eChapters

Long used as beasts of burden, animals in India are still regarded today as little more than commodities rather than sentient beings who feel pain, stress, exhaustion and depression. Low literacy rates and the tendency to follow traditional practices and cultural traditions have left India surely lacking in the area of animal welfare. India holds the majority of the world’s working animal population. Animals that are forced to work in India include bullocks, horses, ponies, mules, donkeys, camels, elephants and dogs. Despite a proud heritage and a culture rooted in tolerance and compassion, animal welfare is largely ignored by Indian society, leading to extreme animal suffering. Our villages and to a great extent, the urban population still allow working animals to be mercilessly exploited in fields, on the streets, in brick kilns, on battlefields, on playgrounds and in timber areas – all in the name of agriculture, entertainment, security and any number of other “justified” pursuits.

ABSTRACT

This paper is an attempt to draw the attention of readers of this volume to understand the importance of documentation and preservation of earth’s resources.  

Introduction

Earth has innumerable treasures that were and are being exploited by mankind. Geo-resources in the form of minerals, non-renewable hydrocarbons, paleontologic relics and related evidences indicate earth’s evolutionary history. Continuum of biological activity albeit varying rates of proliferation had resulted in accumulation of natural resources embedded in Earth and distributed at specific geographical niches. Biological perspective of earth history is significant in unraveling bio-geo-sphere interactions through geological time. Fossil records of invertebrates, vertebrates and plants are signals of major changes in earth’s evolutionary history. These constitute unparalleled windows to understand continental separation. Intercontinental and intra continental migration of living forms are also traced through fossil study.

Introduction
The Protection of Civil Rights Act, 1955 (PCRA) is a crucial piece of legislation in India, designed to address social discrimination and uphold the equality enshrined in the Constitution. Originally known as the Untouchability (Offences) Act, 1955, it was later amended to become the Protection of Civil Rights Act. This law aims to eliminate the social stigma associated with untouchability and caste-based discrimination, providing a framework to ensure that all individuals enjoy civil rights, regardless of caste, creed, or background.
Historical Background
India’s caste system has deep historical roots, often leading to various forms of social exclusion and discrimination, especially for those belonging to the lower castes or “Dalit” communities. Recognizing the need for legal safeguards, India’s Constituent Assembly introduced Article 17 in the Constitution, abolishing untouchability. However, legal provisions were needed to enforce these rights effectively, leading to the enactment of the Untouchability (Offences) Act in 1955. With amendments and broader interpretation, it evolved into the Protection of Civil Rights Act, 1955.
Objectives of the Protection of Civil Rights Act
The PCRA aims to
1. Abolish untouchability in all forms.
2. Protect and promote civil rights, ensuring equal access to public places and services.
3. Prohibit discrimination in any social, economic, or educational sphere based on caste or religion.
4. Penalize those who commit acts of discrimination or deprive others of their rights based on caste or other prejudicial factors.

Introduction

A generator is subjected to electrical stresses imposed on the insulation of the machine, mechanical forces acting on the various parts of the machine, and temperature rise. These are the main factors which make protection necessary for the generator or alternator. Even when properly used, a machine in its perfect running condition does not only maintain its specified rated performance for many years, but it does also repeatedly withstand certain excess of overload.

Preventive measures must be taken against overloads and abnormal conditions of the machine so that it can serve safely. Even ensuring an efficient design, construction, operation, and preventive means of protection – the risk of a fault cannot be completely eliminated from any machine. The devices used in generator protection, ensure that when a fault arises, it is eliminated as quickly as possible.

Abstract

GIS has several products that can be protected in India as copyright. The paper describes major provisions of copyright Act, 1957 that has been amended five times. GIS products qualify to be registered within various sections of the copyright Act. Patenting of software is permitted in some countries but not in India, a comparative account of these two forms of protection has been discussed with practical examples. General public licensing of open source software, translation, exclusive licensing and data sharing & accessibility policy are other areas covered in the present study and analytical learning.

Key words : copyright, patent, GIS, software, data sharing

1.0 Introduction

The copyright Act 1957 was mainly amended in 1994 and1999 to bring into complete harmony with Trade Related Aspects of Intellectual Property Rights (TRIPs), further major amendment has been done in 2012, that include wide range changes in ownership, term of protection, copyright security, cinematography and broadcasting provisions. In the same year government of India has initiated ‘National Data Sharing and Accessibility Policy (NDSAP)’ which came into operation from February 2012. At present the copyright Act and data sharing policy are the two legal and administrative instruments which facilitate the intellectual property protection and appropriate sharing among stakeholders in the country.

1. PPV & FR ACT

With the signing of TRIPs agreement of the World Trade Organization (WTO), India has agreed to provide protection to plant varieties. The Government of India, therefore, enacted “ The Protection of Plant Varieties and Farmers Rights Act (PPV&FR Act)” in 2001 and the Regulations for implementing the Act were formulated in 2003.

The Ministry of Agriculture, Government of India, has been identified as the nodal agency for implementing the PPV& FR Act through an Authority known as “The Protection of Plant Varieties and Farmer Rights Authority”. The Authority came into force in November, 2005.

PPV & FR act has 11 chapters, 4 schedules and 97 sections.

14.1. PPVFR Act

TRIPS (Trade Related Aspects of Intellectual Property Rights) agreement of WTO (World Trade Organization) was signed.

Enacted PPVFR act in 2001.

Regulation formulated in 2003.

Protection of Plant Varieties and Farmers’ Rights Act-2001 (PPVFRA-2001) was enacted by government of India in 2001 to create a sui generis system of providing a legal framework as an IPR mechanism to protect the varieties of crop plants in India along with grant of breeders’ and farmers’ rights in the interest of all the stakeholders and also to comply with WTO guidelines to which India is a legal signatory. The salient features of this act are as follows.

The Protection of Plant Varieties and Farmers’ Rights Act-2001, Act no. 53 of 2001, (30th october, 2001)

This act is in place to provide for the establishment of an effective system for protection of plant varieties, the rights of farmers and plant breeders and to encourage the development of new varieties of plants. It recognizes and protects the rights of the farmers in respect of their contribution made at any time in conserving; improving and making available plant genetic resources for the development of new plant varieties; it encourages accelerated agricultural development in the country; it protects plant breeders’ rights to stimulate investment for research and development, both in the public and private sector, for the development of new plant varieties; it facilitates the growth of the seed industry in the country which will ensure the availability of high quality seeds and planting material to the farmers. To give effect to the aforesaid objectives, it is necessary to undertake measures for the protection of the rights of farmers and plant breeders; as India, has ratified the Agreement on Trade Related Aspects of Intellectual Property Rights should, inter alia, make provision for giving effect to sub-paragraph (b) of paragraph 3 of article 27 in Part II of the said Agreement relating to protection of plant varieties. Accordingly, this act is in place.

Protection of Plant Varieties and Farmers’ Rights Act-2001 (PPVFRA-2001) was enacted by government of India in 2001 to create a sui generis system of providing a legal framework as an IPR mechanism to protect the varieties of crop plants in India along with grant of breeders’ and farmers’ rights in the interest of all the stakeholders and also to comply with WTO guidelines to which India is a legal signatory.

Chapter I
Preliminary 
1. Short Title and Commencement 
    (1)    These rules may be called the Protection of Plant Varieties and Farmers’ Rights Rules, 2003.   
    (2)    They shall come into force on the date on which the Act shall come into force.   

2. Definitions 
        In these rules, unless the context otherwise requires, -   
    (a)    “Act” means the Protection of Plant Varieties and Farmers’ Rights Act, 2001(53 of 2001);

The plant variety is related to intellectual property that needs to be protected for controlled commercial unitization for a specified period. New plant varieties development requires scientific skill, knowledge, significant investment of land and specialized equipments. These varieties are cultivated by farmers in their adaptive region. In development of new variety 10 to 15 years are required. If developed variety successfully commercialized on farmer’s field, then the market business is much higher than input investment. The major issue in developed variety that can be easily reproduced by someone without permission of original breeder. Thus, the original breeder deprived for their f inancial benefit. It is, therefore, critical to provide an effective system of plant variety protection, which encourages the development of new varieties of plants thereby benefiting the breeder and society at large. The sustainable and long-term breeding effort for development of new varieties needs encouragement and financial reward for investing time and skills. To secure legal protection of the developed variety breeder needs to obtained exclusive right from government.After establishment of WTO in 1995 under TRIP the right to protect plant varieties become mandatory for signatory country. It has also realised that existence of the private seed industry, the protection of plant varieties become necessity.

In order to provide for the establishment of an effective system for protection of plant varieties, the rights of farmers and plant breeders and to encourage the development of new varieties of plants it has been considered necessary to recognize and protect the rights of the farmers in respect of their contribution made at any time in conserving, improving and making available plant genetic resources for the development of the new plant varieties. Moreover to accelerate agricultural development, it is necessary to protect plants breeders’ rights to stimulate investment for research and development for the development of new plant varieties.

India is known for its rich wildlife heritage. In fact, the country is considered one of the best among available mega-biodiversity areas in the world. The landscape and agro-climatic conditions of India make it a unique habitat for wild animals. The government of India has made attempts to conserve wildlife through different projects like Corbett tiger, Elephant projects etc. The wild life management in India is at present ‘preservation-oriented’. This is bound to remain so for a long time because of the significantly depleted status of our wild life population. Indian government in pursuit of conserving the heritage of wilderness has made committed efforts with several wildlife and Forest Acts to ensure protection by declaring the areas as protected under the spectrum of national park and wildlife sanctuaries. The eco-tourism and conservation awareness has created environmental consciousness among the people.

Duff and  Daly (2002) briefly describes the commonly found orchid diseases. These include the Bacterial soft rot caused by Erwinia carotovora, black leg or slow decline caused by Fusarium spp., petal blight caused by Curvularia eragrostidis [PseudocochlioBolus eragrostidis] and Alternaria alternata, top rot/shoot rot caused by Phytophthora parasitica, and stem rot caused by Sclerotium rolfsii [Athelia rolfsii].

ABSTRACT

The present study was designed to evaluate the effect of α-mangostin on cardiac markers against isoproterenol-induced myocardial infarction in rats. Induction of rats with Isoproterenol (150 mg/kg body wt, i.p) for 2 days resulted in a marked reduction in the activities of cardiac tissue marker enzymes (LDH, CPK, GOT and GPT). Pretreatment with α-mangostin (200 mg/kg of body wt/day) orally for 6 days significantly prevented these alterations and restored the enzyme activities to near normal levels. In conclusion, these findings demonstrate the protective effect of α-mangostin on isoproterenol induced experimental myocardial infarction in rats.

1. The protein is derived from Greek word “Proteins” which means
   1. Principal
   2. Prime
   3. Holding the first place
   4. All of the above
2. The name protein to the group of organic compounds that are utmost important to life is suggested by
   1. Mulder
   2. Berzelius
   3. Kulim
   4. Sanger

1. Protein Estimation

The method developed by Lowry et al. (1951) is largely followed for the estimation of soluble protein. This method is quite sensitive and gives a moderately constant value.

Protein may also be estimated by analyzing the total nitrogen content. Multiplying total nitrogen value with 6.25 will give the crude protein content. However, the nitrogen per cent varies with amino acid composition of proteins. For more refined expression of protein percentage, different factors are used for different samples. Some of the conversion factors for cereal and oilseed are given below:

Objective: To separate protein fragments by using SDS-PAGE method.

Background information

Electrophoresis is the motion of dispersed particles relative to a fluid under the effect of limited uniform field of electricity (Dukhin, and Derjaguin, 1974). Ferdin and Frederic Reuss (Reuss, 1809) first time observed such phenomena of electrokinetic nature in 1807. They found that the clay particles move in the water by giving constant supply of current. It happened due to interface of charged particles surface and the surrounding media. The technique is used widely in chemistry, biochemistry and molecular biology to separate different molecules based on difference in charge, binding affinity and size. Electrophoresis may be of two types viz., Anaphoresis and Cataphoresis. Anaphoresis is the electrophoresis of anions (-ve charged ions) while, Cataphoresis is electrophoresis of cations (+ve charged ions). Electrophoresis is a major analytical technique in biochemistry that is used for separation of macromolecules like protein, enzymes and nucleic acids. Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size.

PAGE is abbreviated for Polyacrylamide gel electrophoresis that describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

• All green leafy materials in nature have Mg, because it is the structural component of chlorophyll. Wherever such green colour is there, carotenes would be associated. The colour of carotenes is red, but this
red colour is masked by green colour of chlorophyll. Wherever above green colour is there, vitamin K would also be invariably associated. Therefore Mg, vitamin A and vitamin K would be available by – default or what!! {α, β, ϒ, δ – carotene (s)}. Hence, grazing animals generally would be receiving these nutrients. [we humans may also eat fresh green leafy vegetables {spinach (palak), bathua, methi, coriander (dhania), cabbage, knol – khol, capsicum (bell pepper, Shimla mirch) to get above nutrients]
• Vitamins help regulate body functions, keeping the body healthy and promoting resistance to dis – eases.
• Supplementation of micro – nutrients (vitamins and minerals) as pre – mix, have + ve effects on maintenance, growth, and milk production.
• Biotin, niacin, choline improves milk production in dairy cattle.
• Multivitamin tablet = A to Z0NS may be given (preferably natural sources would always be suggested).
• Vitamin ‘B’ complex, vitamins C, K are very much synthesized by rumen microbes.
Importance of vitamins and their requirements in animal health and production:
Tables 2 & 3

Crude protein of feeds contain true protein and non – protein nitrogen. True protein is made up of amino acids and for maximum efficiency, feed must have essential acids in correct proportion, correct balance as well as non – essential amino acids should also be in sufficient amounts. About digestion in simple stomached animals, the true protein is degraded to oligopeptides (less than 10 peptides) in the stomach, subsequently to mono peptide amino acids in the small intestine. Thereafter amino acids are assimilated in the small intestine.
But in ruminants, the situation is complex in the sense that feed proteins get digested in the rumen, but even amino acids are also broken down by the microbes, and thereafter, amino and carboxylic groups are released. Secondly, synthesis of new amino acids or proteins takes place for formation of their own microbial body coat. Because of these reasons, the approach for protein evaluation or expression is different in ruminant animal. Further, non-protein portion of the feed cannot be utilized effectively by non – ruminants like swine and poultry. These animals are usually fed with oil cakes, cereals and
cereal by products, which are poor in non-protein compounds, however, young succulent fodders, clover etcetera are rich in such compounds. In the laboratory, crude protein may be estimated by kjeldahl’s method and true protein may be precipitated from non – protein nitrogen fraction by treating with cupric hydroxide or trichloro acetic acid. The precipitate is filtered off and subjected to kjeldahl’s process. For experimental evaluation of non – ruminants: albino or wistar rats, rabbits, guinea pigs or poultry birds are taken. Casein (milk protein) or albumen (egg protein) are fed to these animals for a period of 4
weeks and any of the following methods are undertaken to look for the protein quality based on response of animals.

A. Answer the following Question

1.    Why availabilityof bacterial protein is low?

Ans: Because, bacterial protein is protected by cell wall (mucopeptide polymer) which are resistant to enzymatic action.

2.    What is isoelectric pH?

Ans: The pH at which an amino acid exists in solution as a zwitter ion is called isoelectric pH that is the pH at which the amino acid is electrically neutral and has no tendency to migrate towards either electrode.

3.    What is ketogenic amino acid?

Ans: Ketogenic amino acid refers to those amino acids which give rise to acetyl CoA and consequently the potential fatty acids producers. e.g. leucine

4.    Why chicks require higher level of arginine?

Ans: Since they do not have urea cycle and feathers are very high inarginine.

5.    Why chicks require high level of glycine?

Amino acids serve as the building blocks of proteins and as precursors of many other important biomolecules such as hormones, purines, pyrimidines, porphyrins and some vitamins. They also serve as sources of energy when present in excess amounts and when they are used as fuel, amino acids undergo deamination reactions. The remaining carbon skeleton will either convert itself into glucose or undergoes oxidation to CO₂ via TCA cycle.

Central dogma: The Central dogma of molecular biology, which has been formulated as a simple linear progression of information from DNA to RNA to Protein, is summarized in the illustration. The replication process on the left consists of   passing  information from a parent DNA molecule to daughter molecules. The middle  transcription process copies this information to a mRNA molecule.  Finally, this information is used by the chemical machinery of the ribosome to make polypeptides.

Introduction

Proteins are the most abundant organic compounds constituting major part of the body. Chemically, proteins are nitrogen containing macromolecules and polymers of L α-amino acids, which serve a wide range of structural and biological functions in animal body. For example, connective tissue made up of collagen, metabolicenzymes, neuro transmitters,signal in gmolecules, structural components of cell membranes and cell organelles, etc. all are chemically proteins.

Before goinginto the details of protein metabolism, first, we need to understand that there are majorly two sources of proteins in our body: dietary protein and body protein. Dietary protein is taken in diet, while body protein indicates the protein and amino acids synthesized in animal body to fulfill the requirements for growth and development. The digestion of proteins will not be discussed here as it has been described in detail in chapter 12. Dietary protein is digested in presence of several enzymes such as trypsin, pepsin, chymotrypsin, aminopeptidases and carboxypeptidases and is converted into mono-, di- and oligopeptides in stomach and small intestine; and finally converted into amino acids. These amino acids are transported to the systemthrough intestinal mucosa via active transport, from where they enter into the cells for further metabolism. Therefore, ‘metabolismof proteins’ is more appropriatelylearnt as ‘metabolism of amino acids’.

Summary

Researchers face a series of similar-yet unique problems to isolate proteins. To solve these they must have dip knowledge to select an appropriate technique. The mechanism, to study the way in which a living system works, is necessary to dismantle the machine and to isolate the component parts so that they may be studied, separately and in their interaction with other parts. The knowledge that is gained in this way may be put to practical use, for example, in the design of medicines, diagnostics, pesticides, or industrial processes. In every case a pure protein is desirable as impurities may either be misleading, dangerous or unproductive, respectively. Protein isolation is, therefore, a very common, almost central, procedure in biotechnology. The purpose of this chapter is thus to fill the beginners with techniques relevant to protein isolation, hopefully to improve their knowledge.

Introduction

All the amino acids have in common an amino group and a carboxyl group attached to the -carbon atom; however, they differ in having distinctive side chains, usually called R groups, as is shown in the following  generalised structural formula

Abstract

The field of bioinformatics emerged as a tool to facilitate biological discoveries more than 10 years ago. Structure prediction is fundamentally different from the inverse problem of protein design. Protein structure prediction is one of the most important goals pursued by bioinformatics and theoretical chemistry; it is highly important in medicine and biotechnology. Structure prediction is fundamentally different from the inverse problem of protein design. While the genome of many organism have been sequenced over the last few year, transforming such raw sequence data into knowledge remains a hard task. A great number of prediction program for genes have been developed hat try to address this problem, which consists of locating the genes along a genome.

1. Introduction

Protein–protein interactions serve as the hub of the functioning of a living cell, coordinate biological processes as well as form core for interactomics system of living organisms. Various biological processes as signal transduction, cell-cell communication, ingestion or expulsion of any macromolecule, host parasite interaction, diseases as cancer and many other metabolic networks employ protein–protein interactions. During such a process two or more proteins interact transiently with each other or form complex for a long period of time. Thus attempts have been made for years to study comprehensive mechanism of such protein–protein interactions by various branches of sciences such as biochemistry, quantum chemistry, molecular dynamics, chemical biology and signal transduction. The availability of three-dimensional (3D) structure of proteins is a prerequisite for understanding functions of protein complexes at a molecular level as well as amino acid interactions within a complex.

Abstract

The field of bioinformatics emerged as a tool to facilitate biological discoveries more than 10 years ago. The ability to capture, manage, process, analyze and interpret data became more important than ever. Bioinformatics in agricultural science has many complex aspects and problems that must be resolved due to the involvement of a wide variety of fields and subjects. Consequently, the establishment of interdisciplinary research areas is required, which should include collaboration among experimental and informatics researchers in different fields within industry, academia, and government. Thus, the Agricultural Bioinformatics Special Interest Group was established to promote interaction among researchers and provide an opportunity to report on bioinformatics studies in agricultural science: design and analysis of functional foods, scientific testing of food safety, breeding of agricultural organisms, development of environmental friendly agricultural chemicals, bioremediation and production of useful chemical materials using microorganisms, measuring and monitoring biodiversity, statistical genetics in resource ecology, and so on. The activities of the Agricultural Bioinformatics Special Interest Group include (1) Studies of applied bioinformatics in the agricultural science (2) Development of new research areas in agricultural science using bioinformatics (3) Practical application of bioinformatics technologies in the field of agricultural science (4) Development of new bioinformatics technologies characteristic of agricultural science.

Abstract

Proteins are complex molecules composed of one or more polypeptide chains made up of amino acid residues. These molecules perform a variety of essential functions in living organisms, including DNA replication, molecule transport, catalyzing metabolic processes, and providing structural support to cells. In this chapter, we explore the four levels of protein structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the specific sequence of amino acids in a polypeptide chain, while secondary structure describes the folding of the chain into alpha-helices, beta-sheets, or other patterns. Tertiary structure is the three-dimensional shape of a single polypeptide chain, and quaternary structure refers to the interactions between multiple polypeptide chains to form a larger protein complex. Additionally, this chapter covers various methods used to predict protein structure, including homology modeling, ab initio prediction, and threading algorithms.

Abstract

Protein-protein interactions play a major role in most cellular processes. Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. The study of protein-protein interactions is of great importance in determining the biological function and impact of proteins. The experimental techniques remain expensive, time-consuming and labor-intense. Computational methods play an important role at all the stages of determining protein-protein interactions. In this chapter, we describe the different types of protein-protein interactions, computational methods, currently available databases and tools, protein-protein docking and as well as protein-protein interactions as drug targets.

Proteinase inhibitors (PIs) are small proteins that are quite common in nature. They are natural, defense-related proteins often present in seeds and induced in certain plant tissues by herbivory or wounding (Koiwa et al., 1997). PIs are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms. In plants they can be counted among the defensive mechanisms displayed against phytophagous insects and microorganisms. The defensive capacities of plant PIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development. PIs are of common occurrence in the plant kingdom. Plant PIs (PPIs) are generally small proteins that have mainly been described as occurring in storage tissues, such as tubers and seeds, but they have also been found in the aerial parts of plants (De Leo et al., 2002). One of the important defense strategies that are found in plants to combat predators involve PIs which are in particular effective against phytophagous insects and microorganisms. The defensive capabilities of PPIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development (Lawrence and Koundal, 2002).

Proteins are large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalysing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity. A polypeptide is a single linear polymer chain derived from the condensation of amino acids. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and, in certain archaea-pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes.

Like other biological macromolecules such as pollysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals’ diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism.

INTRODUCTION

Plants are rich source of nutrients for most of the organisms including microbes' arthropods and vertebrates. There are various biotic viz. insects, weeds and pathogens and abiotic factors viz. temperature, humidity, salinity etc. that causes harm to plants. For yield loss due to pest EIL (Economic injury levels) calculation is important (Stern et al, 1959). EIL calculation will define economic damage (yield losses equal to management costs) in calculable terms. Since plants are sessile they cannot move to avoid the menace of these factors, but they have developed a unique way of structural, chemical, and protein-based defences which are specifically designed to detect and inhibit invading organisms before they are able to cause extensive damage. In response to the incitations by the pathogens some genes are activated which in turn codes for defence proteins (Grennan, 2006). Understanding how plants defend themselves from pathogens and herbivores is essential in order to protect our livelihood and nutritional security. In addition to proteins, plant anatomy and some of the ecological relationship also plays an important role in plant defense mechanism. Induction of plant resistance against fungi and bacteria was reported in 1900.

The word protein was first coined in 1838 to emphasize the importance of this class of molecules. The word is derived from the Greek word proteios which means “of the first- rank”. Proteins are polymers/macro molecules made up of several amino acids. When there are more than 50 amino acids in a chain, the polypeptide is normally called a protein. Each protein in the cells of living beings has a unique sequence of amino acids that determines its biological function. The proteins are folded into specific defined structures, which are maintained by a large number of relatively weak bonds. The  three  dimensional structure is well defined and suited to it’s specific function.

Very small changes in the structure can modify the function. Hence, it is important to know the structure of proteins to understand their behavior and function. The biological activity of proteins depends on maintenance of folded conformation. Proteins fold into well defined three dimensional shapes and they are able to recognize their corresponding substrates or antigen molecules and bind them tightly. The protein structure has been classified into four different levels based on the folded conformation of the protein.

1.    The basic building block(s) of proteins is/are

(a) Amino acids

(b) Phenolics

(c) Carboxylic acids

(d) Fatty acids

2.    Alpha helix and ?- pleated sheet are characteristic of protein structure?

(a) Primary

(b) Secondary

(c) Tertiary

(d) Quaternary

1. INTRODUCTION

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Proteins are the important constituents of foods for a number of reasons. They are a major source of energy, as well as containing essential amino-acids, like lysine, tryptophan, methionine, leucine, isoleucine and valine which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability.

Proteins are complex organic molecules made up of carbon, hydrogen, oxygen, and nitrogen. Most proteins also contain sulphur and phosphorus and also play an important role in food preparation. The final quality of many food products are influenced by how the proteins are treated while food is being prepared.

Proteins are composed of amino acids and are large molecules that play a crucial role in various biological processes. They are considered the fundamental building blocks of life due to their abundance in the body, constituting approximately 60% of the dry weight of cells. These complex biomolecules are intricately folded and twisted strands of amino acids, actively participating in vital life processes, including metabolism, locomotion, immune response intercellular signaling, and molecular identification. As macronutrients proteins are present in all living organisms and directly contribute to numerous metabolic pathways. It is important to note that proteins are unique to each organism, exhibit species specificity, and vary within different organs of the same organism. With 22 different amino acids, the properties of a protein molecule are determined by the specific arrangement of these amino acids. While plants can synthesize all the necessary amino acids for protein production, animals rely on external sources for certain amino acids. Proteins consist of amino acids connected through peptide bonds, formed by the linkage between the amino (NH2 ) group of one amino acid and the carboxyl (COOH) group of another amino acid.

Introduction

Aquatic ecosystems are critical components of the global environment and provide a variety of resources for human population, including food, water for drinking and irrigation, recreational opportunities, and habitat for economically important fisheries. Fish is the main source of animal protein for 3 billion people worldwide, providing a valuable protein complement to the food platter worldwide. Fish is also an important source of essential vitamins and fatty acids. Furthermore, it provides an important source of cash income for many poor households and holds a great potential as a source of foreign exchange. However, aquatic ecosystems are increasingly being threatened directly and indirectly by climate change. Global warming with consequent climate variability and extreme weather conditions has a great impact on the aquatic ecosystems resulting in increased temperature variation, ocean acidification and freshwater salinization etc. As per report of IPCC 2018, global warming of 1.5 ºC above pre-industrial levels has been declared (IPCC, 2018) and expected to rise further in future. Thus, variations in temperature, salinity, acidity and dissolved oxygen along with the effects of other anthropogenic activities may exert strong in?uences on aquatic ecosystems and the aquatic animls will be facing these climate-related stressors in near future. Such environmental stressors is projected to alter fundamental ecological processes, geographic distribution of aquatic species, reproductive and immunological performance of species etc.

The genera Proteus and Providencia belong to the tribe Proteae of the family Enterobacteriaeceae. The members of both these genera are gram negative, motile bacilli, aerobes and facultative anaerobes and can grow on basic media. A characteristic feature which distinguishes tribe Proteae from other members of Enterobactereaceae is the presence of the enzyme phenylalanine-deaminase which converts phenylalanine to phenylpyruvic acid (PPA reaction).They also produces a powerful urease enzyme which rapidly hydrolyses urea to ammonia.
Genus Proteus
They are gram-negative bacilli, 1-3 μm long and 0.6 μm wide. They are noncapsulated and are actively motile by peritrichous flagella. The name ‘Proteus’ refers to their pleomorphism, after the Greek God Proteus who could assume any shape. Four species: Proteus mirabilis, P.vulgaris, P.penneri and P.myxofaciens are recognized. Proteus mirabilis, P.vulgaris are widely recognised as human pathogens.
Culture Characteristics
These can grow on ordinary media like nutrient agar with a characteristic fishy or seminal odour. On MacConkey and Teepol lactose agar, lactose nonfermenting pale colonies, around 2-3 mm in size are formed. On non-inhibitory solid media such as blood and nutrient agar Proteus mirabilis and P vulgaris show characteristic swarming growth in the form of a uniform film, which spreads over the whole surface of the plate. In young swarming cultures, many of the bacteria are long, curved and filamentous, sometimes reaching upto 80 μm in length. When two different strains of swarming proteus mirabilis encounter one another on an agar plate, swarming ceases and a visibile line of demarcation forms. This is known as the Dienes phenomenon. In liquid medium (peptone water, nutrient broth), Proteus produces uniform
turbidity with a slight powdery deposit and an ammoniacal odour.

1.    Collect the bone marrow from iliac crest of rabbit, dog, goat and sheep; sternum of pigs by 16-18 G bone marrow biopsy needle under sedation/anesthesia and maintain strict asepsis protocol
2.    To aspirate bone marrow, negative pressure should create in the syringe by pulling back the plunger. Syringe should be first flushed with heparin before bone marrow collection
3.    Collect approximately 2.5-5 ml of bone marrow from each animal
4.    Mix the bone marrow sample with equal volume of low DMEM having 10% FBS and 1 ml of PS and then layer on 5 ml of histopaque/Ficol-Paque Plus in a falcon tube
5.    Centrifuge at 3000 rpm for 30 min
6.    Collect stem cells from the interface with the help of a micropipette
7.    Cells wash thrice with DPBS 
8.    Centrifuged at 2000 rpm for 5 min

Collect the femur and tibia and clean them very well from blood, tendons, muscle, skin and other tissues (using scalpel)

Cut off epi-and metaphysis from the long bones with electric/manual bone saw
Transfer the open bones to a falcon tubes containing cell culture medium/DPBS without FBS but containing 1% PS (Penicillin-streptomycin)
Transfer the bones to a Petri dish containing 5 ml medium without serum (FBS) and without antibiotic (40-50 mm Petri dish)
Remove the pteri dish to the laminar cabinet
Collect the bone marrow by flushing out to a 50 ml falcon tube (use syringes and needles to flush out the bone marrow; flush out the bone marrow with complete (10% FBS, 1% PS, 90% DMEM-LG) culture medium
Centrifugation: 300rpm, 8 min (in the meanwhile prepare 5 ml of Ficoll into a 15ml falcon tube)
Aspire the medium

1.    Collect fat tissues from euthanized animals into a Falcon tube containing DPBS + 1% PS ( for transferring lab to lab)
2.    Wash 3 times with DPBS + 1% PS
3.    Inside a laminar cabinet, transfer the fat to a Petridis (use an sterile spatula to collect the fat)
4.    Cut the fat into small (mm) pieces using scalpel and forceps or scissors
5.    Transfer the fat into a Falcon tube (eg into a 50 ml Falcon tube you can add upto 10 ml fat tissues)
6.    Wash the fat by adding approx 40 ml DPBS; Centrifugation: 430g, 10 min (without break)
7.    Transfer the fat into a fresh 50 ml Falcon tube (with the help of sterile spatula); Repeat steps 5 to 7 for 2-3 times

Characterization of Oct-4/Sox-2 Transcription Factors by Immunostaining for Characterization of Stem Cells
Protocol
1) The cultured cells at a density of 70,000/well in a 6 well plate or 35,000/ well in a 12 well plate are used for immunostaining.
2) The cells are washed with Dulbecco’s phosphate buffered saline (DPBS) for two times and the cells are fixed using 4% paraformaldehyde for 20 minutes at room temperature.
3) After fixation, the spent Paraformaldehyde is removed and the fixed cells are washed with 1000μl of 1x DPBS for 2 times by clockwise and anti-clockwise rotation of the plate slowly.
4) About 400μl of 3% blocking buffer was added to the wells and plate was covered with aluminum foil to prevent the exposure of light and stored at room temperature for 90 minutes.
5) After blocking, the cells are again washed once with DPBS. About 500 μl of primary antibody Oct-4 in the dilution of 1:40 is added to the well and stored at 4°C over night.
6) After overnight staining with primary antibody, the antibody solution is removed from the wells and washed with DPBS for 2 times.
7) Take 500 μl of secondary antibody anti-rabbit-FITC diluted with antibody buffer solution is added in the ratio of 1:200 and cover with aluminium foil and incubated for 90 minutes.
8) Secondary antibody is removed and washed with DPBS.

1.    Remove the growth media
2.    Wash 2 times with warm PBS (w/o Ca and Mg)
3.    Add 5ml of trypsin /EDTA 0.35 % (Invitrogen), place at 370C in a CO2 incubator for 4-5 min
4.    Wait until cells are detached/lifted and make sure by examining on microscope
5.    Add equal volume (5ml-1:1) of growth medium as appropriate and mix with 5 strokes
6.    Transfer into 15 ml centrifuge tube
7.    Centrifuge at room temperature for 4 min in 980 rpm
8.    Remove supernatant complete but carefully without touching the cell aliquot
9.    Re-suspend in 10ml DPBS (with Ca and Mg)-5 strokes
10.    Immediately after mixing transfer 100µl of cell suspension into Casy vial, containing 10ml Casyton

1. Fix the organ or the animal in 5-10 % formalin preferably neutral buffered formalin. If the organ is an isolated thoracic limb, for example, then cannulate the axillary artery and perfuse the formalin. I find sometimes it is advantageous to pump some buffered saline solution to flush out any blood before pumping in the formalin. Organs, such as the heart, could be nicely flushed with water through major blood vessels, and immersed in formalin to fix. The same can be done with liver, kidney, etc. It takes a week for fixing a large structure (e.g., 10 kg) by immersing in formalin.
2. Dissect the specimen as needed. Remove all fat and clean as much as possible so the specimen looks nice.
3. If the specimen is dark brown, it can be lightened by leaving 1-2 days in 5% hydrogen peroxide. Check the specimen 1-2 times during the day to make sure desired light tone is achieved. Leaving it too long in the hydrogen peroxide may result in tissue degradation.
4. Drain the specimen well, and dehydrate in acetone. The dehydration should be thorough. To achieve this, use 100% acetone, at least 2-3 changes. The acetone can be recycled by repeated distillation. Acetone tends to evaporate, so use a container that has a tight lid. Acetone is also flammable, so use required precautions when handling acetone. If concentration of acetone remains up to 99% even after leaving organ for 4-5 days it is sure that dehydration is complete. If you are using any thin sheets of organ or Brain for dehydration keep slice between glass plates and then dehydrate.
5. Immerse the specimen in glycerin. Buy industrial glycerin, which may be cheaper. It may take 2-3 weeks for the glycerin to permeate into the tissues.

6.1 Designing primers

Designing correct primers are very important to get the expected result in PCR experiment. There are two primers (Forward & Reverse) required for the amplification of the desired DNA fragment. One primer should anneal to the sense strand (oriented to the 5'→3' direction) and another primer should complement the antisense strand (oriented in the 5'→3' direction).

There are various characteristics to be considered to design a good primer including Tm, length, GC content, secondary structure and specificity.

There are commercially available softwares besides free online tools at NCBI (primer blast) for designing PCR primers. In addition, primer sequences published in peer reviewed literature can also be used.

For RT-PCR, the extracted total RNA is first reverse transcribed to cDNA using reverse transcriptase enzyme. Following are the protocols for cDNA sysnthesis and RT-PCR.

Note: cDNA synthesis and RT-PCR can be performed according to the manufacturer protocol from where the specific reagents for the above methodologies are purchased.

8.1 Purpose

To lay down a uniform protocol for the synthesis of c-DNA from total RNA

8.2 Scope

The scope of this Standard Operating Procedure is to lay down a standard procedure for the synthesis of c-DNA from total RNA

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.