogen sulfide, thiocyanates, thiosulfates, etc.;

vMacro-sol ids such as sanitar y napkins, nappies/diapers, condoms, needles, children's toys,

vEmulsions such as paints, adhesives, mayonnaise, hair colorants, emulsified oils, etc.;

vToxins such as pesticides, poisons, herbicides, etc.

Wastewater quality indicatorsAny oxidizable material present in a natural waterway or in an industrial wastewater will be oxidized both by biochemical (bacterial) or chemical processes. The result is that the oxygen content of the water will be decreased. Basically, the reaction for biochemical oxidation may be written as:

Oxidizable material + bacteria + nutrient + O2 ? CO2 + H2O + oxidized in organics such as NO3

or SO4

Oxygen consumption by reducing chemicals such as

sulfides and nitrites is typified as follows:

--–S + 2 O2 → SO4

- -NO2 + ? O2 → NO3

Since all natural waterways contain bacteria and

nutrients, almost any waste compounds introduced into

such waterways will initiate biochemical reactions (such

as shown above). Those biochemical reactions create what is measured in the laboratory as the Biochemical

oxygen demand (BOD). Such chemicals are also liable to be broken down using strong oxidizing agents and these chemical reactions create what is measured in the

laboratory as the Chemical oxygen demand (COD).

Both the BOD and COD tests are a measure of the

relative oxygen-depletion effect of a waste contaminant. Both have been widely adopted as a measure of pollution

effect. The BOD test measures the oxygen demand of biodegradable pollutants whereas the COD test

measures the oxygen demand of oxidizable pollutants.

The so-called 5-day BOD measures the amount of oxygen consumed by biochemical oxidation of waste

contaminants in a 5-day period. The total amount of oxygen consumed when the biochemical reaction is allowed to proceed to completion is called the Ultimate

BOD. The Ultimate BOD is too time consuming, so the

5-day BOD has almost universally been adopted as a

measure of relative pollution effect.

There are also many different COD tests of which the 4-

hour COD is probably the most common.

The laboratory test procedures for the determining the above oxygen demands are detailed in many standard


texts. American versions include the "Standard Methods

for the Examination of Water and Wastewater"

Sewage disposal

Industrial wastewater effluent with neutralized pH from

tailing runoff. Taken in Peru.

In some urban areas, sewage is carried separately in sanitary sewers and runoff from streets is carried in

storm drains. Access to either of these is typically

through a manhole. During high precipitation periods a

sanitary sewer overflow can occur, causing potential

public health and ecological damage.

Sewage may drain directly into major watersheds with

minimal or no treatment. When untreated, sewage can have serious impacts on the quality of an environment

and on the health of people. Pathogens can cause a

variety of illnesses. Some chemicals pose risks even at very low concentrations and can remain a threat for long periods of time because of bioaccumulation in animal or

human tissue.

TreatmentThere are numerous processes that can be used to clean up waste waters depending on the type and extent of

contamination. Most wastewater is treated in industrial-

scale wastewater treatment plants (WWTPs) which may

include physical, chemical and biological treatment

processes. However, the use of septic tanks and other

On-Site Sewage Facilities (OSSF) is widespread in rural

areas, serving up to one quarter of the homes in the U.S. The most important aerobic treatment system is the

activated sludge process, based on the maintenance and

recirculation of a complex biomass composed by micro-organisms able to absorb and adsorb the organic matter

carried in the wastewater. Anaerobic processes are widely applied in the treatment of industrial wastewaters

and biological sludge. Some wastewater may be highly

treated and reused as reclaimed water. For some waste waters ecological approaches using reed bed systems

such as constructed wetlands may be appropriate. Modern systems include tertiary treatment by micro

filtration or synthetic membranes. After membrane

filtration, the treated wastewater is indistinguishable

from waters of natural origin of drinking quality. Nitrates can be removed from wastewater by microbial

denitrification, for which a small amount of methanol is typically added to provide the bacteria with a source of

carbon. Ozone Waste Water Treatment is also growing

in popularity, and requires the use of an ozone

generator, which decontaminates the water as Ozone

bubbles percolate through the tank.

Disposal of wastewaters from an industrial plant is a

difficult and costly problem. Most petroleum refineries, chemical and petrochemical plants have onsite facilities to treat their wastewaters so that the pollutant concentrations in the treated wastewater comply with

the local and/or national regulations regarding disposal of wastewaters into community treatment plants or into

rivers, lakes or oceans. Other Industrial processes that

produce a lot of waste-waters such as paper and pulp production has created environmental concern leading to development of processes to recycle water use within

plants before they have to be cleaned and disposed of.

RECENT TRENDS

NONBIOLOGICAL:Combination of physical and chemical methods are used

for effective decolorisation process.photocatalytic decolorisation in presence of DegussaP25 titanium d i ox i d e wa s u s e d t o t r e a t mu n i c i p a l

wastewater(Robert.J.Davis,1991) was proved to be

effective.Fentons reagent ,an inorganic flocculent

removed 93% of color in molasses derived from alcohol

distillery industry(Veroniap,1993).coagulation,


flocculation with microfiltration or ultra filtration and

adsorption on powdered activated carbon removed 80%

of the color(Farida Harreelkas,2009).Nano filtration

also proved effective (A.Bespia,2002).Electodialysis and reverse osmosis removed color and heavy metals

from tannery effluent(Wenroizuo,2008).Ozonation and

UV/H202 treatment proved 100% efficient in color

removal(Hung-yeeshu,2006). Electro chemical o x i d a t i o n a l s o p r o v e d t o b e

effective(A.G.Vlyssides,1999).electro coagulation followed by ion exchange process of textile dye effluent proved to be promising methods in decolorisation

process(S.Raghu,2007).

BIOLOGICAL:Under anaerobic and facultative conditions

Lactobacillus plantarum removed 68%of the

color(Tusanee Tondee,2008).Many dyes including

Remazol red, blue, golden yellow was degraded 100% by many bacteria under aerobic and anaerobic

conditions(Poonam nigam,1996).Bacil lus sp strainMD32was found todecolorize molasses wastewater under thermpphillic and anaerobic

conditions(Toshiaki Nakajima,1998).Coriolus

versicolor(white rot fungi) was found to decolorize high mol wt chromophores released by kraft mill

bleacheries under aerobic conditions (Frederick

Archibard,1990).Submerged bioreactor with g r a v i t a t i o n a l f i l t r a t i o n p r o v e d

effective(Taksukiueda,1999).

Future prospects including all these techniques with conventional techniques will help mankind to solve the

water crisis, but the best method is to create awareness among people about water management skills and to

support afforestation wherever possible.

ACKNOWLEDGEMENT

We are grateful to our Chairman, Dr. Joseph V.G.,

Dr.K.C.John and Dr.P.Chary for their excellent

guidance and support.

REFERENCES:

vA. Bes-Piá, J. A. Mendoza-Roca, M. I. Alcaina-Miranda, A. Iborra-Clar, M. I. Iborra-Clar Reuse of

wastewater of the textile industry after its treatment with a combination of physico-chemical treatment and membrane technologies

Desalination, Volume 149, Issues 1-3, 10 September 2002, Pages 169-174

vFarida Harrelkas, Abdelaziz Azizi, Abdelrani Yaacoubi, Ahmed Benhammou, Marie Noelle PonsTreatment of textile dye effluents using coagulation–flocculation coupled with membrane processes or adsorption on powdered activated carbon

Desalination, Volume 235, Issues 1-3, 15 January 2009, Pages 330-339

vG. Vlyssides, M. Loizidou, P. K. Karlis, A. A. Zorpas, D. Papaioannou Electrochemical oxidation of a textile dye wastewater using a Pt/Ti electrode

Journal of Hazardous Materials, Volume 70, Issues 1-2, 23 December 1999, Pages 41-52

S. Raghu, C. Ahmed BashaChemical or electrochemical techniques, followed by ion e xch a n g e , f o r r e c y c l e o f t e x t i l e d y e wastewaterJournal of Hazardous Materials, Volume 149, Issue 2, 22 October 2007, Pages 324-330

Oliver J. Hao; Hyunook Kim; Pen-Chi Chiang Critical Reviews in Environmental Science and Technology, Volume 30, Issue 4 October 1999 , pages 449 – 505

vPoonam Nigam, Ibrahim M. Banat, Dalel Singh, Roger Marchant Microbial process for the decolorization of textile effluent containing azo, diazo and reactive dyes

Process Biochemistry, Volume 31, Issue 5, June 1996, Pages 435-442

vRobert J. Davis, John L. Gainer, Gilbert O'Neal and I-Wen Wu Photocatalytic Decolorization of Wastewater Dyes Water Environment Research, Vol. 66, No. 1 (Jan. - Feb., 1994), pp. 50-53

vS. H. Mutlua, U. Yetisb, T. Gurkana and L. Yilmaz, Decolorization of wastewater of a baker's yeast plant by membrane processes

vTatsuki Ueda, Kenji HataDomestic wastewater treatment by a submerged membrane bioreactor with gravitational filtration

Water Research, Volume 33, Issue 12, August 1999, Pages 2888-2892


Faisal Ibney Hai, Kazuo Yamamotob and Kensuke Fukush Development of a submerged membrane f u n g i r e a c t o r f o r t e x t i l e w a s t e w a t e r treatmentDesalination

Volume 192, Issues 1-3, 10 May 2006, Pages 315-322

vToshiaki Nakajima-Kambe, Mifumi Shimomura, Nobuhiko Nomura, Thalerng Chanpornpong and Tadaatsu NakaharaDecolorization of molasses wastewater by Bacillus sp. under thermophilic and anaerobic conditions Journal of Bioscience and Bioengineering

Volume 87, Issue 1, 1999, Pages 119-121

vTusanee Tondeea and Suntud Sirianuntapiboon Decolorization of molasses wastewater by Lactobacil lus plantarum No. PV71-1861 Bioresource Technology

Volume 99, Issue 14, September 2008, Pages 6258-6265

vVeronica P. Migo, Masatoshi Matsumura, Ernesto J. Del Rosario, Hiroshi Kataoka Decolorization of molasses wastewater using an inorganic flocculant

Journal of Fermentation and Bioengineering, Volume 75, Issue 6, 1993, Pages 438-442

vWenrui Zuo, Guoliang Zhang, Qin Meng, Hongzi Zhang, Characteristics and application of multiple membrane process in plating wastewater reutilization

Desalination, Volume 222, Issues 1-3, 1 March 2008, Pages 187-196


Rupa Das1, Kirana Shekar*2 1. P.G. Student, Department of Microbiology, Garden City College, Bangalore.

2. Lecturer, Department of Microbiology, Garden City College, Bangalore.* e-mail: [email protected]

Title- Ayurvastra: Herbal Couture ForImproving Health

Clothes are one among the basic amenities of mankind. The clothes we drape around our body. not only act as a covering and protect us but also help us make a personal statement – in short,

they project the image of ourselves that we want others to see. Now, they can also do more than just provide an outer covering, clothes can now heal our physical body and bring about emotional and mental changes.These clothes are known as ‘Ayurvastra’. One just have to wear these clothes to get rid of many diseases. Ayurvastra is an ancient technique of dyeing textiles in medicinal herbs. Textiles will be dyed using natural vegetable dyes, especially extracted from medicinal plants. The Directorate of Handloom, has revived this tradition and uses medicinal herb dyes for its handloomed textiles. When exposed to skin, the herbs are absorbed into the body and may function as a means of providing ayurvedic treatment for a broad range of diseases including diabetes, skin infections, asthma, arthritis, and hypertension. It is also known to strengthen the immune system.

Overview of Ayurvastra:Ayurvastra is a branch of Ayurveda, the ancient 5,000 year old Indian system of Vedic healthcare. Ayur is health in sanskrit, veda means wisdom, and vastra is cloth

or clothing. Every step in the preparation of Ayurvastra fabric is carefully and precisely controlled. Ayurvastra, begins with 100% handloomed organic cotton. It does not require machine processing, niether chemical additives to prepare the cotton fibres for spinning and weaving, nor a craving for chemical finishes is needed. The organic cotton yarn or fibres are then dyed in a carefully controlled mixture of herbal dyes depending upon the disease or ailment being treated. For diabetes, mimosa pudica (touch-me-not), Cuminum cyminum (cumin) seeds, Michaelia champaka (champa) flower and Hibiscus (shoe flower) are combined in the herbal dye. The main herbs used in the herbal dye for arthritis are Murraya koenigii, (curry) leaves and apocynceae. For skin diseases, the herbs used are Curcuma longa (turmeric), Azardiractha indica (neem) and Santalum album (sandalwood) are preferred. Bamboo hypoallergenic fibres are the ideal alternative for individuals with sensitive skin (1). Dyes for Ayurvastra fabric typically contain between 40 and 60 specifically blended and carefully prepared medicinal herbs, plants, flowers, roots and barks. Such cloth is completely free of synthetic chemicals and toxic irritants and is totally organic and biodegradable, offering a proactive solution to deal with global warming (2 )

This traditional weaving and dyeing is 600 years old and has been preserved by the society.

This type of dyeing is for general well being like

immune booster , Making Aroma , Skin Condition ..

This type of dyeing is for tonic or wellbeing and will prevent skin diseases and

boost the immune system .

The ayurvedic dyeing technology is done by the Handloom weavers society

Balaramapuram .


Ayurvedic Dyed Products from the Ayurvastra Manufacturer

General Well Being Product Disease Based Products Dyeing Technology

Usage of the cloth is based on the principle of touch. By coming in contact with Ayurvastra, the body loses toxins and its metabolism is enhanced. The most effective time to wear Ayurvastra clothing is when the body is most at rest such as during sleep or meditating because this is when the body is naturally healing and re-establishing balance. Ayur vastra cloth is often used for sleepwear, bed sheets, towels, meditation clothes and coir mats. For coir mats, the fibers are soaked in Ayurvastra dyes and then woven into coir mats (3).

The inherent antibacterial properties of bamboo fabric commonly called“bamboo kun” is a natural antifungal and antibacterial agent. Bamboo does not require the use of pesticides because of this. It is rarely attacked by pests or infected by pathogens. The same natural substance that protects bamboo growing in the field functions in the spun bamboo fibres. These fibres constitute the bamboo fabric.

Ayur vastra cloth is also being used for sarees, the traditional flowing dress of Indian women, and in purdahs(4). There is a budding interest among ladies in the Middle East, particularly in Jordan and Saudi Arabia, for all-covering Ayurvastra dress (burkhas). The spread of Ayurvastra and Ayurveda into other cultures and regions of the world indicates the growing interest in more traditional and natural healthcare systems that are based upon restoring balance and health through natural methods rather than through Western medicines (2)

Skin infections reflect an imbalance in the health of an

individual. Ayurvastra clothing is believed to help restore balance within the body’s systems and strengthen the immune system. If Ayur vastra cloth and clothing can improve the skin’s ability to act as a barrier to external and environmental toxins, the wearer’s health might be improved.

Fast-growing bamboo makes unlikely but soft colourful linens and towels. Manufacturers have discovered that bamboo cloth mixed with a dash of cotton is silky to the touch. These are Bamboo linens Towels and sheets have been made with a mix of either 70 percent bamboo, 30 percent cotton material or 65 percent bamboo to 35 percent cotton. It absorbs colour faster than cotton (1).

Mongolians use many species of medicinal plants to cure all kinds of chronic illnesses

and have traditionally used pillows made from a wide variety of aromatic plants. The

Mongolian company Khuvsgul-Ikh Taiga has produced aromatic pillows, using timehonoured

methods. These pillows are now sold in shops in Ulaanbaatar. The aromatic pillow has wormwood (Artemisia), thyme and juniper contents. It has a soothing effect and acts as a

precaution against coughs and influenza (5).

Dyeing Process:Due to excessive use of chemicals the duration and number of the dye soaks, the blend of herbs and oils, the


equipments used needs to be carefully sterilized.

1.Selecting Organic Fabrics: Certified organic cotton yarn and fabrics, jute ,wool, silk, linen. Hemp, bamboo is selected.

2.Natural Bleaching: The selected fabrics or yarn are then bleached using cow urine which has medical values and removes toxins.

3. Selecting Colour Shade: Ayurvedic dyeing process is followed using any one out of more than 25 dyes.

4. Selecting type of dyeing: Fabrics are dyed in two types --

a) Disease based dye which cures disease.

b) Tonic /Well being dye which prevents diseases. (Table 1)

5. Dyeing Process: Dyes for Ayurvastra cloth typically contain between 40 and 60 specifically blended and carefully prepared medicinal herbs, plants, flowers, roots and barks. The temperatures of the dyes, the duration and number of the dye soaks, are carefully controlled.

6. Finished Ayurvastra Products: After the dyeing process the fabrics are made up in to finished garments and products according to the customers requierments.

Bamboo fibres are processed using a patented manufacturing technology of

hydroalkalization. Processed without the addition of caustic additives, according to ISO 9000 and ISO 14000 principles (1).

Ayurvedic Dyeing (Disease Based)

Arthritis

Osteo arthritis

arthritis rheumatism

Dermatitis atopica

Dermatitis From contact

Allergies in general

Tunnel carpal

Rheumatics

Diabetic

Psoriasis

Asthma

Blood Pressure

Ayurvedic Dyeing (Well Being)

Immune Booster

Weight Loss

Well-Being (Give Energy)

Sleeping condition

Making Aroma(Sweet Smell)

Making Color(Skin color)

blood Purification

Skin Shining

Body Relaxation

Ayurvedic dyed (finished Products)

Bed Sheets

Pillows cover

Mattress

Burqhas

Bathing soap

Curtain cloth

Shirtings

Scarf

Sarees

Over Coat


Table 1: Ayurvedic Dyeing

Research work:A trial conducted by Kerala’s Government Ayurveda College concluded that after using bedding, rugs and towels made from ‘Ayurvastra’ for one month, patients suffering from eczema, psoriasis and rheumatism showed remarkable improvement.

Experimental research conducted by Ayurvastra College in Trivandrum, Kerala confirms that Ayurvastra may help treat skin diseases, arthritis, and lower blood pressure.

A quantitative antibacterial capability test was performed by the China Industrial

Testing Centre from 7 to11 July 2003. One hundred percent bamboo fabric was tested over a 24–hour incubation period with bacterial strain type Staphylococcus

aureus. After the 24–hour period the numbers of live bacteria were counted in

each sample. The results showed that 100 percent bamboo fabric exhibits a 99.8

percent antibactericidal rate (6)

Value-added performance fabrics are the latest trend in textiles. Jonäno™, a division

of Sami Designs LLC, is concentrating on one particular area of study in hightechnology

fabrics: a new generation of antimicrobial and antibacterial textiles. This technology incorporates built-in abilities to fight harmful microbes that cause odour and can lead to infection (7).

The State of Kerala Coir Department reported on a six-month clinical trial initiated by the Ministry of Health at t h e G o v e r n m e n t A y u r v e d a C o l l e g e i n Thiruvananthapuram India on patients suffering from rheumatism, allergy, hypertension, diabetes, psoriasis and other skin ailments. For the study, all clothes, bed linens and mattresses for the resident subjects were dyed in Ayurvastra herbs and the walls, floors and ceilings in the patient/subjects’ rooms were lined with Ayurvastra coir mats so that the patient were surrounded by Ayurvastra medicated materials. According to Dr. Vishwanathan, the former Dean of the Drug Research Department at Ayurveda College, "We treated around 40 people. And the response was remarkably good, especially in cases of arthritis and skin ailments."

Experiments to test the clinical effectiveness of Ayurvastra textiles – dyed

in medicinal herb concoctions was held at the G o v e r n m e n t A y u r v e d i c C o l l e g e i n Thiruvananthapuram on 22 August 2006. The outcome of these experiments proved that Ayurvastra aimed at establishing a niche for the ecofriendly handloom fabric. Results of experiments carried out by the Ba la ramapuram-based Handloom Weavers ’


Development Society also supported the fact that handloom fabrics dyed in herbal medicines can actually be used as part of Ayurvedic treatment (8).

The Handloom Weavers’ Development Society had initiated certain experiments in

this direction and the novel idea of Ayurvedic clothes as a project was proposed. Implementation was entrusted to the state department of handlooms and textiles, with assistance for research and development. It was as part of this project that the Dravyaguna Department of the Ayurvedic

College started work on creating Ayurvastra (9). In an earlier similar project, Ayurvedic dyes were developed to add colour to coir products. The Society also had a role in this venture. A Chirayinkeezh-based coir matting company had manufactured coir mats using the medicated coir fibres developed by the Society. The Dravyaguna Department used these coir mats to create an “Ayurvedic environment”, which various trials found can help those suffering from skin diseases, arthritis and blood pressure (10)

A clinical trial initiated by the Ministry of Health at the Government Ayurveda College in Thiruvananthapuram, Kerala, suggest that Ayurvastra does indeed work remarkably good, especially in the cases of arthritis and skin ailments.There are more than 100 finished products (11)

So in this time and age when the world is slowly getting

eroded due to various natural

and man-made calamities, we can do our bit to save the world. Let’s take the first

step with organic clothing!

References:( Please note that some of the following references have been taken from journals published by FAO, funded by WHO, and therefore authors are not indicated.)

1. The Columbus Dispatch, vol.5, March 2006.

2. Clothes that heal, in life positive, january 2009, pg15.

3 . www.ayurvastra.in

4. News letter V & K, vol. 5, spring, 2008.

5. Mongolia Web News, vol.11, April 2006.

6. PR.Com, vol. 12, September 2006.

7. o r g a n i c clothing.blogs.com/my.../02/ayurvastra_herb.html

8. Non Wood News, vol. 14, January 2007, pg 1.

9. www.ayurvedam.com/htm/newtoayurveda.htm

10. The Hindu, vol 24, August 2006.

1 1 .www.associatedcontent.com/.../ayurvastra_a_fabric_from_the_past_ with.html


“Treasure Trove of Sea”- A Key to Human Curses

Nisha Talukdar* (Lecturer)Moumita Sanyal, Priyanka Mukherjee, Rituparna Poria

Department of Microbiology, Garden City College,Bangalore -49.

email ID: [email protected]*

Review Article

ABSTRACT:The treasure of sea, the Sea creature produces certain type of protein and other compounds which can cure or control different disorders and diseases in human beings. There is a very good possibility that the answer to some of the human curses like diabetes, cancer, Alzheimer’s, Parkinson's, Huntington's disorders and infertility may be found in ocean. For example; Sea Urchin's gene which is responsible for the production of "NG Peptides", the protein that helps the creatures to release their egg and sperms at the same time, is similar to mutant gene which causes Diabetes Insipidus in humans.NG Peptides genes enables to understand faulty Vasopressin gene in humans. Likewise, Sponges and Acorns produce Nortopsentin, that has the anti-malarial activity. Sea Cucumber also secretes a protein in their gut, which inhibits malarial parasites. Another compound produced by Sea creature called Eribulin, that also has lot of medical significance.

Keywords*: Sea Urchin ,Sponges, Acorns, Sea Cucumber, NG Peptide ,Nortopsentin, Eribulin, Vasopressin, Diabetes Insipidus , Malaria, Alzheimer’s disorder, Parkinson's disorder, Huntington's disorder, Infertility.

INTRODUCTION:Sea creatures are the main components of life in the ocean. Their course of evolution points out same ancestry with humans thus making them an important model organism for studying various disorders found in human. The resemblance to human genome to a certain extent holds a key to the secrets of many human

disorders like Alzheimer’s (1), Parkinson’s , Huntington ’s disease (2), Diabetes mellitus (3,4), Breast, Colon &Urinary Tract Cancer (5,6,7,8). It also holds the answer too many unanswered questions for malarial diseases. There are around 7000 genes from sea creatures’ e.g. sea urchin which are similar to human genes. For example sea urchin gene produces NG peptides (neurophysin-associated neuropeptides) similar to mutant human vasopressin gene (9,10,11). So sea urchin can be a good model organism for studying diabetes insipidus caused by faulty vasopressin gene (3,4,10,11,12). In a similar way other sea creatures like acorn worms also produces NG peptides. Other studies highlights sponges belonging to phylum Porifera produces Nortopsentin , an alkaloid metabolite and Eribulin have the anti malarial and anti-cancer properties respectively (13,14). Studies have also found out that these sea creatures have a long life span around 100 years so their immune system is very strong so that could be used as an antibiotic and antiviral agents. Thus the sea creatures are indeed treasure trove of sea –a key to human curses.

SEA CREATURES:

SEA URCHIN:• Small, spiny, dominant echinodermata. First

deuterostome outside the chordate includes starfish, sea cucumber, sea lilies and brittle star; usually seen in North Pacific Ocean. They have no eyes and they eat Kelp and Algae, but still the Sea Creature's genome is remarkably similar to humans' and may hold the key to preventing and curing several human diseases.

• Nearly 7000 genes are similar with human pointing to a “common ancestry”, produce quite a few


matching amino acid sequences as well as certain genes involved in vision, hearing and smelling are also similar with humans (Sodergren).The genome of Sea Urchin is more similar to human than fruit fly and worms, thus acting as a “model organism” for studying biological pathway (Francis Collins) and gene regulation (4).

• The recent sequencing of the sea urchin genome establishes homology between sea urchin and vertebrate immune system-related genes. Sea urchins code for at least 222 Toll-like receptor (TLR) genes and over 200 genes related to the vertebrates' Nod-like-receptor (NLR) family found in vertebrates. This increases its usefulness as a valuable model organism for studying the evolution of innate immunity.

• Although it has similar gene family with humans it is usually half the size reflecting in part two whole genome duplication event in vertebrate evolution .Humans have similar innate and acquired immune system but Sea Urchins have 10-20 times greater innate immunity.

• Sea Urchins produce “NG Peptide”- a sex protein that helps the creature to release their egg and sperm at the same time (4,10).

SEA CUCUMBER: Another Echinodermata has gained great medical importance after many humans have become resistant to anti-malarial drug (9).

SPONGES AND ACORNS: • They are usually found in Florida and Great Barrier

Reef of Australia (9).

• The sponges belong to the phylum Porifera while acorns represents Hemichordate

• The sponges are known to produce “Nortopsentin” (Chakraborti and Wright) . It is a marine alkaloid metabolite.

• It has been found that, from Inactive Nortopsentin D (8,14) a Novel Bis (indole) Alkaloid Isolated from the Axinellid Sponge Dragmacidon sp. from Deep Waters South of New Caledonia,which has the stronge cytotoxic effect (8).

• Certain sponges also produce eribulin. Structurally, eribulin is a fully synthetic macrocyclic ketone analogue of the marine sponge natural product

halichondrin B, a potent mitotic inhibitor with a unique mechanism of action found in the Halichondria genus of sponges (9,13).

• Halichondrin B is a naturally-occurring compound originally isolated from the marine sponge Halichondria okadai (Hirata and Uemura I, 1986.) It is also reported that the exquisite anticancer activity of halichondrin B against murine cancer cells both in culture and in in vivo studies. Halichondrin B was highly prioritized for development as a novel anticancer therapeutic (9).

• Acorn Worms produce NG Peptides: a novel family of neurophysin-associated neuropeptides in the same way as the human brain cells produce Vasopressin hormone (10).

HOW SEA CREATURES CAN BE USED AS A KEY TO HUMAN CURSES:• Sea Urchins act as a Model Organism to study

human diseases like Alzheimer’s, Parkinson’s, Huntington’s diseases and other cancer related problems along with Infertility disorders (1,2,3,4,8).

• NG Peptide made by a gene of Sea Urchin, which is similar to a mutant gene that causes Diabetes Insipidus (inability to produce Vasopressin Hormone, that controls the amount of production of urine)in human beings(Prof. Maurice Elphick) (4,12).

• NG Peptides genes enable to understand faulty Vasopressin gene in human. By researching further into how Sea Urchins produce NG Peptides, we will understand better. Why the faulty human vasopressin gene can cause this form of diabetes in humans (Elphick) (5,11).

• Nortopsentin is useful in curing Malaria (Chakraborty and Wright) (9).

• Another compound “Eribulin”, (Japan) is of great medical significance. Eribulin is a mechanistically-unique inhibitor of microtubule dynamics, exerting its anti-cancer effects by triggering apoptosis of cancer cells following prolonged mitotic blockage (5,8,13).

• Sea Cucumber secretes a protein in its guts which inhibits malarial parasites (7,9).


FUTURE ASPECTS:• Sea Urchin feels a large gap in the sequence genome

and thus producing a sneak in the path of evolution between the ancestors that give rise to insect and humans.

• It allows noting the recent innovation in the human genome comparing with ancestor gene also evident in sea urchin. Thus pointing out which are more susceptible to natural selections (5,14).

• Sea creatures have many potential to be used as a model organisms to study cancer related disorders like colon cancer, breast cancer & urinary cancer (5,7,8).

• It is also a building block of the picture of extinct ancestor which gave rise to animal life from worms to human looked like. Thus, it is an ideal model organism for sequencing, analyzing and annotation supported by NIH and NSF.

• Thus in the end sea urchin reminds us of unity underlying of life on Earth.

• Sponges and Acorn Worms has a huge future potential in the study of Breast, Colon and Urinary Cancer due to the healing powers of the unusual compound Eribulin secreted by them(Australia) (5,7,8,13).

• They act as super Antibiotic especially as anti-fungal agents (1,6).

CONCLUSION:These Sea Creatures could become a very powerful tool in the field of medicine. With this study the humans can find the answers for different disorders like diabetes, Alzheimer’s, Parkinson's, Huntington's, cancer related and infertility disorders.

Sea Urchins have been used for many years as a research model to study embryonic development. With their multiplication study, scientists began to understand the complexity involved in human development. Using a very simple creature like Sea Urchin embryo, we can begin to figure out how to intercede to repair and to heal in case of human system.

Sea Creatures have a long life span; some can even live up to 100 years, so their immune system must be very powerful. In this regard, we can say that the Sea Creatures could very well provide a new set of Antibiotic and Antiviral compound to fight various infectious

diseases. Thus sea creatures have the potential to open new dimensions in the field of medicine.

REFERENCES:1. www.bendermedsystems.com

2. www.ParkinsonsDisease-Guidebook.com

3. Jordan MA, Kamath K, Manna T, Okouneva T, Miller HP, Davis C, Littlefield BA, Wilson L (July 2005). "The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth". Mol. Cancer Ther. 4 (7): 1086–95. doi:10.1158/1535-7163.MCT-04-0345. PMID 16020666.

4. Queen Mary, University of London (2010, March 22). Sea creatures' sex protein provides new insight into diabetes. ScienceDaily. Retrieved July 21, 2010, f r o m http://www.sciencedaily.com/releases/2010/03/100322101659. htm

5. Hirata Y, Uemura D (1986). "Halichondrins - antitumor polyether macrolides from a marine sponge". Pure Appl. Chem. 58 (5): 701–710. doi:10.1351/pac198658050701.

6. www.ambrygen.com/nextgensequencing

7. www.taconic.com/KORmice

8. Yu MJ, Kishi Y, Littlefield BA (2005). "Discovery of E7389, a fully synthetic macrocyclic ketone analogue of halichondrin B". in Newman DJ, Kingston DGI, Cragg, GM. Anticancer agents from natural products. Washington, DC: Taylor & Francis. ISBN 0-8493-1863-7.

9. University of Central Florida (2010, July 20). Underwater sponges and worms may hold key to cure for malaria. ScienceDaily. Retrieved July 21, 2 0 1 0 , f r o m http://www.sciencedaily.com/releases/2010/06/100628124601.htm

10. Maurice R. Elphick. NG peptides: A novel family of neurophysin-associated neuropeptides. Gene, 2010; DOI: 10.1016/j.gene.2010.03.004

11. Ines Mancini, Graziano Guella, Francesco Pietra *, Cécile Debitus, Jean Waikedre 25 Oct 2004

12. www.diabeteslibrary.org

13. Okouneva T, Azarenko O, Wilson L, Littlefield BA, Jordan MA (July 2008). "Inhibition of centromere


dynamics by eribulin (E7389) during mitotic metaphase". Mol. Cancer Ther. 7 (7): 2003–11. doi:10.1158/1535-7163.MCT-08-0095. PMID 18645010.

14. Anstrom J. 1989. Sea urchin primary mesenchyme cells: ingression occurs independent of

microtubules. Dev Biol. 131(1): 269-75.

15. Diana P, Carbone A, Barraja P, Martorana A, Gia O, DallaVia L, Cirrincione G. 2007


Role of Neurotransmittersin Mitochondrial Disease

Shruti Awasthi , Parvathi Chary, Pooja AwasthiDepartment Of Biochemistry,

Garden City College, Bangalore.email : [email protected], [email protected]

Neural synapses signifies a special type of polarized junction of two nerve cells which has the particular property of transmitting nervous impulses from one cell to the other. Messages travel within the neuron as an electrical action potential. The space between two cells is known as the synaptic cleft. To cross the synaptic cleft requires the act ions of neurotransmitters. Neurotransmitters are stored in small synaptic vessicles clustered at the tip of the axon. Arrival of the action - potential causes some of the vesicles to move to the end of the axon and discharge their contents into the synaptic cleft. Released neurotransmitters diffuse across the cleft, and bind to receptors on the other cell's membrane, causing ion channels of that cell to open. Some neurotransmitters cause an action – potential ; while others are inhibitory e.g .Acetylcholine and nor - epinephrine. Once the chemicals is in the cleft, neurotransmitters are activated only for a short time. Enzymes in the cleft inactivate the neurotransmitters. Inactivated neurotransmitters are taken back into the axon and recycled. Diseases that affect the function of signal transmission can have serious consequences. Parkinson's disease has a deficiency of the neurotransmitter dopamine. Alzheimer's disease is a group disorder involving the parts of the brain that control thought, memory, and language. Mitochondrial myopathies are a group of neuromuscular diseases caused by the damage to this organelle nerve cells in the brain and muscles require a great deal of energy, and thus appear to be particularly damaged when mitochondrial dysfunction occurs. Some of the more common mitochondrial myopathies include Kearns-Sayre syndrome, myoclonus epilepsy with ragged-red fibres ,mitochondrial encephalomyopathy and ataxia.

Keywords :- Neurotransmitters, Mitochondrial myopathies, Kearns-Sayre syndrome , Acetylcholine, Nor - epinephrine.

ROLE OF NEUROTRANSMITTERS

IN

MITOCHONDRIAL DISEASEThe neuron is the functional unit of the nervous system which have three parts. Dendrites receive information from another cell and transmit the message to the cell body. The Cell body contains the nucleus, mitochondria and other organelles typical of eukaryotic cells. The Axon conducts messages away from the cell body. An action potential is part of the process that occurs during the firing of a neuron. The plasma membrane of neurons, like all other cells, has an unequal distribution of ions and electrical charges between the two sides of the membrane. The outside of the membrane has a positive charge, inside has a negative charge. This charge difference is a resting potential and is measured in millivolts . Passage of ions across the cell membrane passes the electrical charge along the cell. The voltage potential is - 65 mV of a cell at rest. Resting potential results from differences between sodium and potassium positively charged ions and negatively charged ions in the cytoplasm. Sodium ions are more concentrated outside the membrane, while potassium ions are more concentrated inside the membrane. This imbalance is maintained by the active transport of ions to reset the membrane known as the sodium potassium pump. The sodium-potassium pump maintains this unequal concentration by actively transporting ions against their concentration gradients.

Changed polarity of the membrane, the action potential, results in propagation of the nerve impulse along the membrane. An action potential is a temporary reversal of the electrical potential along the membrane for a few milliseconds. Sodium gates and potassium gates open in the membrane to allow their respective ions to cross. Sodium and potassium ions reverse positions by passing


through membrane protein channel gates that can be opened or closed to control ion passage. Sodium crosses first. At the height of the membrane potential reversal, potassium channels open to allow potassium ions to pass to the outside of the membrane. Potassium crosses second, resulting in changed ionic distributions, which must be reset by the continuously running sodium-potassium pump. Eventually enough potassium ions pass to the outside to restore the membrane charges to those of the original resting potential. The cell begins then to pump the ions back to their original sides of the membrane. The action potential begins at one spot on the membrane, but spreads to adjacent areas of the membrane, propagating the message along the length of the cell membrane. After passage of the action potential, there is a brief period, the refractory period, during which the membrane cannot be stimulated. This prevents the message from being transmitted backward along the membrane.

Neurotransmitters are stored in small synaptic vessicles clustered at the tip of the axon. Arrival of the action - potential causes some of the vesicles to move to the end of the axon and discharge their contents into the synaptic cleft. Released neurotransmitters diffuse across the cleft, and bind to receptors on the other cell's membrane, causing ion channels of that cell to open. Some neurotransmitters cause an action – potential ; while others are inhibitory e.g .Acetylcholine and nor - epinephrine. Once the chemicals is in the cleft, neurotransmitters are activated only for a short time. Enzymes in the cleft inactivate the neurotransmitters. Inactivated neurotransmitters are taken back into the axon and recycled. Diseases that affect the function of signal transmission can have serious consequences. Parkinson's disease has a deficiency of the neurotransmitter dopamine. Alzheimer's disease is a group disorder involving the parts of the brain that control thought, memory, and language.

Mitochondrial diseases are a group of disorders relating to the mitochondria, the organelles that are the "powerhouses" of the eukaryotic cells that compose higher-order life-forms (including humans). The mitochondria convert the energy of food molecules into the ATP that powers most cell functions. Mitochondrial diseases comprise those disorders that in one way or another affect the function of the mitochondria or are due to mitochondrial DNA. Mitochondrial diseases take on unique characteristics both because of the way the

diseases are often inherited and because mitochondria are so critical to cell function. The subclass of these diseases that have neuromuscular disease symptoms are often referred to as a mitochondrial myopathy. Symptoms include poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, learning disabilities, mental retardation, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, and dementia. The effects of mitochondrial disease can be quite varied. Since the distribution of the defective mitochondrial DNA may vary from organ to organ within the body, and each mutation is modulated by other genome variants, the mutation that in one individual may cause liver disease might in another person cause a brain disorder. The severity of the specific defect may also be great or small. Some minor defects cause only "exercise intolerance", with no serious illness or disability. Defects often affect the operation of the mitochondria and multiple tissues more severely, leading to multi-system diseases.

Mitochondrial diseases as a rule are worse when the defective mitochondria are present in the muscles, cerebrum, or nerves, because these cells use more energy than most in the body. Although mitochondrial diseases vary greatly in presentation from person to person, several major clinical categories of these conditions have been defined, based on the most common phenotypic features, symptoms, and signs associated with the particular mutations that tend to cause them. An outstanding question and area of research is whether ATP depletion or reactive oxygen species are in fact responsible for the observed phenotypic consequences. Mitochondrial disorders may be caused by mutations, acquired or inherited, in mitochondrial DNA (mtDNA) or in nuclear genes that code for mitochondrial components. They may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes (see MeSH).

Mitochondrial DNA inheritance behaves differently from autosomal and sex-linked inheritance. Nuclear DNA has two copies per cell except for sperm and egg cells, and one copy is inherited from the father and the other from the mother. Mitochondrial DNA, however, is strictly inherited from the mother and each mitochondrial organelle typically contains multiple mtDNA copies (see Heteroplasmy). During cell division


the mitochondrial DNA copies segregate randomly between the two new mitochondria, and then those new mitochondria make more copies. If only a few of the mtDNA copies inherited from the mother are defective, mitochondrial division may cause most of the defective copies to end up in just one of the new mitochondria. Mitochondrial disease may become clinically apparent once the number of affected mitochondria reaches a certain level; this phenomenon is called "threshold expression".

Mitochondrial DNA mutations occur frequently, due to the lack of the error checking capability that nuclear DNA has (see Mutation rate). This means that mitochondrial DNA disorders may occur spontaneously and relatively often. Defects in enzymes that control mitochondrial DNA replication may also cause mitochondrial DNA mutations.

Most mitochondrial function and biogenesis is controlled by nuclear DNA. Human mitochondrial DNA encodes only 13 proteins of the respiratory chain, while most of the estimated 1,500 proteins and components targeted to mitochondria are nuclear-encoded. Defects in nuclear-encoded mitochondrial genes are associated with hundreds of clinical disease phenotypes including anemia, dementia, hypertension, lymphoma, retinopathy, seizures, and neuro developmental disorders. Although research is ongoing, treatment options are currently limited; vitamins are frequently prescribed, though the evidence for their effectiveness is limited. Pyruvate has been proposed recently as a treatment option.

Spindle transfer, where the nuclear DNA is transferred to another healthy egg cell leaving the defective mitochondrial DNA behind, is a potential treatment procedure that has been successfully carried out on monkeys. Using a similar pronuclear transfer technique, researchers at Newcastle University successfully transplanted healthy DNA in human eggs from women with mitochondrial disease into the eggs of women donors who were unaffected. No humans have been produced by any "three person in vitro fertilisation" technique to date. Embryonic mitochondrial transplant and protofection have been proposed as a possible treatment for inherited mitochondrial disease, and allotopic expression of mitochondrial proteins as a radical treatment for mtDNA mutation load Mitochondria (singular: mitochondrion) are structures within cells that convert the energy from food into a

form that cells can use. Each cell contains hundreds to thousands of mitochondria.

These energy producing mitochondria have their own DNA molecules that are used to create a DNA profile, which is called mitochondrial DNA or mtDNA. In humans, the mitochondrial DNA genome consists of about 16,000 DNA building blocks (base pairs), representing just a fraction of the total DNA in cells. Because mitochondria are structurally strong and protect the DNA they contain, mitochondrial DNA is useful for identifying victims of mass disasters, like the Tsunami, where the nuclear DNA in the cells could have been degraded or damaged. It is also often used in Cold Cases. Most cells in our bodies contain between 500 and 1000 copies of the mtDNA molecule, which makes it a lot easier to find and extract than nuclear DNA.

Usually, when we speak about DNA, we are talking about what is technically known as nuclear DNA or nDNA. It controls most aspects of our physical appearance and physical make-up. We know that every cell in our bodies contains two copies of it in the cell nucleus. What sets mtDNA apart is that, unlike nuclear DNA which is equally inherited from both father and mother, mtDNA is inherited only from the mother, because all our mitochondria are descended from those in our mother's egg cells. This means that Mitochondrial DNA is passed from a mother to her children, which also makes it useful for tracing individuals’ maternal lineage. So, that while both sons and daughters inherit mtDNA from their mothers, only daughters can pass their mtDNA to their children.DNA plays an important role in modern forensic science. Today, DNA fingerprinting has become one of the primary methods of identifying people and solving crimes.

Imagine the mitochondrial DNA of all women living today, then that of all their mothers, and their mothers’ mothers. It is obvious that each set will be as small as or smaller than the previous set. Eventually the set will contain only the mitochondrial DNA of one woman - "Mitochondrial Eve". The idea of a "Mitochondrial Eve" came from a 1987 paper by Rebecca Cann and coworkers supporting the Out-of-Africa theory that says that all modern humans descended from a common African ancestral population. Cann's group reported that genetic diversity in mitochondrial genes of all living humans could be traced back to one woman who lived in Africa approximately 200,000 years ago.

Compared with Traditional nuclear nDNA analysis,


Mitochondrial mtDNA offers three primary benefits to forensic scientists:

• Its structure and location in the cell make mtDNA more stable, enabling investigators to test older or degraded samples

• mtDNA is available in larger quantities per cell – smaller samples can be tested

• mtDNA can be extracted from samples in which nDNA cannot, especially hair shafts and bone fragments.

mtDNA sequence analysis is a valuable tool for determining whether individuals are biologically related through their mothers’ side of the family. This is commonly referred to as a maternal lineage test. An mtDNA maternal lineage test works by comparing the mitochondrial DNA (mtDNA) sequences of two or more individuals. People who are biologically related in this way will have similar mtDNA sequences, while individuals who are not will have dissimilar mtDNA sequences. The rise of mtDNA testing in the field of forensics means that cases that were previously thought hopeless, may now be resolved. Mitochondrial DNA in human cells is often more robust and more plentiful than nuclear DNA. MtDNA typing can be performed on hair shafts, bone, and teeth. As a result, mtDNA testing has been widely utilized by investigators in "cold case" police units.

Parkinson’s disease : Loss of dopaminergic neurons of the pars compacta in the substantia nigra and other areas, with reduced levels of dopamine and metenkephalin, altering the dopamine/acetylcholine balance and resulting in striatal acetylcholine overactivity.

L-dopa reaches the synaptic cleft, is taken up by the axon, and is decarboxylated to dopamine ,which is secreted into the cleft to activate dendritic dopamine receptors. Amantadine increases the presynaptic release of dopamine; dopamine agonists stimulate dopamine receptors, although

bromocriptine, pramipexole, and ropinirole bind only to D2, D3, and D4 dopamine receptor subtypes. Anticholinergic drugs reduce activity of the cholinergic system, restoring the balance of dopamine and acetylcholine. MAO-B inhibitors prevent reuptake of dopamine, increasing its levels. Selegiline, an MAO-B inhibitor, blocks dopamine breakdown and thus prolongs the response to levodopa and allows the dosage of carbidopa/ levodopa to be reduced. Catechol O-

methyltransferase (COMT) inhibitors also inhibit dopamine breakdown.

Alzheimer’disease : Extracellular ß -amyloid deposits,intracellular neurofibrillary tangles,

and senile plaques, particularly in the limbic system, in

the association area of the cortex, and in neurons that

synthesize and use acetylcholine (eg, in the basal nucleus of Meynert and its wide projections to the

cortex).Cholinesterase inhibitors (donepezil,

rivastigmine, galantamine) delay synaptic degradation of acetylcholine and thus modestly improve cognitive

function and memory. Memantine, anNMDA-receptor

antagonist, may slow progression of the disease and

increaseautonomy.

Autism : Possible hyperserotonemia, which occurs in

30–50% of autistic people, with no evidence of central

5-HT abnormalities SSRIs and risperidone may be

helpful.

Kearns-Sayre syndrome : Kearns-Sayre syndrome is a rare neuromuscular disorder with onset usually before

the age of 20. It is characterized by progressive external

ophthalmoplegia and mild skeletal muscle weakness. Because mitochondria are found in cells throughout the

body, Kearns Sayre syndrome may affect many different

organs and body systems. Symptoms may

include:Muscle weakness ,Difficulty walking or moving

,Dementia, brain damage, or both , Deafness , Heart

disease , Short stature (38% of individuals) ,Small sex

organs (20% of individuals) If a diagnosis of Kearns

Sayre syndrome is suspected, two tests can be done. One is to check the protein and lactate levels in the

cerebrospinal fluid; if the syndrome is present, the levels

are elevated. The other test would be to examine a

muscle sample (biopsy), looking to see if the DNA in the

mitochondria is abnormal. There is no treatment which

can sure or stop Kearns Sayre syndrome, and

unfortunately its effects become worse over time. Many

complications can develop, such as diabetes, heart

failure, and blindness. Problems are treated as needed.

Exercise may help maintain or increase muscle strength.

The drug ubidecarenone (Coenzyme Q10) may help


relieve symptoms. All individuals with Kearns Sayre syndrome should be monitored by an ophthalmologist

and a cardiologist.

ACKNOWLEDGEMENT

We are greatful to our beloved chairman Dr. Joseph V.

G. and Principal Mr. K.C. JOHN for providing us with

an opportunity to work on this project.

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12. Transmission of an action potential. The above i m a g e i s f r o m http://eleceng.ukc.ac.uk/~sd5/pics/research/big/actpot.gif.

LEGENDS:Figure 1 : NEURON

Figure 2 : TRANSMISSION OF ACTION POTENTIAL

Figure 3 : NEUROTRANSMISSION WITH THE HELP OF NEUROTRANSMITTER

Figure 4 : MITOCHONDRIAL DISEASE

Figure 5 : ALZEIMERS DISEASE

Figure 6 : PARKINSON DISEASE

Figure 7 : KEARNS – SAYRE SYNDROME


Figures:

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7


Alzheimer’s DiseaseM. Obulesu,

R. Venu, R. Somashekhar

Department of Biotechnology, Capital College, Garden City Group of Institutions, Bangalore.

INTRODUCTIONAlois Alzheimer, a German neurologist for the first time identified a series of synonymous symptoms which lead to the memory loss in one of his patients in 1906. Since then this deleterious neurodegenerative disorder has been known as Alzheimer’s Disease. This being the fifth major cause of death in the US, has been reposing burgeoning burden on the health care system due to the ever rising number of patients. The number of Alzheimer’s Disease patients in the world are 12 million currently with one fourth population present in United States alone. Recent reports accentuate that 10% of Americans above the age of 65 and 50% of those above 85 years have been suffering from this disease presently. This number has been expected to augment leaps and bounds and may quadruple by 2050 due to the considerable accrual of aging population in the western world. Although appreciable research has been progressing for more than a century yet neither the etiology of the disease is completely unraveled nor has a potential drug been discovered to put down this deleterious disorder with a heavy hand. This has not only been a biological riddle but also has been a major Achilles heel prevailing in the field of neurobiology for more than a century.

Pathology:The pathology of Alzheimer’s Disease has been intricate and abstruse for almost a century. The hallmark features of this disease are extracellular deposition of amyloid beta peptides, for mation of intraneuronal neurofibrillary tangles, hyperphosphorylation of tau, oxidative stress etc. The etiology has been complicated entailing manifold aspects such as oxidation of biomolecules like DNA, proteins and lipids, mutations in genes such as amyloid precursor protein, presenilins and apolipoprotein E, impaired metabolic pathways such as lipid metabolism, calcium homeostasis etc. The diagnosis of this disease has been a problem of

considerable concern because the usual memory loss occurring during normal aging camouflages the primary event of memory loss in this disease.

Enzymes:ß, ?- secretases are the enzymes which play a pivotal role in the processing of amyloid precursor proteins. These proteins are very long containing more than 400 amino acids. They are cleaved to small peptides ranging from 39-43 amino acids. They are popularly called A ß peptides and they play significant role in senile plaque formation. These senile plaques generate reactive oxygen species which cause tremendous havoc in the cell eventually leading to cell death.

Oxidative Stress:The reactive oxygen species like OH radicals, superoxide radicals are usually generated during normal metabolic pathways in mitochondria of the cell. Because the cells are endowed with substantial antioxidant enzymes and a few antioxidants are ingested by people, well-timed scavenging of these radicals takes place in the cell. Elevated production of these radicals occurs in Alzheimer’s Disease thus failing the cell to counter them potentially.

Oxidation of Biomolecules:Elevated oxidative stress leads to oxidation of biomolecules like nucleic acids (DNA, RNA), proteins and lipids which aggravate apoptosis. Guanosine, a primary component of nucleic acids has been found to be susceptible for oxidation compared to other nucleotides. Brain being rich in lipids is more susceptible for peroxidation of lipids instigated by free radicals like OH, 4 hydroxynonenal.

Metals:Metals also play a crucial role in the etiology of Alzheimer’s Disease. Aluminium, Iron, Copper and Zinc are a few metals which cause considerable havoc in


neurons, despite their vital role in the physiology. These metal ions act as cofactors in paramount enzymes entailed in crucial biological pathways. Epidemiological studies showed that the disease has been prevalent in the areas where potable water is rich in Aluminum. It has been estimated that the nations which are enriched with Aluminium industries anticipate more citizens with Aluminium exposure which results in the onset of this dreadful neurodegenerative disorder. Citizens of United States have been constantly exposed to this Aluminium. They also stand first in consumption of antacids in the world although their population is less compared to the world’s population. These antacids are rich in Aluminium, hence driving the consumers towards the onset of this disease. Despite the employment of a few metal chelators as therapeutic agents, they did not give considerable protection from the disease. Henceforth, there is a growing need to develop appropriate drugs to combat this disease.

Conclution.Multi -dimentional therapeutic approach is required to target disease by using different drugs so that target site

and biochemical interference should be traced ,ultimately age related Alzheimer’s and early onset AD should be treated.

REFERENCES.Carlos A.Saura.,2010. Presenilin/ã-secretase and inflammation. Frontiers in Aging Neuroscience .2: 1-11

Knowles,R.G., Monocada, 1994. Nitric oxide synthase in mammals. Biochem.J. 298(2): 249-258.

Ravinder Pannu, and Inder j i t S ingh.2006. Pharmacological strategies for the regulation of inducible nitric oxide

Selkoe,D.J. 2002. Alzheimer’s disease is a synaptic failure. Science. 298: 789-791.

synthase: Neurodegenenerative versus neuroprotective mechanisums. Neurochemistry International. 49:170-182.

Thomas D Bird,MD 2008. Genetic Aspects of Alzheimer Disease . Genet Med.10(4):231-239.

16th KM, Old Madras Road, Bangalore - 49Tel: 080-66487600, Fax: 080-66487667, E-mail: [email protected] / [email protected]

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