Giant Freshwater Prawn Farming

Farming of giant freshwater prawn, Macrobrachium rosenbergii is a growing industry in India.  Generally prawn ponds are shallow having depth of 1.5 to 2.00 meters. 

Preparation of Pond:

Pond preparation includes checking and repair of sluice gates and eradication of unwanted species, liming, placing of hide outs, manuring and filling of water.  In older ponds, pumping out the water and drying helps in elimination of unwanted species.  Drying helps mineralization of bottom soil and improves the productivity of the bottom sole.  Disease spreading micro organisms and parasites also can be eliminated by drying.  Ploughing of bottom soil after drying helps removal of obnoxious gases.  In undrainable ponds, eradication can be done by poisoning.  Poisons of plant origin, like mahua oil cake, tea seed cake derris root powder are advisable.  After eradication, liming and ploughing can be done.  Liming helps in disinfection of pond bottom and improves the fertility of the bottom soil.  The quantity of lime could be around 200 kg to 1000 kg based on the pH of the soil.  After drying, liming and ploughing, the pond bottom should be kept in a moist condition for a week to facilitate bacterial action.  Then manure can be applied.  Manuring helps the development of worms like tubifex, zooplankton, algae, etc. 

Laying of shelters: Freshwater prawn are cannibalistic.  They grow by moulting.  Moulting is a process by which the old exoskeleton is shed and new exoskeleton is formed over the body.  It takes nearly 24 hours for hardening of the new exoskeleton.  During that period moulted prawns require protection from other prawns.  Tile, bricks, etc., can be provided in the pond as hide outs.  Discarded PVC pipes, earthern pipes can also be used as hide outs.  Apart from hide outs, it is advisable to keep some floating weeds like Eichornia, Pistia and duck weed. 

Stocking the pond: 7 to 10 days old (PL 7 to 10) post larvae is the ideal age group for transportation and stocking the pond.  Before stocking the PLs are to acclimatized to freshwater, preferably with the pond water itself.  The density of stocking depends on the intensith of management.  For semi-intensive culture with a targeted production of 3 to 3.5 tonnes per year per hectatre, 75000 PLs or 60000 juveniles can be stocked. 

During the rearing period, artificial diet is given to the growing prawns.  Water quality parameters are to be checked regularly.  Disease control measures are to be followed by maintaining a healthy environment, avoiding over stocking, etc.  Harvesting should be done by netting the pond, after removing the hide outs.  Harvesting should be done during early hours when the temperature is less.  After harvesting the prawns are cleaned in freshwater and are iced and transported. 

Hatchery Technology of Giant Freshwater Prawn

Hatchery Technology of Giant Freshwater Prawn

Macrobrachium rosenbergii is the giant freshwater prawn usually cultured in India.  It is euryhaline and eurythermal in habit, so it is a suitable candidate species for culture.  Macrobrachium rosenbergii matures when they are months old.  The female after a premating moult mates with a hard shelled male.  The fertilized eggs are carried by the female in a brood pouch in the ventral side of the abdominal region.  The fertilized eggs hatch out in 18 to 23 days.  The newly hatched larvae require estuarine water but it can survive in fresh water for three to five days.  The larvae after passing through many stages in estuarine region become postlarvae which then migrate to freshwater. 

Brood stock collection and hatching:

Female prawn carrying fertilized eggs in the abdominal pouch is called berried female.  It can be collected either from rivers or culture ponds.  While collecting, care should be taken to see that the eggs are not lost due to rough handling.  The berried females are transported with oxygen packing.  Hatching can be done either in larval tanks or in a separate hatching tank using freshwater or 12 ppt saline water.  Usually hatching of egg takes place during night hours.  The hatching period lasts for 3 or 4 days.  Major quantity of larvae is released on the second day of the hatching.  The newly hatched-out larvae can survive for about 5 days in fresh water.  After hatching the mother prawn should be removed from the hatching tank.  The larvae pass through 11 stages to become post larvae.  For collecting the larvae they can be concentrated at one portion of the tank by putting a light at the collection point after covering other parts with a black cloth or black sheet as they are very much attracted by light.

Feed and feeding:

Feeding of larvae starts from second day onwards.  Artemia is the most ideal and widely used feed for larval rearing.  Artemia is a small crustacean seen in hyper saline water.  Its eggs are collected, packed and supplied by several companies.  The cysts are hatched in separate tanks and the hatched out naupli are fed to larvae. 

RNA

RIBOSE NUCLEIC ACID

Most cellular RNA is single stranded, although some viruses have double stranded RNA (Wound tumor plant virus, Reo animal virus). The single RNA strand is folded upon itself, either entirely or in certain regions. In the folded region a majority of the bases are complementary, and are joined by hydrogen bonds. This helps in the stability of the molecule. In the unfolded region the bases have no complements. Because of this RNA does not have the purine-pyrimidine equality that is found in DNA.

RNA also differs from DNA in having ribose sugar instead of deoxyribose. The common nitrogenous bases are adenine, guanine, cytosine and uracil. Thus the pyrimidine uracil substitutes thymine of DNA. In region where purine-pyrimidine pairing takes place, adenine pairs with uracil and guanine with cytosine.

Three types of cellular RNA have been distinguished:
1. messenger RNA (mRNA) or template RNA,
2. ribosomal RNA (rRNA) and
3. transfer RNA (tRNA) or soluble RNA (sRNA).

Ribosomal RNA: Ribosomal RNA, as the name suggests, is found in the ribosomes. Ribosomes contain about 60% ribonucleic acid, which is ribosomal RNA and 40% protein. It comprises of about 80% of the total RNA of the cell. Ribosomal RNA consists of a single strand twisted upon itself in some regions. In these twisted regions the base pairs are complementary. But there is no purine-pyrimidine equality. rRNA is synthesized on the part of the chromosome traversing the nucleolus or the nucleolar organizer. The DNA associated with this is called ribosomal DNA. The 70s ribosome of prokaryotes consists of a 30s sub unit and a 50s sub unit. The30s sub unit contains 23s and 5s rRNA. The 80s eukaryote ribosome consists of a 40s and a 60s sub unit. In vertebrates the 40s sub unit contains 18s rRNA while the 60s sub unit contains 28-29s, 5.8s and 5s rRNA. In plants and invertebrates the 40s sub unit contains 16-18s rRNA, while the 60s sub unit contains 25s and 5s and 5.8s rRNA. Thus there are three types of rRNA, based on sedimentation and molecular weight. Two of these classes are high molecular weight RNAs, while the third is a low molecular weight RNA. The three classes are –
1. high molecular weight rRNA with molecular weight of over a million(21s-29s rRNA)
2. high molecular weight rRNA with molecular weight below a million(12s-18s rRNAs)
3. low molecular weight rRNA (5s rRNA)

The function of rRNA is in protein synthesis, but it’s role is not yet completely clear.

Messenger RNA: Jacob and Monod (1961) proposed the name messenger RNA for the RNA carrying information for protein synthesis from the DNA (genes) to the sites of protein formation (ribosomes). It consists of only 3 to 5% of the total cellular RNA. Messenger RNA is always single stranded. It contains mostly the bases adenine, guanine, cytosine, and uracil. There are few unusual bases. There is no base pairing as base pairing in the mRNA strand destroys its biological activity. mRNA is synthesized on a DNA strand through the enzymatic action of RNA polymerase. The mRNA has the following structural features:
1. Cap: at the 5’ end of the mRNA molecule in most eukaryote cells and animal virus molecules is found a ‘cap’. This is blocked methylated structure. The rate of protein synthesis depends upon the presence of the cap. Without the cap mRNA molecules bind very poorly to the ribosomes.
2. Non-coding region 1: The cap is followed by a region of 10 to 100 nucleotides. This region is rich in A and U residues, and does not translate protein.
3. The initiation codon is AUG in both prokaryotes and eukaryotes.
4. The Coding region consists of about 1500 nucleotides on the average and translates protein.
5. Termination Codon: translation on mRNA is brought about by a termination codon. In eukaryotes the termination codons are UAA, UAG or UGA.
6. Non-coding region 2 consists of 50-100 nucleotides and does not translate proteins. This region contains an AAUAAA sequence in all the examples sequenced.
7. Poly (A) sequence: At the 3’end is a polyadenylate or poly (A) sequence, which initially consists of 200 – 250 nucleotides, but which becomes shorter with age. The poly (A) sequence is added in the nucleus before the mRNA reaches the cytoplasm.

It is now established that when the code has been transcribed from DNA on to mRNA, the later leaves the nucleus passes through the nuclear membrane into the cytoplasm. Here it moves to the ribosomes, the site of protein synthesis, the mRNA molecules attach reversibly to the surface of ribosome always binding to the smaller sub-units.

Transfer RNA (tRNA) or Soluble RNA (sRNA): After rRNA the second most common RNA in the cell is transfer RNA. It is also called sRNA because it is small to be precipitated by ultracentrifugation. It constitutes about 10-20% of the total RNA of the small. Transfer RNA is synthesized in the nucleus on a DNA template. Only 0.025% of DNA codes for tRNA. The function of tRNA is to carry amino acids to mRNA during protein synthesis. A specific tRNA carries each amino acid. Since 20 amino acids are coded to form proteins, it follows that there must be at least 20 types of tRNA. However, it has been proved that there are at least two types of tRNA for each amino acid. Thus there are many tRNA molecules than amino acid types.
Holley et al (1965) first worked out the structure of tRNA. He proposed the cloverleaf model for tRNA. According to the cloverleaf model the single polynucleotide chain of tRNA is folded upon itself to form 5 arms. As a result of the folding the 3’ and the 5’ ends of the chain come near each other. An arm consists of a stem and a loop. In the double helical stems there is internal base pairing which follows the A-U and G-C combinations. There is no base pairing in the loops. One of the arms has stem but not a loop and is called the acceptor stem. The other arms are called the D arm, the anti codon arm, the variable arm and the TjC arm. The variable arm may or may not have a stem. The acceptor stem consists of 7 base pairs and 4 unpaired nucleotide units. The latter include a constant 3’ terminal-CCA sequence and a fourth nucleotide, which is a variable purine (A or G). The amino acid molecule attached to the 3’terminal of the –CCA sequence is known as the amino acid binding site. The 5’ end of tRNA is either guanine (G) or Cytosine (C).
The second arm is called the D arm. It consists of 15-18 nucleotides with 3 – 4 base pairs in the stem and 7 – 11 unpaired nucleotides in the loop. The loop of the D arm is called Loop I or dihydrouridine (DHU) loop or the D loop. The synthetase site that recognizes the amino acid activating enzyme is located on a part of the D loop.
The third arm or anti codon arm consists of an anti codon stem of 5 base pairs and a loop, called Loop II or the anti codon loop. This loop consists of 7 unpaired nucleotides of which the middle three form the anti codon. The anti codon recognizes the 3 complementary bases, which constitute the codon of mRNA.
The variable arm (the lump, the mini loop, Loop III) is of two types. In one type there is a loop containing 4 – 5 bases but no stem. In the other type, the arm consists of 13-21 residues, and both the stem and the loop can be distinguished.
The TjC arm consists of a stem having 5 base pairs and a loop of 7 nucleotides. The outer most of the 5 pairs of the stem is C –G. The TuC loop contains a constant TjC sequence (ribothymine-pseudouracil-cytosine). All tRNAs have a ribosome recognition site on the TuC loop consisting of a G-T-u-C sequence. (Ribothymidine(T), Pseudouridine(j), are unusual base pairs).

FRESHWATER PRAWN HATCHERY AND FARMING

Hatchery Technology of Giant Freshwater Prawn

Macrobrachium rosenbergii is the giant freshwater prawn usually cultured in India. It is euryhaline and eurythermal in habit, so it is a suitable candidate species for culture. Macrobrachium rosenbergii matures when they are months old. The female after a premating moult mates with a hard shelled male. The fertilized eggs are carried by the female in a brood pouch in the ventral side of the abdominal region. The fertilized eggs hatch out in 18 to 23 days. The newly hatched larvae require estuarine water but it can survive in fresh water for three to five days. The larvae after passing through many stages in estuarine region become postlarvae which then migrate to freshwater.

Brood stock collection and hatching:
Female prawn carrying fertilized eggs in the abdominal pouch is called berried female. It can be collected either from rivers or culture ponds. While collecting, care should be taken to see that the eggs are not lost due to rough handling. The berried females are transported with oxygen packing. Hatching can be done either in larval tanks or in a separate hatching tank using freshwater or 12 ppt saline water. Usually hatching of egg takes place during night hours. The hatching period lasts for 3 or 4 days. Major quantity of larvae is released on the second day of the hatching. The newly hatched-out larvae can survive for about 5 days in fresh water. After hatching the mother prawn should be removed from the hatching tank. The larvae pass through 11 stages to become post larvae. For collecting the larvae they can be concentrated at one portion of the tank by putting a light at the collection point after covering other parts with a black cloth or black sheet as they are very much attracted by light.

Feed and feeding:
Feeding of larvae starts from second day onwards. Artemia is the most ideal and widely used feed for larval rearing. Artemia is a small crustacean seen in hyper saline water. Its eggs are collected, packed and supplied by several companies. The cysts are hatched in separate tanks and the hatched out naupli are fed to larvae.

Giant Freshwater Prawn Farming

Farming of giant freshwater prawn, Macrobrachium rosenbergii is a growing industry in India. Generally prawn ponds are shallow having depth of 1.5 to 2.00 meters.

Preparation of Pond:
Pond preparation includes checking and repair of sluice gates and eradication of unwanted species, liming, placing of hide outs, manuring and filling of water. In older ponds, pumping out the water and drying helps in elimination of unwanted species. Drying helps mineralization of bottom soil and improves the productivity of the bottom sole. Disease spreading micro organisms and parasites also can be eliminated by drying. Ploughing of bottom soil after drying helps removal of obnoxious gases. In undrainable ponds, eradication can be done by poisoning. Poisons of plant origin, like mahua oil cake, tea seed cake derris root powder are advisable. After eradication, liming and ploughing can be done. Liming helps in disinfection of pond bottom and improves the fertility of the bottom soil. The quantity of lime could be around 200 kg to 1000 kg based on the pH of the soil. After drying, liming and ploughing, the pond bottom should be kept in a moist condition for a week to facilitate bacterial action. Then manure can be applied. Manuring helps the development of worms like tubifex, zooplankton, algae, etc.

Laying of shelters: Freshwater prawn are cannibalistic. They grow by moulting. Moulting is a process by which the old exoskeleton is shed and new exoskeleton is formed over the body. It takes nearly 24 hours for hardening of the new exoskeleton. During that period moulted prawns require protection from other prawns. Tile, bricks, etc., can be provided in the pond as hide outs. Discarded PVC pipes, earthern pipes can also be used as hide outs. Apart from hide outs, it is advisable to keep some floating weeds like Eichornia, Pistia and duck weed.

Stocking the pond: 7 to 10 days old (PL 7 to 10) post larvae is the ideal age group for transportation and stocking the pond. Before stocking the PLs are to acclimatized to freshwater, preferably with the pond water itself. The density of stocking depends on the intensith of management. For semi-intensive culture with a targeted production of 3 to 3.5 tonnes per year per hectatre, 75000 PLs or 60000 juveniles can be stocked.

During the rearing period, artificial diet is given to the growing prawns. Water quality parameters are to be checked regularly. Disease control measures are to be followed by maintaining a healthy environment, avoiding over stocking, etc. Harvesting should be done by netting the pond, after removing the hide outs. Harvesting should be done during early hours when the temperature is less. After harvesting the prawns are cleaned in freshwater and are iced and transported.

Role of DNA and RNA in Protein Synthesis

The central dogma of protein synthesis is that DNA makes RNA, which in turn makes protein. This can be expressed as follows:

Replication Transcription Translation
DNA------------------à DNA-----------------à RNA---------------à PROTEIN

Protein synthesis consists of two main events, transcription and translation. Transcription is the copying of a complementary messenger RNA strand on a DNA strand. The DNA strand unwinds and one of the two strands forms a mRNA strand. This strand is called anti-sense strand or template strand. The nucleotides of mRNA are complementary to those of the DNA strand. However, in mRNA uracil (U) is replaces thymine (T) of DNA. As a result the complementary pairing is A-U and G-C. The mRNA thus formed makes its way through the pores in the nuclear membrane to the cytoplasm. Here it forms a complex with a group of ribosomes.

Transcription requires along with a template strand, ribonucleotide triphosphate (ATP, GTP, UTP, CTP), the enzyme RNA polymerase and divalent metal ions. RNA polymerase consists of a core enzyme (with sub units α, α, β, β and ω) and a sigma (σ)factor. The sigma factor initiates transcription of mRNA on the DNA template and the core enzyme continues transcription. All the RNA chains start with either pppG or pppA and are synthesized in the 5’ – 3’ direction. Chain termination is brought about by a rho factor.

Translation: During translation the genetic information present in mRNA directs the order of specific amino acids to from a polypeptide or protein. The mRNA has a series of triplet bases, each triplet forming a codon. The codons pair with anticodons of the tRNA molecule. Each anticodon consists of three free bases. This pairing follows the A-U and G-C combination. Thus the codon GUC pairs with the anticodon CAG to tRNA. Thus the series of codons on mRNA determines the series of anticodons of the different tRNA molecules, and hence of the amino acids. Since the triplets of mRNA in turn depend upon the series of bases in DNA, it follows that the DNA molecule determines the sequence of amino acids, and thus the structure of the protein molecule.

The translation process consists of –
1. activation of amino acids
2. transfer of the activated amino acid to tRNA
3. initiation of polypeptide chain synthesis
4. chain elongation and
5. chain termination

1. Activation of amino acids: The 20 amino acids (aa) used in protein synthesis are activated by ATP in the present of specific activating enzymes (E) called aminoacyl synthetases to from aminoacyl adenylates (aaa), also called aminoacyl AMP. Pyrophosphates (PPi) are released.

aa + ATP + E --------à E-aa-AMP + PPi

2. Transfer of activated amino acids to tRNA: The activated amino acid is transferred to a specific tRNA with the release of AMP and activating enzyme.

E.aa.AMP + E + tRNA -----------à aa-tRNA + AMP + E

3. Initiation of chain synthesis: This requires the ribosome subunits, mRNA, an energy source (GTP), activated amino acids attached to tRNA (aa-tRNA) and initiation factors (IF). These factors are called IF-1, IF-2, IF-3 in prokaryotes and eIF-2, e-IF2’, eIF-2a1 eIF-2a2 , eIF-2a3 and eIF-3 in eukaryotes.
a. The 30S ribosomal subunit attaches to mRNA to form an mRNA-30S complex. The process requires IF-3 and Mg++
b. the starting amino acid is methionine (Met) in eukaryotes and N-formyl methionine in prokaryotes. The amino acid-tRNA complex (fMEt-tRNA) attaches to the initiation codon, AUG, on mRNA through its anticodon UAC to form the 30S initiation complex. The process requires initiation factors IF-2 and IF-1 as well as GTP.
c. The larger ribosomal subunit (50S in prokaryotes) joins to the 30S initiation complex to form the complete initiation complex (70S).
d. The larger ribosomal subunit has two binding sites for tRNA, an A or acceptor site and a P or peptidyl site. fMet-tRNA binds to the P site.

4. Chain Elongation: Elongation of the polypeptide chain requires elongation factors (EF). These are EF-Tu. EF=Ts and EF-G in prokaryotes and EF-1 and EF-2 in eukaryotes.
a. The second amino acid-tRNA complex (aa2-tRNA) now occupies the A site. There is enzymatic recognition of internal codons. The process requires EF-Tu, GTP and Mg++.
b. Formation of a peptide bond takes place by transfer of fMet to the second amino acid (aa2). Mg++ and K+ are required. The catalyzing enzyme is pepitdyl transferase.
c. Translocation: The aa2-tRNA complex moves from the A site to the P-site. This process is called translocation and requires EF-G, GTP and Mg++. Translocation involves movement of the ribosome relative to mRNA in the 5’--à3’ direction. The third amino acid-tRNA (aa3-tRNA) complex now occupies the vacant A-site.

5. Chain termination: Chain elongation continues until a termination codon (UAA, UAG, UGA) reaches the ribosome. The chain is then terminated and released from the ribosome. This process requires release factors RF-1, RF-2 and RF-3 in prokaryotes and RF in eukaryotes.
a. The termination codon provides signals to the ribosome for the attachment of release factors.
b. the release factors interact with peptidyl transferase causing hydrolysis of the bond between tRNA and the polypeptide chain, and the chain is released from the ribosome.
c. Hydrolysis of GTP results in the dissociation of the release factors from the ribosome. The tRNA is also unloaded. The ribosomal subunits dissociate and mRNA is released, for breakdown to nucleotides.
d. Processing of the polypeptide chain, e.g., cleavage of the formyl residue or of methionine, takes place after release.

REPLICATION OF DNA

REPLICATION OF DNA

One of the most important properties of DNA is that it can make exact copies of itself. This process is called replication. The two stands of DNA double helix are united by hydrogen bonds between the purine and pyrimidine base pairs. When the hydrogen bonds break the two strands separate and unwind. The nucleus contains free nucleotides, which form the nucleotide pool. These free nucleotides pair with the nucleotides of the two separated strands by means of hydrogen bonds. Free adenine nucleotide pairs with thymine nucleotide of the strand and free guanine nucleotide pairs with the cytosine nucleotide of the strand, etc. In this way a new strand is formed around each of the old strand. The result of replication is the formation of tow double helices, each identical to the original.

Replication:
1. Replication takes place during the interphase between two mitotic cycles.
2. Replication is semi-conservative process in which each of the two double helices formed from the parent double strand have one old and one new strand. Repair replication is non-conservative.
3. DNA replication requires a DNA template, a primer, deoxyribonucleoside triphosphates (dATP, dGTP, dTTP and dCTP), Magnesium ions, DNA unwinding protein, superhelix relaxing protein, a modified RNA polymerase to synthesize RNA primer, the products of dnaA, dnaB, dnaC-D, dnaE and dnaG genes and polynucleotide ligase, a joining enzyme.
4. Replication starts at a specific point called the origin.
5. According to one model replication starts with a ‘nick’ or incision made by an incision enzyme (endonuclease).
6. The two strands of the DNA double helix unwind with the help of a DNA unwinding protein (also called the DNA binding protein) which binds to single DNA strands.
7. The unwinding of the strands imposes strain, which is relieved by the action of a superhelix relaxing protein.
8. Initiation of DNA synthesis requires an RNA primer. The primer is synthesized by the DNA template close to the origin of replication. A special form of RNA polymerase catalyzes the synthesis.
9. Deoxyribose nucleotides are now added to the 3’ end of the RNA primer and the main DNA stand is synthesized on the DNA template. This strand is complementary to the DNA strand and is synthesized by DNA polymerase III.
10. The enzyme DNA Polymerase I now degrades the RNA primer and simultaneously catalyses the synthesis of a short DNA segment to replace the primer. This segment is then joined to the main DNA strand by a DNA ligase.
11. Replication takes place discontinuously and short pieces called Okazaki fragments are synthesized. One strand may synthesize a continuous strand and the other Okazaki fragments, or both strands may synthesize Okazaki fragments. Thus one strand is synthesized forwards and the other backwards.
12. The Okazaki pieces are joined by polynucleotide ligase, a joining enzyme, to form continuous strands.
13. Replication may be in one direction (unidirectional) from the point of origin or in both directions (bidirectional).

DNA

Deoxyribose Nucleic Acid

DNA is present in all plant cells, animals, few prokaryotes and in a number of viruses. In eukaryotes it is combined with proteins to form nucleoproteins. In prokaryotes it is without any proteins. The DNA of all plants and animals and many viruses is double stranded. In the bacteriophage phi 174, however it is single stranded.

The widely accepted molecular model of DNA is the double helix structure proposed by Watson and Crick. The DNA molecule consists of two helically twisted strands connected together by ‘steps’. Each strand consists of alternating molecules of deoxyribose (a pentose sugar) and phosphate groups. Each step is made up of a double ring purine base and single ring pyrimidine base. The purine and pyrimidine bases are connected to deoxyribose sugar molecules. The two strands are intertwined in a clockwise direction i.e., in a right hand helix and run in opposite directions. The twisting of the strands results in the formation of deep and shallow spiral grooves.

The DNA molecule is a polymer consisting of several thousand pairs of nucleotide monomers. Each nucleotide consists of the pentose sugar deoxyribose, a phosphate group and a nitrogenous base which may be either a purine or a pyrimidine. Deoxyribose and a nitrogenous base together for a nucleoside and a nucleoside with a phosphate together form a nucleotide.

Deoxyribose is a pentose sugar with five carbon atoms. Four of the five carbon atoms plus a single atom of oxygen form a five-membered ring. The fifth carbon atom is outside the ring and forms a part of a –CH2 group. The four atoms of the ring are numbered 1’, 2’, 3’ and 4’. The carbon atom of the –CH2 is numbered 5’. There are three –OH groups in positions 1’, 3’ and 5’. Hydrogen atoms are attached to carbon atoms 1’, 2’, 3’ and 4’.

Ribose the pentose sugar of RNA, has an identical structure except that there is an –OH group instead of H on carbon atom 2’. All the sugars of one strand are directed to one end, i.e, the strand has polarity. The sugars of the two strands are directed in opposite directions.

Nitrogen bases: there are two types of nitrogenous bases, pyrimidines and purines. The pyrimidines are single ring compounds, with nitrogen in positions 1’ and 3’ of a 6 membered benzene ring. The two most common pyrimidines of DNA are cytosine and thymine. The purines are double ring compounds. A purine molecule consists of a 5-membered imidazole ring joined to a pyrimidine ring at position 4’ and 5’. The two most common purines of DNA are adenine and guanine.

Base Paring: Each step of the DNA ladder is made up of a purine and a pyrimidine pair, i.e., of a double ring and a single ring compound. Two purines would occupy too much space, while two pyrimidines would occupy too little. Because of the purine-pyrimidine pairing the total number of purines in a double stranded DNA is equal to the total number of pyrimidines. Thus A/T = 1 and G/C = 1 or A+G = C+T. The ratio of A+T/G+C, however, rarely equals to 1, and varies with different species from 0.4 to 1.9. The purine and pyrimidine bases pair only in certain combinations. Adenine pairs with thymine and guanine with cytosine. A and T are joined by two hydrogen bonds through atoms attached to positions 6’ and 1’. Cytosine and guanine are joined by three hydrogen bonds through positions 6’, 1’ and 2’. The pyrimidine and purine bases are linked to the deoxyribose sugar molecules. The linkage in pyrimidine nucleosides is between position 1’ of deoxyribose and 3’ of the pyrimidine. In purine nucleosides it is between position 1’ of deoxyribose and position 9’ of the purine.

Phosphate: In the DNA strand the phosphate group alternate with deoxyribose. Each phosphate is joined to carbon atom 3’ of one deoxyribose and to carbon atom 5’ of another. Thus each strand has a 3’end and a 5’ end. The 3’ end of one strand corresponds to the 5’ end of the other. Consequently the oxygen atoms of deoxyribose point in opposite directions in the two strands.

Forms of DNA: DNA can exist in the A. B. C and D forms. Sugar puckering is the most important characteristic for distinguishing the DNA forms. The A form has 3’-endo puckering and one turn of the helix consists of 11 base pairs (11-fold helix). The B form is with 3’-exo puckering and 10 base pairs (basic structure proposed by Watson and Crick). C form is with 2’-endo puckering with a helical symmetry of 91/3 i.e., there are fewer residues per turn than in the B form. The D form is with 3’-exo puckering with 8 base pairs per turn of the helix.

RL helix and Z DNA: According to the Watson and Crick model DNA exists in the form of right handed helix. But G.A. Rodley’s group working in New Zealand and V. Sasisekharan’s group working in India have independently proposed a structure of B-DNA radically different from the Watson and Crick model. According to them, the DNA duplex is formed by alternating right and left handed helices arranged side by side. Each strand of the DNA duplex has 5 base pairs in the right handed helix alternating with 5 base pairs in the left-handed helix.

Chromosomes

Chromosomes were first seen by Hofmeister (1848) in the pollen mother cells of Tradescantia in the form of darkly stained bodies. The term chromosomes (Gr: chrom = colour, soma = body) was used by Waldeyer (1888) to designate their great affinity to basic dyes. W. S. Sutton (1900) described their functional significance.

Chromosomes are the most significant components of the cell, particularly they are apparent during mitosis and meiosis. A chromosome can be considered as a nuclear component having special organisation, individuality and function.

Morphology

The morphology of chromosome can be best studied at the metaphase or anaphase of mitosis when they are present as definite organelles, being most condensed or coiled.
Number: The number of chromosomes in a given species is usually constant containing diploid number (2n) of chromosomes in their somatic cells and haploid (gametic or reduced) number (n) of chromosomes in their sex cell. The number of chromosomes in variable from one to several hundreds among different species. For e.g., in Ascaris megalocephala it is 2, while in certain protozoans (Aulocantha), there are 1600 and in man there are 46.
Size: Chromosomes range, on an average from 0.5 to about 30 microns in length and from 0.2 to 3microns in diameter. The salivary gland chromosomes of Diptera are 2mm long. Usually all the chromosomes in a cell are of the same size. Plant cell normally posses larger chromosomes than animal cells. Among the animals, grasshoppers, crickets, mantids newts and salamanders have large chromosomes. Variation in size of the chromosomes can be induced by a number of environmental agents like temperature, rate of cell division, etc.
Shape: The shape of the chromosomes is changeable from phase to phase in the continuous process of the cell growth and cell division. In the resting phase or interphase stage of the cell, the chromosomes occur in the form of thin, coiled, elastic and contractile, thread like stainable structures, called the chromatin threads.
Structure: In the earlier light microscopic description of chromosome was thought to consist of a coiled thread called the chromonema lying in a matrix. The chromosome was supposed to be covered by a membrane called pellicle. However electron microscopic studies revealed that there is no pellicle. During metaphase a chromosome appears to possess two threads called chromatids.
Chromonema: The chromatids are really spirally coiled chromonemata (singular chromonema). They may be composed of 2, 4, or more fibrils depending on the species. The fibrils of the chromonema are coiled with each other. The coils are of two types:
1. Paranemic coils: When the chromonemal fibrils are easily separable from each other.
2. Plectonemic coils: When the chromonemal fibrils are closely interwined and they cannot be separated easily.

Chromomeres: The chromonema of thin chromosomes of mitotic and meiotic prophases have been found to contain alternating thick and thin regions and thus giving the appearance of a necklace in which several beads occur on a string. The thick or bead like structures of the chromonema are known as the chromomeres and the region in between the chromomeres is termed as the inter-chromomeres. Actually the chromomeres are regions of the super-Imposed coils.
Centromere: (Kinetochore or Primary Constriction) It is indispensable part of the chromosome and forms the primary constriction of metaphase. The position of the centromere is constant for a particular chromosome. The structure and function of the centromere is different form that of the rest of the chromosome. During division the centromere is functional, while the rest of the chromosome is genetically inactive. The position of the centr4omere varies in different chromosomes. Since the spindle fibers are attached to the centromere, the shape of the chromosome during anaphase will depend on the position of the centromere. Four different categories of chromosomes can be recognized, based on the position of the centromere. They are – Metacentric, Submetacentric, Acrocentric and Telocentric.
In Metacentric chromosomes the centromere is near the middle of the chromosome. The two arms of the chromosome are nearly equal and the chromosome appears ‘V’ shaped during anaphase.
In Submetacentric chromosome the centromere is situated some distance away from the middle; one arm of the chromosome is shorter than the other. Such a chromosome will appear L-shaped during anaphase.
In Acrocentric chromosome the centromere is situated near the end of the chromosomes. It appears rod-shaped.
In Telocentric chromosomes the centromere is truly terminal, i.e., situated at the tip of the chromosome.
Secondary constriction: In addition to the primary constriction or centromere the arms of the chromosome may show one or more secondary constrictions called Secondary Constriction II. These differ from nucleolar organizers called Secondary Constriction I. The location of secondary constriction II is constant for a particular chromosome, and is, therefore, useful for in identification of chromosomes.
Nucleolar Organizer: Normally in each diploid set of chromosomes, two homologous chromosomes have additional ‘constrictions’ called nucleolar organizers. These are so called because they are essential for the formation of nucleolus. The nucleolar organizer appears as a ‘constriction’ near one end of chromosome. The part of the chromosome beyond the nucleolar constriction is very short and appears like a sphere or satellite. In man chromosomes 13, 14, 15, 21, 22, and Y have nucleolar organizers and satellites. Chromosomes bearing satellites are called SAT-Chromosomes. The prefix Sat stands for ‘Sine Acid Thymonucleionico’ (without thymonucleic acid or DNA), since the chromosomes on staining shows relative deficiency of DNA in the nucleolar region.
Euchromatin and Heterochromatin: Certain segment of the chromosomes or the entire chromosomes, are more condensed than the rest of the karyotype during various stages of the cell cycle. Such difference in thickening has been called heteropycnosis. Heteropycnosis may be positive, where there is overcondensation or negative, where there is undercondensation. Chromosomes, which remain condensed during interphase, are called heterochromosomes and the non-condensed chromosomes, which extend during interphase, are called euchromosomes.
Chromatin material is of two types- heterochromatin and euchromatin. Chromatin material showing heteropycnosis at any stage is called heterochromatin. Regions of the chromosomes, which never show heteropycnosis, consist of euchromatin. Heterochromatin stains deeply whereas euchromatin stains less deeply. Heterochromatin is found in the condensed region of the chromosome, and is associated with tight folding and coiling of the chromosome fibre. Euchromatin consists of the diffused or less tightly coiled regions. There are two types of heterochromatin, constitutive heterochromatin and facultative heterochromatin. Constitutive heterochromatin shows heteropycnosis in all cell types and throughout the cell cycle. Facultative heterochromatin on the other hand is hyperpycnotic only in some special cell types, or at some particular stages of the life cycle.

ULTRA STRUCTURE OF CHROMOSOME

Two views have been proposed for ultra structure of chromosome.
1. Multistranded view: This was proposed by Ris (1966). By electron microscope the smallest visible unit of the chromosome is the fibril which is 100Ao in thickness. This fibril contains two DNA double helix molecules. Next largest unit is the half chromatid. The half chromatid consists of four 100Ao fibrils so that it is 400Ao in thickness and contains eight double helixes of DNA. Two half chromatids form a complete chromatid consisting of 16 double DNA helix molecules. As the chromosome consists of two chromatids, thus total number of helixes will be 32 and diameter 1600Ao thick before duplication or synthesis. In summary, the chromosome is composed of numerous micro fibrils, the smallest of which is a single nucleoprotein molecule.
2. Folded - Fibril Model: Dupraw (1965) presented this model for the fine structure of chromosome. According to this model, a chromosome consists of single long chain of DNA and protein forming what is called a fibril. The fibril is folded many times and irregularly entwined to form the chromatid. This measures 250 – 300Ao in thickness. Dupraw’s model of DNA association is now considered unlikely by the discovery that DNA itself looped around histone beads to form nucleosomes.
3. Nucleosome: The chromatin is formed of repeating units called nucleosomes. The term was given by Oudet et al, (1975). The nucleosome is made up of DNA and histone proteins. The proteins form a core particle, which consists of two molecules of each of four histone proteins – H2a, H2b, H3 and H4. The surface of core particle is surrounded by 1.75 turns of DNA (200 base pairs). The DN linking the core particle is called linker DNA. An other histone protein H1, is bounded to the linker DNA.

Welcome to new batch of students

Welcome to the new batch of 2008-2009 students. You are the 20th batch of students under autonomy. You are the 55th batch of 'LOYOLITES'. I also welcome the fresh batch of IMMUNOLOGY students. Wish you all a bright career

G. Mathew Srirangam

Head, Dept. of Zoology

& Dean, Admin

Ag- Ab INTERACTIONS

ANTIGEN – ANTIBODY INTERACTIONS
Precipitation Reactions
Agglutination Reactions
ELISA
Western Blotting
Introduction:
The antigen-antibody interaction is a bimolecular association similar to an enzyme-substrate interaction but with the important distinction that it does not lead to an irreversible chemical alteration in either the antibody or antigen and therefore is reversible.

1. Precipitation Reactions:
The interaction between an antibody and a soluble antigen in aqueous solution forms a lattice that eventually develops into a visible precipitate. Antibodies that thus aggregate soluble antigens are called precipitins. Although formation of the soluble Ag-Ab complex occurs within minutes, formation of the visible precipitate occurs more slowly and often takes a day or two to reach completion.
Precipitate reactions in fluids: A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amounts of antigen to the tubes. After the precipitate forms, each tube is centrifuged to pellet the precipitate, the supernatant is poured of, and the amount of precipitate against increasing antigen concentrations yields a precipitin curve.
Precipitation reactions in Gels: immune precipitates can form not only in solution but also in agar matrix. When antigen and antibody diffuse toward one another in agar or when antibody is incorporated into the agar and antigen diffuse into the antibody-containing matrix, a visible line of precipitation will form.

2. Agglutination reactions:
The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reactions are similar in principle to precipitation reactions. Just as antibody excess inhibits precipitation reactions, an excess of antibody inhibits agglutination reactions; this inhibition is called the prozone effect.
Hemagglutination: Agglutination reactions are routinely performed to type red blood cells. In typing for the ABO blood groups, RBCs’ are mixed on a slide with antisera to the A and B blood-group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide.
Bacterial agglutination: A bacterial infection often elicits the production of serum antibodies specific for surface antigens on the bacterial cells. The presence of such antibodies can be detected by bacterial agglutination reactions. Serum from patients thought to be infected with a given bacterium is serially diluted in a series of tubes to which the bacteria is added. The last tune showing visible agglutination will reflect the serum antibody titer of the patient. For example, if serial dilutions of serum are prepared and if the dilution of 1/20 shows agglutination but the dilution of 1/40 does not, then the agglutination titer of the patient’s serum is 40. This is widely used in typing Salmonella typhi.
3. ELISA

4. Western Blotting:
Identification of a specific protein in a complex mixture of proteins can be done by a technique called Western Blotting, named for its similarity to Southern Blotting, which detects DNA fragments and Northern blotting, which detects mRNAs’. In Western blotting a protein mixture is electrophoretically separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS), a dissociating agent. The protein bands are transferred to a nitrocellulose membrane by electrophoresis and the individual protein bands are identified by flooding the nitrocellulose membrane with radiolabeled polyclonal or monoclonal antibody specific for the protein of interest. The Ag-AB complexes that form are visualized by autoradiography. If labeled specific antibody is not available, Ag-AB complexes can be detected by adding a secondary anti-isotype antibody that is either radiolabeled or enzyme labeled; in this case the band is visualized by autoradiography or substrate addition.Western blotting can also identify a specific antibody in a mixture. In this case, the separated antibody bands are visualized with a labeled antigen. For example, this technique has been used to identify the envelope and core proteins of HIV and the antibodies to these components in the serum of HIV-infected individuals

IMMUNOGLOBULINS - STRUCTURE AND FUNCTION

P. Sanjana DBZ 8

I. DEFINITION
Immunoglobulins (Ig):These are the molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field.

II. GENERAL FUNCTIONS OF IMMUNOGLOBULINS
A. Antigen binding
Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host. The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all antibodies is at least two and in some instances more.
B. Effector Functions
Frequently the binding of an antibody to an antigen has no direct biological effect. Rather, the significant biological effects are a consequence of secondary "effector functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. Not every immunoglobulin will mediate all effector functions. Such effector functions include:
1. Fixation of complement - This result in lysis of cells and release of biologically active molecules.
2. Binding to various cell types - Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

III. BASIC STRUCTURE OF IMMUNOGLOBULINS
The basic structure of the immunoglobulins is illustrated in the. Although different immunoglobulins can differ structurally they all are built from the same basic units.
A. Heavy and Light Chains
All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)
B. Disulfide bonds
1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.
2. Intra-chain disulfide bnds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions
After the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids) and CL (110 amino acids)
2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)
D. Hinge Region
This is the region at which the arms of the antibody molecule form a Y. It is called the hinge region because there is some flexibility in the molecule at this point.
E. Domains
Three dimensional images of the immunoglobulin molecule show that it is not straight.
Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond. These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides
Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations.

IV. STRUCTURE OF THE VARIABLE REGION
A. Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons of the amino acid sequences of the variable regions of immunoglobulins show that most of the variability resides in three regions called the hypervariable regions or the complementarity determining regions. Antibodies with different specificities (i.e. different combining sites) have different complementarity determining regions while antibodies of the exact same specificity have identical complementarity determining regions (i.e. CDR is the antibody combining site). Complementarity determining regions are found in both the H and the L chains.
B. Framework regions
The regions between the complementary determining regions in the variable region are called the framework regions. Based on similarities and differences in the framework regions the immunoglobulin heavy and light chain variable regions can be divided into groups and subgroups. These represent the products of different variable region genes.

V. IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin fragments produced by proteolytic digestion have proven very useful in elucidating structure/function relationships in immunoglobulins.
A. Fab
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 4. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain. Antigen binding - These fragments were called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. An antibody is able to bind a particular antigenic determinant because it has a particular combination of VH and VL. Different combinations of a VH and VL result in antibodies that can bind a different antigenic determinants.
B. Fc

Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.
Effector functions - The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment. Normally the ability of an antibody to carry out an effector function requires the prior binding of an antigen; however, there are exceptions to this rule.
C. F(ab')2
Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites. This fragment was called F(ab')2 because it was divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab')2 binds antigen but it does not mediate the effector functions of antibodies.

VI. HUMAN IMMUNOGLOBULIN CLASSES, SUBCLASSES, TYPES AND SUBTYPES
A. Immunoglobulin classes
The immunoglobulins can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences).
1. IgG - Gamma heavy chains
2. IgM - Mu heavy chains
3. IgA - Alpha heavy chains
4. IgD - Delta heavy chains
5. IgE - Epsilon heavy chains
B. Immunoglobulin Subclasses
The classes of immunoglobulins can de divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a subclass will have very similar heavy chain constant region amino acid sequences. Again these differences are most commonly detected by serological means.
1. IgG Subclasses
a) IgG1 - Gamma 1 heavy chains
b) IgG2 - Gamma 2 heavy chains
c) IgG3 - Gamma 3 heavy chains
d) IgG4 - Gamma 4 heavy chains
2. IgA Subclasses
a) IgA1 - Alpha 1 heavy chains
b) IgA2 - Alpha 2 heavy chains
C. Immunoglobulin Types
Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. These differences are detected by serological means.
1. Kappa light chains
2. Lambda light chains
D. Immunoglobulin Subtypes
The light chains can also be divided into subtypes based on differences in the amino acid sequences in the constant region of the light chain.
Lambda subtypes
a) Lambda 1
b) Lambda 2
c) Lambda 3
d) Lambda 4
E. Nomenclature
Immunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass, types and subtypes are present. IgG means that all subclasses and types are present.
F. Heterogeneity
Immunoglobulins considered as a population of molecules are normally very heterogeneous because they are composed of different classes and subclasses each of which has different types and subtypes of light chains. In addition, different immunoglobulin molecules can have different antigen binding properties because of different VH and VL regions.

VII. STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES
A. IgG
1. Structure:
All IgG's are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.
2. Properties
Most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.
a) IgG is the major Ig in serum - 75% of serum Ig is IgG. b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement
e) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. The antibody has prepared the antigen for eating by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells result in the activation of other functions.
B. IgM
1. Structure:
IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.
2. Properties
a) IgM is the third most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig
Surface IgM exists as a monomer and lacks J chain but it has extra 20 amino acids at the C-terminus to anchor it into the membrane. Cell surface IgM functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B cell called Ig-alpha and Ig-beta as indicated in Figure 10. These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an antigen is required before a signal can be transduced by the Ig-alpha and Ig-beta chains. In the case of T-independent antigens, contact between the antigen and surface immunoglobulin is sufficient to activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent antigens, a second signal provided by helper T cells is required before B cells are activated.
C. IgA
1. Structure
Serum IgA is a monomer but IgA found in secretions is a dimmer. When IgA exits as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions. The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.
2. Properties
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
D. IgD
1. Structure:
IgD exists only as a monomer.
2. Properties
a) IgD is found in low levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.
c) IgD does not bind complement.
E. IgE
1. Structure:
IgE exists as a monomer and has an extra domain in the constant region.
2. Properties
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.
b) Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells result in the release of various pharmacological mediators that result in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.

SECONDARY LYMPHOID ORGANS
- Mathew Srirangam

Lymphatic System
As blood circulates under pressure, the fluid component of the blood (plasma) seeps through the thin wall of the capillaries into the surrounding tissues. This fluid is called the interstitial fluid or extra cellular fluid. Much of this fluid returns to the blood through the capillary membranes. The reminder of the interstitial fluid now called lymph flows from the connective tissue spaces into a network of thin open lymphatic capillaries and then into a series of progressively larges collecting vessels called lymphatic vessels. The largest lymphatic vessel called the thoracic duct opens into the left subclavian vein near the heart. In this way lymphatic system functions to capture fluid lost from the blood and returns it to the blood, thus ensuring the fluid steady state of the blood.

As the lymph is draining the connective tissues of the body, when a foreign antigen gains entrance into the tissues, it is picked up by the lymphatic system, and carried to various organized lymphatic tissues, which trap the antigen. Various types of organized lymphoid tissues are located along the vessels of the lymphatic system.

Some lymphoid tissues in the lung and lamina propria of the intestinal wall consist of diffuse collections of lymphocytes and macrophages. Other lymphoid tissue is organized into structures called lymphoid follicles. In the absence of antigen stimulus, a lymphoid follicle called primary follicle, comprises a network of follicular dendritic cells and small resting B-cells. Following an antigenic challenge, a primary follicle becomes a large secondary follicle – a ring of concentrically packed B lymphocytes surrounding a center called the germinal center in which proliferating B lymphocytes, memory cells and plasma cells are interspersed with macrophages and follicular dendritic cells.

Dendritic cells in lymphoid organs are two types.
In T-cell areas they are
called interdigitating dendritic cells
and in B-cell areas they are
called follicular dendritic cells.

The germinal center is a site of intense B-cell activation. Here the B-cells that interact with antigen displayed on the membrane of follicular dendritic cells are induced to proliferate and differentiate into plasma and memory cells. In the absence of antigen activation, the B-cells appear to undergo programmed cell death with in the germinal center.

In order to maintain steady state levels of cells, the cells undergo programmed
cell death. These cells exhibit morphological changes collectively called
apoptosis. Apoptosis includes, decrease in cell volume, modification of
the cytoskeleton resulting in pronounced membrane blebbing (pinching off small
pieces).

Lymph Nodes:
Lymph nodes are encapsulated bean shaped structures containing a reticular network packed with lymphocytes, macrophages and dendritic cells. Clustered at junctions of the lymphatic vessels, lymph nodes are the first organized lymphoid structure to encounter the antigens that enter the tissue spaces. As lymph percolates through a node, the cellular network of phagocytic cells and dendritic cells will trap any particulate antigen that is brought in with the lymph.

Morphologically, a lymph node can be divided into three roughly concentric regions – the cortex, the paracortex and the medulla. The outermost layer, the cortex consists of lymphocytes (mostly B cells) macrophages, and follicular dendritic cells arranged in primary follicle. With antigenic challenge, the primary follicles enlarge into secondary follicles, each containing la germinal center. Intense B-cells activation and differentiation into plasma and memory cells occur in the germinal centers. Beneath the cortex is the paracortex, which is populated largely with T-lymphocytes and also contains interdigitating dendritic cells. These dendritic cells express high levels of Class II MHC molecules, which are necessary for antigen presentation to TH cells. The innermost layer of lymph node is the medulla, which is sparsely populated with lymphocytes, but many of these are plasma cells.

Afferent lymphatic vessels pierce the capsule of lymph node at numerous sites and empty lymph into the sub capsular sinus. Lymph coming from the tissues percolates slowly inward through the cortex, the paracortex and medulla; allowing phagocytic cells and dendritic cells to trap any bacteria or particulate material, (like antigen-antibody complexes) carried by the lymph.

The trapped antigen is processed and presented together with Class I MHC molecules by interdigitating dendritic cells in the paracortex resulting in TH cell activation. Followed by the activation of B cells. TH and B cells bring about their immunological responses respectively.

Spleen
Spleen is a large, ovoid, secondary lymphoid organ situated high in the left abdominal cavity. Unlike lymph nodes, which are specialized to trap localized antigen from regional tissue spaces, the spleen is adapted to filtering blood and trapping blood-borne antigens, and thus can respond to systemic infections.

The spleen is surrounded by a capsule that sends a number of projections (trabeculae) into the interior to form a compartmentalized structure. The compartments are of two types, the red pulp and the white pulp, which are separated by a diffuse marginal zone. The splenic red pulp consists of a network of sinusoids populated with macrophages and numerous red blood cells (erythrocytes). It is the site where old and defective red blood cells are destroyed and removed. Many of the macrophages within the red pulp contain engulfed red blood cells or iron pigments from degraded hemoglobin. The splenic white pulp surrounds the arteries, forming a periarteriolar lymphoid sheath (PALS) populated mainly by T-lymphocytes. The marginal zone, located peripheral to the PALS, is rich in B cells organized into primary lymphoid follicles.
The main immunological function of the spleen is to filter the blood and trap blood borne microorganisms and producing an immune response to them.

e-Mail ID of the Department of Zoology for correspondence

The following is the e-mail ID of the Department of Zoology for all correspondence.
andhraloyolazoology@gmail.com

Attn: To all Immunology students

As agreed earlier, no one came out with their presentation. I request all those who have been informed earlier to come out with their presentations (oral) for the next class, i.e., on 6th July 2007.

Srirangam. G. M.

Intro' Ecology - Light

Introductory remarks on Ecology and Light

By G. Mathew Srirangam

origin of word: oikos = the family household logy = the study of
interesting parallel to economy = management of the household many principles in common – resources allocation, cost benefit ratios
definitions: Haeckel (German zoologist) 1870: “By ecology we mean the body of knowledge concerning the economy of Nature - the investigation of the total relations of the animal to its inorganic and organic environment.”
Burdon-Sanderson (1890s): Elevated Ecology to one of the three natural divisions of Biology: Physiology - Morphology – Ecology
Andrewartha (1961): “The scientific study of the distribution and abundance of organisms.”
Odum (1963): “The structure and function of Nature.”
Definition we will use (Krebs 1972):
“Ecology is the scientific study of the processes regulating the distribution andabundance of organisms and the interactions among them, and the study of how theseorganisms in turn mediate the transport and transformation of energy and matter in the biosphere (i.e., the study of the design of ecosystem structure and function).”
The goal of ecology is to understand the principles of operation of natural systems and to predict their responses to change.
LIGHT
PENETRATION OF SOLAR RADIATION
Solar radiation is the primary determinant of global climate. The amount of energy reaching the outer atmosphere from the sun is known as the solar constant: 2.0 cal/cm2/minute. Most, but not all, of solar radiation is visible light.
Of this light:
21% is reflected by clouds
5% reflected by dust, aerosols, and soot.
6% reflected by earth
32% total reflected (due to albedo)
18% of radiation is absorbed by dust, water vapor, clouds, carbon dioxide, and soot.
Total radiation removed by atmosphere: 50%. However, different wavelengths are removed differently. Nearly all ultraviolet light is removed.
albedo: Reflection of solar radiation by the earth. Snow and ice have high albedos, forests have low albedos.
Solar radiation is lower at poles than at equator because of the curvature of the earth.





LIGHT

Introduction. Light from the sun supports photosynthesis, permits vision, and heats the earth.
Solar constant = 1.94 cal/cm2 striking upper atmosphere perpendicular to sun at mean distance from sun Fate of 100 units striking atmosphere
Reflected Absorbed
Atmosphere 25 25
Earth 5 *45

*29% = thermals & evaporation, 16% = long wave IR)
88% of IR light is reflected back to earth (12% passes through IR windows)
Low intensity. Low intensity of light reduces photosynthesis. Some animals are adapted to darkness.
Winter: angle of sun greater, more air absorption, shorter day length
In shade: ferns, mosses survive in low light
Under water
Absorption in water is logarithmic for a given wave length
Affected by water color, suspended materials, plankton
Photosynthesis down to 5-50 m in fresh water, 100 m in ocean
Compensation intensity is about 1% of surface intensity for terrestrial and aquatic plants
At night
Carnivores and ungulates have a reflector behind their retina that sends light back through it.
Insects, owls, see in low light
Pit viper snakes, boas detect infra red

High intensity. Plants must adapt to high amounts of light that can destroy their enzymes. When sun directly overhead, and toward equator
Photosynthesis decreases
Photo-oxidation of enzymes, increased respiration
Photosynthesis maximal at 10-20% of full sunlight for leaf perpendicular to sun
5% efficient at high light, 20% at low light
Adaptations
Move to low intensity areas (phytoflagellates)
Turn angle of leaves from sun
Sun vs. shade leaves

Wave length. Visible light is only a small portion of the electromagnetic spectrum that ranges from short wave length gamma radiation to long wave length radio waves.
Electromagnetic spectrum
Gamma: danger in space travel, bombs, radioisotopes, reactors
X-ray: danger from x-ray machines
Ultra violet: kills surface cells (germicidal lamps)
Visible: photosynthesis, vision
Infra red: heat, laser = burns
Microwave: radar, microwave ovens cellular phones TV
Radio: no known effects
Citizens
Amateur
Long wave (submarines)
Ecological considerations
Photosynthesis: chlorophyll mainly uses blue and red
Sight
Color vision in some arthropods, fishes, birds, mammals
Insects see UV to orange
Absorption by water
IR, UV, red, orange absorbed first
Yellow = intermediate
Blue, green, violet = penetrate deepest (red algae use these wavelengths)

Duration. Animals use variation in day length or phase of the moon coordinate their periodicity.
Daily variation: diurnal, crepuscular, nocturnal
Monthly: moon light used to coordinate reproductive periods
Seasonal: day length varies with seasons, used in timing of migration, reproductive cycles, etc.

Structure of Immunoglobulin-Fig.

Immunoglobulins

IMMUNOGLOBULINS

G. Mathew Srirangam

Antibodies are glycoproteins that bind antigens with high specificity and affinity (they hold on tightly). They are molecules, originally identified in the serum, which are also referred to as ‘immunoglobulins’; a term often used interchangeably with antibodies. In humans there are five chemically and physically distinct classes of antibodies – IgG, IgA, IgM, IgD, IgE). Immunoglobulins are synthesized by plasma cells and to some extent by lymphocytes also. All antibodies are immunoglobulins but all immunoglobulins may not be antibodies. Immunoglobulins constitute 20-25 percent of the total serum proteins.

Basic Structure of Immunoglobulin:

Immunoglobulins when digested by papain in the presence of cysteine, will split into two fractions – an insoluble fraction which crystallized in the cold called – Fc (for crystallizable) fragment, and a soluble fragment which is unable to precipitate and is called – Fab (for antigen binding) fragment. Each molecule of immunoglobulin is split by papain into three parts, one Fc and two Fab pieces. When treated with pepsin, a 5S fragment is obtained, which is composed essentially of two Fab fragments held together in position. This fragment is called F(ab)2. The Fc portion is digested by pepsin into smaller fragments.

Immunoglobulins are glycoproteins, each molecule consisting of two pairs of polypeptide chains of different sizes. The smaller chains are called ‘light’ (L) chains and larger ones ‘heavy’ (H) chains. The L chain has a molecular weight of approximately 25000 and the H chain of 50,000. The L chain is attached to the H chain by a disulfide bond. The two-H chains are joined together by 1-5 S-S bonds, depending on the class of immunoglobulins.

The H chains are structurally and antigenically distinct for each class and are designated by the Greek letter corresponding to the immunoglobulin class, like –
IgG g(gamma)
IgA a(alpha)
IgM m(mu)
IgD d(delta)
IgE e(epsilon)
The L chains are similar in all classes of immunoglobulins. They occur in two varieties kappa ( k ) and lambda ( l ). A molecule of immunoglobulin may have either kappa or lambda chains, but never both together.

The antigen-combining site of the molecule is at its aminoterminus. It is composed of both L and H chains. Of the 214-aminoacid residues that make up the L chain, about 107 that constitute the carboxyterminal half occur only in a constant sequence. This part of the chain is therefore called the ‘constant region’. Only two sequence patterns are seen in the constant region- those determining the kappa and lambda specificities. On the other hand the aminoacid sequence in the aminoterminal half of the chain is highly variable, the variability determining the immunological specificity of the antibody molecule. It is therefore called the ‘variable region’. The H chain also has ‘constant’ and ‘variable’ regions. While in the L chain the two regions are of equal length, in the H chains the variable region constitutes approximately only a fifth of the chain and is located at its aminoterminus. The infinite range of the antibody specificity of immunoglobulins depends on the variability of the aminoacid sequences at the ‘variable regions’ of the H and L chains, which form the antigen combining sites.

The aminoacid sequences of the variable regions of the L and H chains are not uniformly variable along their length but consist of relatively invariable and some highly variable zones. The highly variable zones numbering three in the L and four in the H chains are called hypervariable regions or hot spots and are involved with the formation of the antigen binding sites.

The Fc fragment is composed of the carboxyterminal portion of the H chains. It does not possess antigen-combing activity but determines the biological properties of the immunoglobulin molecule. The portion of the H chain present in the Fab fragment is called the Fd piece.

Each immunoglobulin peptide chain has internal disulfide links in addition to interchain disulfide bonds, which bridge the H and L chains. These intrachain disulfide bonds form loops and each of the loop is compactly folded to form a globular domain, each domain having a separate function. The variable region domains, VL and VH are responsible for the formation of a specific antigen-binding site. The area of the H chain in the C region between the first and second C region domains (CH1 and CH2) is the hinge region. It is more flexible and is more exposed to enzymes and chemicals.

Organs of Immune system

ORGANS OF THE IMMUNE SYSTEM

G. Mathew Srirangam

The immune system consists of many structurally and functionally diverse organs and tissues that are widely dispersed throughout the body. These organs can be classified based on functional differences into two main groups –

1. The primary lymphoid organs or Central lymphoid organs
2. The secondary lymphoid organs or Peripheral lymphoid organs.

The primary lymphoid organs provide appropriate microenvironments in which the precursor lymphocytes proliferate, develop and acquire immunological capability. And the secondary lymphoid organs trap antigen form defined tissues or vascular spaces and provide sites where mature lymphocytes can interact effectively with that antigen. The thymus and bone marrow constitute the primary lymphoid organs. Spleen, Lymph nodes and various mucosal-associated tissues (MALT) compose the secondary lymphoid organs.

Thymus
Thymus develops from the epithelium of the third and fourth pharyngeal pouches at about the sixth week of gestation and by the eight week, grows into a compact epithelial structure. Mesenchymal stem cells or precursors of lymphocytes from the yolk sac, foetal liver and bone marrow reach the thymus and differentiate into the thymic lymphoid cells (thymocytes). The thymus acquires its characteristic lymphoid appearance by the third month of gestation. It is thus the first organ to become predominantly lymphoid. In human beings, the thymus reaches its maximal relative size just prior to birth. It continues to grow till about the 12th year. After puberty, it undergoes spontaneous progressive involution, indicating that it functions best in early life.

The thymus is a flat, bilobed organ located behind the upper part of the sternum above the heart. Each lobe of thymus is surrounded by a capsule and is divided into lobules, which are separated from each other by strands of connective tissue called trabeculae. Each lobule is organized into two compartments; the outer compartment or cortex, which is densely packed with immature T cells, called thymocytes. The inner compartment or medulla is sparsely populated with thymocytes but have mature lymphocytes between are present Hassall’s corpuscles which are whorl-like aggregations of epithelial cells. Both the cortex and medulla of the thymus are criss-crossed by a three-dimensional stroma-cell network composed of epithelial cells, interdigitating dendric cells and macrophages. Some epithelial cells in the outer cortex, called nurse cells, have long membrane processes that surround as many as 50 thymocytes, forming large multicellular complexes.

The primary function of thymus is the production of thymic lymphocytes or T lymphocytes or Thymus dependent (T) lymphocytes. It is the major site for lymphocyte proliferation. The thymus confers immunological competence on the lymphocytes during their stay in the organ, so that they are capable of mounting cell-mediated immune response against appropriate antigens. Finally the T lymphocytes are selectively seeded into certain sites in the peripheral lymphatic tissues, being found in the white pulp of the spleen, around the central arterioles and in the paracortical areas of lymphoid nodes. A congenital birth defect in humans called DiGeorge’s syndrome and in certain mice (nude mice) involves the failure of the thymus to develop T-lymphocytes and absence of cell-mediated immunity.

Bone Marrow

In birds a lymphoid organ called the bursa of Fabricus is the primary site of B-cell maturation. There is no bursa in mammals and no single counterpart to it as a primary lymphoid organ. Instead, regions of the bone marrow serve as the ‘bursal equivalent’ where B cell maturation occurs. Immature B cells proliferate and differentiate within the microenvironment of the bone marrow. Bone marrow is composed of hematopoietic cells of various lineage and maturity packed between fat cells, thin bands of bony tissue (trabeculae), collagen fibers, fibroblasts and dendritic cells. All the hematopoietic cells are derived from multipotential stem cells which give rise not only to all of the lymphoid cells found in the lymphoid tissue, but also to all of the cells found in the blood. The bone marrow gives rise to all of the lymphoid cells that migrate to the thymus for T-cell maturation as well as to the major population of conventional B cells. B cells mature in the bone marrow and undergo selection for non-self before making their way to the peripheral lymphoid tissues. Stromal cells within the bone marrow interacts directly with the B cells and secretes various cytokines that are required before the B-cell developmental process.

Spleen

Spleen is a large, ovoid, secondary lymphoid organ situated high in the left abdominal cavity. Unlike lymph nodes, which are specialized to trap localized antigen from regional tissue spaces, the spleen is adapted to filtering blood and trapping blood-borne antigens, and thus can respond to systemic infections.

The spleen is surrounded by a capsule that sends a number of projections (trabeculae) into the interior to form a compartmentalized structure. The compartments are of two types, the red pulp and the white pulp, which are separated by a diffuse marginal zone. The splenic red pulp consists of a network of sinusoids populated with macrophages and numerous red blood cells (erythrocytes). It is the site where old and defective red blood cells are destroyed and removed. Many of the macrophages within the red pulp contain engulfed red blood cells or iron pigments from degraded hemoglobin. The splenic white pulp surrounds the arteries, forming a periarteriolar lymphoid sheath (PALS) populated mainly by T-lymphocytes. The marginal zone, located peripheral to the PALS, is rich in B cells organized into primary lymphoid follicles.

The main immunological function of the spleen is to filter the blood and trap blood borne microorganisms and producing an immune response to them.

ACQUIRED (SPECIFIC) IMMUNITY

Acquired or specific immunity reflects the presence of a functional immune system that is capable of specifically recognizing and selectively eliminating foreign microorganisms and molecules (i.e., foreign antigens). Unlike innate immune responses, acquired immune responses are adaptive and display four characteristic attributes:
• Antigen specificity
• Diversity
• Immunological memory
• Self/nonself recognition
The antigen specificity of the immune system permits it to distinguish subtle (exact) differences among antigens. The immune system is capable of generating tremendous diversity in its recognition molecules, allowing it to specifically recognize billions of uniquely different structures on foreign antigens. Once the immune system has recognized and responded to an antigen, it exhibits immunologic memory; that is, a second encounter with the same antigen induces a heightened state of immune reactivity. Finally, the immune system normally responds only to foreign antigens indicating that it is capable of self/nonself recognition. The ability of the immune system to distinguish self from nonself and respond only to nonself-molecules is essential, for the outcome of an inappropriate response to self-molecules can be a fatal autoimmune disease.

Cells of Immune System:
Generation of an effective immune response involves two major groups of cells; lymphocytes and antigen presenting cells. Lymphocytes are one of many types of white blood cells produced in the bone marrow during the process of hematopoiesis. Lymphocytes leave the bone marrow circulate in the blood and lymph system, and reside in various lymphoid organs. Lymphocytes, which possess antigen-binding cell-surface receptors, possess the defining immunologic attributes of specificity, diversity, memory, and self/nonself recognition. The two major populations of lymphocytes are: B – lymphocytes (B cells) and T – Lymphocytes (T cells)

B Lymphocytes
B-lymphocytes mature within the bone marrow and leave the marrow expressing a unique antigen-binding receptor on their membrane. The B – Lymphocyte receptor is a membrane-bound antibody molecule. Antibodies are glycoproteins. The basic structure of the antibody molecule consists of two identical light polypeptide chains. The chains are held together by disulfide bonds. The amino-terminal ends of each pair of heavy and light chains form a cleft within which antigen binds. When a naïve B cell, which has not previously encountered antigen, first encounters the antigen for which its membrane bound antibody is specific, the cell begins to divide rapidly; its progeny differentiate into memory B cells and effector B cells called plasma cells.
Memory B cells have a longer life span and continue to express membrane-bound antibody with the same specificity as the original parent naïve B cell. Plasma cells do not express membrane-bound antibody; instead they produce the antibody in a form that can be secreted. Although plasma cells live for only a few days, they secrete enormous amounts of antibody during the time.
T Lymphocytes:
T lymphocytes also arise from hematopoietic stem cells in the bone marrow. Unlike B cells, which mature within the bone marrow, T cells migrate to the thymus gland to mature. During its maturation within the thymus, the T cell comes to express a unique antigen-binding receptor on its membrane, called the T-Cell receptor. Unlike membrane-bound antibodies on B cells, which can recognize antigen alone, T cell receptors can only recognize antigen that is associated with cell-membrane proteins known as major histocompatibility complex (MHC) molecules. When a naïve T cell encounters antigen associated with a MHC molecule on a cell, the T cell proliferates and differentiates into memory T cell and various effector T cells.
There are two well-defined subpopulation of T cells - T helper (TH) and T cytotoxic (TC) cells. After a TH cell recognizes and interacts with an antigen-MHC molecule complex, the cell is activated and becomes an effector cell that secretes various growth factors know collectively as cytokines. Under the influence of TH -derived cytokines, a TC cell that recognizes an antigen-MHC complex proliferates and differentiates into an effector cell called a cytotoxic T Lymphocyte.

Functions of Humoral and Cell-Mediated Immune Responses:

Immune responses can be divided into humoral and cell-mediated responses.
The term humoral is derived from the Latin humor, meaning “body fluid”; thus humoral immune system involves interaction of B cells with antigen and their subsequent proliferation and differentiation into antibodies. Antibody functions as the effector of the humoral response by binding to antigen and neutralizing it or facilitating its elimination. When an antigen is coated with antibody, it can be eliminated in several ways. For example, antibody can cross-link the antigen, forming clusters that are more readily ingested by phagocytic cells. Binding of antibody to antigen on a microorganism also can activate the complement system, resulting in lysis of the foreign organism. Antibody can also neutralize toxins or viral particles by coating them and preventing their subsequent binding to host cells.

Effector T cells generated in response to antigen are responsible for cell-mediated immunity. Activated TH cells serve as effector cells in cell-mediated immune reactions. Cytokines secreted by TH cells can activate various phagocytic cells, enabling them to phagocytose and kill microorganisms more effectively. This type of cell-mediated immune response is especially important in host defense against intracellular bacteria and protozoa. Cytotoxic T lymphocytes (CTLs) participate in the cell-mediated immune reactions by killing altered self-cells; they play an important role in the killing of virus-infected cells and tumor cells.

Innate Immunity

INNATE (NONSPECIFIC IMMUNITY)
Innate immunity has four types of defensive barriers; anatomic, physiologic, endocytic and phagocytic, and inflammatory.

Anatomic barriers: Physical and anatomic barriers that tend to prevent the entry of pathogens are an organism’s first line of defence against infection.

The skin and the surface of mucous membranes are included in the category because they provide an effective barrier to the entry of most microorganisms. The skin consists of two distinct layers; a relatively thin outer layer- the epidermis and a thicker layer the dermis. The epidermis contains several layers of tightly packed epithelial cells. The outer layer consists of dead cells and is filled with a waterproofing protein called keratin. The dermis, is composed of connective tissue, contains blood vessels, hair follicles, sebaceous glands. The sebaceous glands secrete an oily substance called sebum. Sebum consists of lactic acid, which maintains the pH of the skin between 3 and 5. Thus intact skin not only prevents the penetration of most pathogens but also inhibits most bacterial growth due its low pH.

The conjunctiva and the alimentary, respiratory, and urinogenital tracts are lined by mucous membranes, not by the dry, protective skin covering the exterior of the body. These membranes consist of an outer epithelial layer and an underlying connective tissue layer. The secretions of these mucus membranes wash away potential invaders and also contain antibacterial or antiviral substances. The viscous fluid called mucus, which is secreted by epithelial cells of mucous membrane, entraps foreign microorganisms.

Physiologic barriers: The physiologic barriers that contribute to innate immunity include temperature, pH, oxygen tension and various soluble factors. Many species are not susceptible to certain diseases simply because their body temperature inhibits pathogen growth. Gastric acidity also provides an innate physiologic barrier to infection because very few ingested microorganisms can survive the low pH of the stomach. One reason newborns are susceptible to some diseases that do not afflict adults is that their stomach contents are less acid than that of adults. Among the soluble proteins are lysozyme, interferon, and complement. Lysozyme, a hydrolytic enzyme found in mucous secretions, is able to cleave the peptidoglycan layer of the proteins of the bacterial cell wall. Interferon comprises a group of proteins produced by virus-infected cells. Among the many functions of the interferons is the ability to bind to nearby cells and induce a generalized antiviral state. Complement is a group of serum proteins that circulate in an inactive proenzyme state. These proteins can be activated by a variety of specific and nonspecific immunological mechanisms that convert the inactive proenzymes into active enzymes. The activated complement components participate in a controlled enzymatic cascade that results in damage to the membranes of pathogenic organisms, either destroying the pathogens or facilitating their clearance.



Endocytic and Phagocytic Barriers: Another important innate defense mechanism is the ingestion of extracellular macromolecules via endocytosis and of particulate material via phagocytosis. These tow internalization processes not only bring different types of extracellular material into the cell, they also differ in several other ways.

In endocytosis, macromolecules within the extracellular tissue fluid are internalized by cells via the invagination (inward folding) and pinching off of small regions of the plasma membrane. Phagocytosis involves the ingestion of particulate material, including whole pathogenic microorganisms.

Barriers created by the Inflammatory Response:
Tissue damage caused by a wound or by invasion by a pathogenic microorganism induces a complex sequence of events collectively known as the inflammatory response. In the first century A.D. the Roman Physician Celsus described the four cardinal signs of inflammation are rubor (redness), tumor (swelling), calor (heat) and dolor (pain). Afterwards the fifth sign functio laesa (loss of function) was added in second century A.D. Inflammatory response includes three events:

1. Vasodilation: an increase in the diameter of blood vessels – occurs as the vessels that carry blood away from an affected area constrict, resulting in engorgement of the capillary network. The engorged capillaries are responsible for tissue redness (erythema) and an increase in tissue temperature.
2. An increase in capillary permeability facilitates an influx of fluid and cells form the engorged capillaries into the tissue. The fluid that accumulates (exudate) has a much higher protein content than fluid normally released from the vasculature. Accumulation of exudate contributes to tissue swelling (edema).
3. Influx of phagocytes from the capillaries into the tissues is facilitated by the increased capillary permeability. The emigration of phagocytes involves a complex series of events including adherence of the cells to the endothelial wall (margination) followed by their emigration between the capillary endothelial cells into the tissue (diapedesis or extravasation) and, finally, their migration through the tissue to the site of the inflammatory response (chemotaxis). As phagocytic cells accumulate at the site and begin to phagocytose bacteria, they release lytic enzymes, which can damage nearby healthy cells. The accumulation of dead cells, digested material, and fluid forms a substance called pus.

The events in the inflammatory response are initiated by a complex series of interactions involving a variety of chemical mediators. One of the principal mediators is histamine, a chemical released by a variety of cells in response to tissue injury. Histamine binds to receptors on nearby capillaries and venules, causing vasodialtion and increased permeability. Once the inflammatory response has subsided and phagocytic cells have cleared most of the debris away, tissue repair and regeneration of new tissue occur.