Know Life Sciences

PI3K Pathway

noreply@blogger.com (R Santhosh Kalash) — Fri, 18 Oct 2013 09:02:00 +0000

Phosphoinositide 3-Kinase enzyme

It is a signal transduction system that connects oncogenes and multiple receptor classes to many cellular functions such as cell survival, proliferation and differentiation. The key enzyme family in this pathway is PI3Ks (Phosphoinositide 3-Kinases) which transducer signals from various growth factors and cytokines into intracellular messages by generating phospholipids. These phospholipids activate the serine- threonine protein kinase AKT (also known as protein kinase B (PKB)) and other downstream effector pathways.

PIP2 (PtdIns (4,5) P₂) àPIP3 (PtdsIns (3,4,5) P₃)

The enzyme family is classified into three classes based on the structural characteristics and substrate specificity:

Class I enzyme: à These enzymes are activated directly by the cell surface receptors. They are further classified into two sub classes. i.e. class IA and class IB.

Class IA enzyme à they are heterodimers consisting of a P110 catalytic subunit and p85 regulatory subunit.

p85 regulatory subunit mediates receptor binding, activation and localization of enzyme.

This subunit directly interacts with tyrosine phosphate motif of activated receptor (eg: platelet growth factor receptor) or to adapter proteins associated with receptor (Eg: IRS1)

Activated P110 catalytic subunit generates phosphoinositide 3,4,5 triphosphate which further activates multiple downstream signalling pathway.

Class IB enzyme à It’s also a heterodimer with p110γ catalytic subunit and p101 regulatory subunit. Apart from this they also contain some adapter proteins such as p84, p87.

P110γ subunit is activated by GPCRs through interaction of regulatory subunit with Gβγ subunit of trimeric G proteins. P110γ is expressed in leukocytes but also found in heart, pancreases, liver, and skeletal muscles.

Class II enzyme: à

It consists of only a single catalytic subunit. Phosphatidylinositol 4-phosphate (PtdsIns4P) is used as the substrate.

It is found in three isoforms: a) PI3KC2α b) PI3KC2β c) PI3KC2γ

And these can be activated by receptor tyrosine kinases, cytokine receptors, and integrins.

But still their specific role in cellular function remains unclear.

Class III enzyme: à

It consists of a single catalytic subunit VPS34 (Homologue of the yeast vacuolar protein sorting associated protein 34). It’s also known as PIK3C3 which only producesPtdIns3P, which is an important regulator of membrane trafficking.

The subunit function as a nutrient regulated lipid kinase that mediates signalling throught mTOR (mammalian target of rapamycin).

The negative regulator of this PI3K pathway is a tumor suppressor protein called PTEN (Phosphotase and Tensin homologue). PIP3 (PtdsIns (3,4,5) P₃) is the key second messenger that drives several downstream signalling cascades that regulate cellular processes. The cellular levels of PIP3 are tightly regulated by the opposing activity of PTEN. PTEN functionally antagonizes PI3K activity through its intrinsic lipid phosphatase activity that reduces the cellular pool of PIP3 by converting PIP3 back to PtdIns (4,5) P₂.

Phage Vector

noreply@blogger.com (R Santhosh Kalash) — Fri, 13 Sep 2013 15:17:00 +0000

Bacteriophages

1) Viruses specifically infect bacteria.

2) Phages have simple structure: Genetic material (DNA or RNA) is encapsulated in a protective

proteineous coat called caspid.

3) It replicates within the bacteria and lysis them.

Bacteriophage λ in Gene cloning

* It’s an E.Coli virus that can carry an insert of a range between 15-20 kb

* After Bacteriophage λ infects the E.Coli, two things may happen:

a) Lytic cycle: where the phage replicates within cell and finally lysis the cell inorder to release 100 of phage particles and this occurs in 20 minutes.

b) Lysogenic cycle: the phage DNA can be incorporated into the bacterial genome as a prophage and maintained as a benign guest through successive cell divisions.

But under environmental circumstances, the integrated phage DNA is excised out and therefore the phage enters the lytic cycle.

* The length of the Bacteriophage λ is 50kb and integration-excision (I/E) events occurs at 20 kb respectively.

* This 20 kb I/E region is replaced with 20 kb of cloned DNA.

Bacteriophage as Vector in gene cloning

Ultraviolet Mutagenesis

noreply@blogger.com (R Santhosh Kalash) — Fri, 13 Sep 2013 07:08:00 +0000

Ultraviolet light (UV)

UV light, an electromagnetic radiation with a wavelength range between 400 nm to 10 nm, i.e. shorter and longer than visible light and X-ray.

Natural source of UV light is sun and its artificial emitted by electric arcs or by mercury lamps.

It has a strong genotoxic effects to produce DNA damage, induce mutations and causes tumor development. It also said to be the main cause of skin cancer.

Purines and pyrimidine absorbs UV at 260 nm wavelength, which makes a perfect source for identifying nucleic acids in Electrophoresis.

There three bands of UV classified based on their wavelength: UVA àUVA1 (340-400 nm); UVA2 (320-340 nm), UVB à (290-400nm). UVB is has the strong carcinogenic effect on skin. And UVA has some role in skin carcinogenesis.

UV Induced DNA lesions

A lesion in the DNA means the weak hydrogen bonds in the base pair denatures, which leads to instability in the DNA structure. It can be an abnormal base in the DNA or damage to the sugar-phosphate backbone.

UV induces DNA lesions by the two ways:

By producing photoproducts

UV photons are easily absorbed by the DNA molecules and an excited state is produced which allows rearrangement of electrons resulting in the formation of photoproducts. cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone (64PPs) are the photoproducts formed by the UVB radiation. These photoproducts are found to be the cause of UV-specific mutations.

By producing ROS

And UV can also induce oxidative stress in the irradiated cells through the production of reactive oxygen species (ROS) by activating small molecules such as riboflavin, tryptophan and porphyrin. This will further activate the cellular oxygen.

DNA exposed to these ROS will produce oxidative base damage such as 8-hydroxyguanine (8OH-G) and thymine glycol in DNA or can make strand breaks. ROS also attack cellular nucleotide pools, producing oxidized nucleotides such as 8-hydroxydeoxygunanosine-triphosphate (8OH-dGTP), which can still be used as nucleotide precursors for DNA synthesis.

UV induced mutation

UV photons are absorbed by the DNA molecular and therefore pyrimidine dimers (Thymine-thymine dimers) are formed. During this moment, the DNA repair mechanism works and correct the dimer units. But in some cases mutation occurs, which is basically meant as DNA repair error.

Two types of mutations are induced by UV:

1) Base substitution of Cytosine à Thymine at dipyrimidine site.

2) Tandem base substitution of cytosine-cytosine à thymine-thymine.

These two types of mutations are best known to be UV signatures, because detection of these mutations suggests the past exposure to UV.

UV mutation by Deamination

Cytosine containing CPDs are quite unstable and they are easily deaminated to uracil. So, the uracil containing CPD are the causative agent for DNA damage.

In Deamination process, an amino group is converted to a keto group in cytosine and adenine. In that case, cytosine is converted to uracil and adenine is converted to hypoxanthine. So this will alter the base-pairing specificities of these two bases during replication. i.e Normally Cytosine pairing with thymine, but after deaminated to uracil, it will now pair with adenine. Then further replication process will replace the uracil to thymine. The same case goes for adenine which gets converted to hypoxanthine, which has affinity for cytosine.

Translesion DNA synthesis (TLS)

Actually, these photoproducts such as CPDs, 64PPs and dewar isomer block the DNA synthesis by preventing the replicative DNA polymerase from passing them when they reside on a template strand during DNA replication. Nucleotide excision repair which is a DNA repair mechanism tries to excise these photolesions, but sometimes failure in the repair before replication fork passing would lead to stall and collapse of the fork at the damaged site. Thus it leads to DNA double strand break and cell death eventually.

Inorder to overcome these conditions, Translesion DNA synthesis process will overcome this replication blocks and save the cell from death. In this process, TLS polymerase enzyme restarts the DNA synthesis been stalled at obstructive, damaged bases on a template strand. It is meant to be error prone and it introduces mutation in the genome at high frequency.

But somehow DNA polymerase (pol) η suppresses efficiently the induction of mutations after UV irradiation by performing an error-free TLS opposite CPDs using the base-pairing ability still remaining for CPDs.

Patient with pol η are prone to this UV radiation and they show high photocarcinogenic sensitivity in skin regions exposed to sunlight. Therefore pol η is consider as a suppressor for CPD mediated mutation. But unintentionally, it will produce the UV signature mutation produced by the deamination process.

Cloning Vector : Vehicle for transport foreign DNA into Host cell.

noreply@blogger.com (R Santhosh Kalash) — Wed, 11 Sep 2013 16:22:00 +0000

A DNA molecule that can carry inserted DNA and can be perpetuated in host cell is called cloning vector.

A Vector for cloning should

a) have origin of replication which makes them replicate inside an appropriate host, even though a foreign DNA is present.

b) have a site for inserting the target gene.

c) have restriction enzyme site for cutting and inserting the target gene.

d) have a selectable marker for identify the cloning containing the targeting gene.

e) be small with an ideal size of less than 10 kb. (large size tends to break during purification process)

There four major types of vectors:

1) Plasmids

2) bacteriophages and other viruses

3) cosmids

4) Artificial chromosomes

(Note is incomplete. Will be updated soon. )

Ti plasmid as vector for plants

noreply@blogger.com (R Santhosh Kalash) — Wed, 11 Sep 2013 12:34:00 +0000

It is circular and not always
It’s contains 56%GC content out of which 81% encodes for a protein.
It has various types of opines

Octopine
Nopaline
Succinamopine
Leucinopine

It is found in Agrobacterium tumefacien and Agrobacterium rhizogenes.
There is no pseudogene.
It works only in dicotyledon plants.
The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent.

Genes in the virulence region are grouped into the operons vir ABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.

virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone, syringealdehyde oracetovanillone which leak out of damaged plant tissues.
virB encodes proteins which produce a pore/pilus-like structure.
virC binds the overdrive sequence.
virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border.
virG activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA.

When Agrobacterium infects plants, a region of the Ti plasmid called the T-DNA is taken up by the plant cell and incorporated into one of its chromosomes.

The genes in the T-DNA are referred to as phyto-oncogenes because they induce neoplastic, or tumor-producing, growth. To use the Ti plasmid as a vector for introducing new genes into plants, it is necessary to disarm the plasmid so that it does not cause tumors. The task is accomplished by deleting the genes in the T-DNA that encode the enzymes controlling auxin and cytokinin synthesis. Genes for antibiotic resistance are normally used for this purpose.

A cloned gene can then be inserted into the T-DNA of the engineered Ti plasmid, and the plasmid can be used to infect cultured cells, leaf discs, or root slices. The infected cells are placed on a culture medium that contains auxin and cytokinin (to induce growth) and the antibiotic. Only the transformed cells can grow in the presence of the antibiotic, because they have received the T-DNA containing not only the foreign gene but also the gene for antibiotic resistance.

To obtain a plant containing the foreign gene, it is necessary to regenerate whole plants from the cultured, transformed cells. Fortunately, methods for accomplishing this regeneration have been developed for many plants, although not for all important crop species yet. The regeneration involves adjusting the ratio of cytokinin to auxin to stimulate both shoot and root formation.

Although the transformation of cereal crops with Agrobacterium and their regeneration from cultured cells has been difficult, some remarkable successes have resulted from this approach. Investigators have introduced numerous foreign genes into plants such as tobacco, including soybean storage protein genes. Genes for such desirable characteristics as disease resistance, herbicide resistance, and salt tolerance have been transferred to crop plants by these techniques, and commercial crops now are being grown with these genetically engineered strains.

Arabidopsis can also be transformed directly by exposing whole plants to a solution containing Agrobacterium that is carrying engineered or wild-type Ti plasmids. The plants must be treated in such a way to allow the Agrobacterium to enter tissue, either by applying a vacuum or by treating with detergents. The Agrobacterium penetrates the floral tissue and transforms the developing ovules. Isolation of seeds from these Agrobacterium-exposed plants yields up to 2% of the seeds that are transformed with the T-DNA. This approach is very useful for molecular genetic studies, such as for characterizing DNA sequences involved in the control of gene expression, or constructing large libraries of insertional mutants.

Gene transfer using Ti plasmids

In the Ti plasmid itself, the T-DNA is flanked by 25 bp imperfect direct repeats known as border sequences, which are conserved between Octopine and nopaline plasmids. The border sequences are not transferred intact to the plant genome, but they are involved in the transfer process. The analysis of junction regions isolated from plant genomic DNA has shown that the integrated T-DNA end-points lie internal to the border sequences.

The right junction is rather precise, but the left junction can vary by about 100 nucleotides. Deletion of the right border repeat abolishes T-DNA transfer, but the left-hand border surprisingly appears to be non-essential. Experiments in which the right border repeat alone has been used have shown that an enhancer, sometimes called the overdrive sequence, located external to the repeat is also required for high-efficiency transfer.

Two of these genes, virA and virG, are constitutively expressed at a low level and control the plant-induced activation of the other vir genes. VirA is a kinase that spans the inner bacterial membrane and acts as the receptor for certain phenolic molecules that are released by wounded plant cells.

Acetosyringone, a phenolic compound has been the most widely used in the laboratory to induce vir gene expression

But acetosyringone do not attract bacteria to wounded plant cells. Rather, the bacteria appear to respond to simple molecules, such as sugars and amino acids, and the vir genes are induced after attachment

Activated VirA transphosphorylates the VirG protein, which is a transcriptional activator of the other vir genes.

The induction of vir gene expression results in the synthesis of proteins that form a conjugative pilus through which the T-DNA is transferred to the plant cell. The components of the pilus are encoded by genes in the virB operon.

DNA transfer itself is initiated by an endonuclease formed by the products of thevirD1 and virD2 genes. This introduces either single-strand nicks or a double-strand break at the 25 bp borders of the T-DNA, a process enhanced by the VirC1 and VirC2 proteins, which recognize and bind to the overdrive enhancer element. The VirD2 protein remains covalently attached to the processed T-DNA.

T strands are coated with VirE2, a single-stranded DNA-binding protein. The whole complex, sometimes dubbed the firecracker complex because of its proposed shape, is then transferred through the pilus and into the plant cell. It has been proposed that the VirD2 protein protects the T-DNA against nucleases, targets the DNA to the plant-cell nucleus and integrates it into the plant genome. The protein has two distinct nuclear localization signals, with the C terminal signal thought to play the major role in targeting the T-DNA.

It has been observed that the nucleus of wounded plant cells often becomes associated with the cytosolic membrane close to the wound site, suggesting that the T-DNA could be transferred directly to the nucleus without extensive exposure to the cytosol.

Plasmid

noreply@blogger.com (R Santhosh Kalash) — Wed, 11 Sep 2013 12:33:00 +0000

1) Self- replicating, double stranded, circular DNA molecule.

2) It’s maintained as an independent extrachromosomal entity in bacteria.

3) There are different types of plasmid that has been present in certain for survival and diversification:

F plasmid à Involves in conjugation for transferring information to another bacteria.

* Its total length may be 100 kb.
* It contains genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation.
* It has an insertion sequences that assist plasmid integration into the host cell chromosome.
(Small Picture of F plasmid.)

R plasmid à resistance to antibiotic.

Degradative plasmid à carry specific genes for the utilization of unusual metabolites.

Cryptic plasmid à no functional coding genes.

Col plasmids à colicin production. Bacteriocin gene produces cloacins which kills bacteriophages.

4) High copy number plasmids (relaxed plasmids) à 10-100 copies of plasmids per host cell.
5) Low copy number plasmids (Stringent plasmids) à 1-4 copies of plasmids per host cell.
6) Host range of plasmid is determined by its ori region. And there are two host range of plasmid i.e. Restricted where the plasmid is restricted to replicate in certain bacteria only (eg: Plasmid Col E1 replicates only in enteric bacteria like E.Coli or salmonella) and Broad host range where the plasmid can replicate any group of bacterium (RP4 plasmid can replicate in most of gram negative bacteria).

7) An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

Plasmid exists in three forms: covalently closed circles (CCC) DNA, open circles (OC) DNA, linear DNA.

Plasmids look like a coiled rubber band and . Topoisomerase, an enzyme that regulates the winding (over or under) of the DNA. It relaxes the supercoiled plasmid and brings CCC form. But if a Endonuclease (Enzyme cleaving the phosphodiester bond of DNA) attacks this uncoiled CCC, then a nick is formed in one of the strands of plasmid DNA. So therefore it forms a open circular DNA. The nick can be cured by another enzyme called DNA ligase which joins both the ends of the DNA strand. This enzyme is one of the important enzyme for joining the fragmented DNA with the plasmid.

Now, DNA gyrase is type of Topoisomerase II which provide negative supercoiling of DNA by cutting the double strands and then joining them together by twisting the ends.

In certain bacteria like streptomyces sp or Borrelia burgdorferi, plasmids are found in linear form. To protect the ends from nuclease digestion, there are two general mechanism involved:
a) terminal contains a hairpin loop structure (Borrelia).
b) terminal are protected by covalent attachment of protein (Streptomyces sp.)

Certain traits exhibited by the plasmid carrying genes

The R plasmid contains antibiotic resistance genes such

The antibiotic resistance genes act as marker for identifying the plasmids containing the gene of interest. For eg: plasmid containing Ampicillin resistant marker will let the host bacteria to grow in a medium containing ampicillin.

Plasmid protects the bacterium for hazardous condition and enable them to survive even in limited resources. They also pass some part or whole gene to another host genome or plasmid through conjugation.

Plasmid in gene cloning

What makes the plasmid a suitable vector for gene cloning?

* Plasmids are easy to isolate, purify and can be reintroduced into a bacterium by transformation.

* They bear antibiotic resistant genes which can be used for selecting the bacterial host during screening process.

* Plasmids with a foreign DNA is said to be chimera.

* They contain origin of replication (ori) region that can allow the plasmid to replicate individually from the bacterial genome.

* plasmids with high copy number, low molecular weight and conjugation property can be produced in more numbers and thus increasing the volume of sample.

pBR322 – First gene cloning vector

1. It contains 4,361 bp and carries two antibiotic resistance genes: Amp^r (Ampicillin) and Tet^r (Tetracycline).

2. Plasmid make up: Within Tet^r (BamH1, HindIII, and SalI RE recognition site), Within Amp^r (PstI site), EcoRI not within any coding DNA, and origin of replication that functions only in E.Coli.

How does this pBR322 plasmid work as cloning vector?

How does the Alkaline phosphatase treated linear plasmid ligate together even with lose of phosphate group at 5' end?

A phosphate group at the 5' end is important for the T4 ligase to join the plasmid and the target DNA. But due to the loss of 5' phosphate group, two nicks are formed at the joining site. Therefore the plasmid with target DNA should become unstable and linear.

Somehow this doesn't happen, because two phosphate group present in the target DNA hold the plasmid together and once they are transformed into the host cell, the two nicks are ligated by the host DNA ligase enzyme.

Other plasmids

Ti plasmids

cDNA Library - A detailed note

noreply@blogger.com (R Santhosh Kalash) — Wed, 11 Sep 2013 12:24:00 +0000

Its a collection of cDNA (Complementary DNA) clones generated from mRNA sequences of a single cell population. cDNA is double stranded DNA complementary to the mRNA sequence synthesized by the enzymes reverse transcriptase and DNA polymerase.

Steps involved in creating cDNA library:

Extraction and purification of mRNA from the cell

CLICK HERE TO READ

Synthesis of cDNA from the total mRNA

CLICK HERE TO READ

cDNA cloning

CLICK HERE TO READ

Screening of the host containing the cDNA

CLICK HERE TO READ

Genomic Library- A detailed note

noreply@blogger.com (R Santhosh Kalash) — Wed, 11 Sep 2013 12:23:00 +0000

It is the collection of total genomic DNA from a single organism. The total genome from an organism is taken and digested into fragments. Then, they are carried into a host organism by a vector DNA and cloned into many copies. Through screening process, the cloned host organism with the gene of interest is taken and preserved for future use.

So, now we will discuss the steps in constructing a genomic library.

1) Isolation and purification of total genomic DNA from the cell (Prokaryote or eukaryote)

CLICK HERE TO READ

2) Restriction digestion of the total genomic DNA using Restriction enzymes.

CLICK HERE TO READ

3) Selection of Vector

CLICK HERE TO READ

4) Insertion of the fragment into the vector using Ligase enzyme.

CLICK HERE TO READ

5) Host Transformation.

CLICK HERE TO READ

6) Selection.

CLICK HERE TO READ

Endocytosis: Import system of the cell

noreply@blogger.com (R Santhosh Kalash) — Sun, 08 Sep 2013 15:38:00 +0000

Endocytosis is the import system of the cell where the macromolecules and particles from the surrounding medium is taken inside the cell. Cells internalize the materials by using plasma membrane components and deliver them to an internal compartment called endosomes. From this endosomes they can be recycled to same or different regions of the plasma membrane or can be delivered to lysosomes for degradation.

There are three types of endosomes in the cell,

Early endosomes

Endosomes which are found just beneath the plasma membrane which consists of the components obtained directly from outside of the cell like carbohydrates, fats etc. The components may go to the Golgi apparatus or may be transferred to the lysosomes via late endosomes. In early endosomes, mild digestion may start, hence many hydrolases are synthesized and delivered there as proenzymes called Zymogens which contain extra inhibitory domains at their N terminus that keep the hydrolases inactive until these domains are proteolytically removed.

Late endosomes

Endosomes which are found close to Golgi apparatus and near the nucleus. These are actually intermediate between early endosomes and lysosomes.

Recycling endosomes

Endosomes which is found near the early endosome will be containing certain membrane proteins or receptor. It will fuse with the plasma membrane when a signaling molecule activates the signaling cascade which inturn activates the endosome to bind to the cell membrane. After finishing their function, they will get back to the cell as vesicle. Best example for this is the GLUT4 proteins which is found inside the cell bounded to the vesicle.

Both the late and early endosomes differ in their protein compositions and transition from early to late endosomes is accompanied by the release of Rab5 and the binding of Rab7.

The acidity inside the late endosomes is higher compared to that of the earlier one. This gradient of acidic environments has a crucial role in the function of these organelles. Endosomes has a vascular H+ ATPase over the membrane which is responsible for making the vesicle acidic by bringing in more amount of H+ ion. The pH basically in early endosomes is 6.

Coated vesicles: They are having a distinctive cage of proteins covering their cytosolic surface. Now before the vesicles fuse with a target membrane, they discard their coat as it is required for the two cytosolic membrane surfaces to interact directly and fuse.

The coat has two functions,

1) It concentrates specific membrane proteins in a specialized patch which then gives rise to the vesicle membrane.

2) The coat molds the forming vesicle.

These proteins are assembles into a curved basketlike lattice that deforms the membrane patch and thereby shapes the vesicle.

Three well characterized types of coated vesicles distinguished by their coat proteins:

Clathrin coated à mediate transport from the endosomal-Golgi compartments and from the plasma membrane.

COPII coated and COPI coated àmediate transport from the ER and Golgi cistern

Clathrin is a coat protein whose subunits consists of three large and three small polypeptide chains that together form a three legged structure called triskelion. These triskelions assemble together into a basketlike convex framework of hexagons and pentagons to form coated pits on the cytosolic surface of membranes.

Adapter protein which is a coat component in Clathrin coated vesicles form a discrete second layer of the coated positioned between the Clathrin cage and the membrane. They bind the Clathrin coat to the membrane and trap various transmembrane proteins which include the transmembrane receptors that capture soluble cargo molecules inside the vesicle so called cargo receptors.

Dynamins: These are the cytoplasmic proteins which helps in regulation of pinch off and uncoating of coated vesicles. The protein consists of PIP2 binding doman which tethers the protein to the membrane and a GTPase domain, which regulates the rate at which the vesicles pinch off from the membrane.

There are three types of Endocytosis:

Phagocytosis
Pinocytosis
Receptor Mediated Endocytosis

Phagocytosis: large particles are ingested via large vesicles called phagosome, which is about > 250 nm in diameter. Phagosome end up in lysosomes and the products of the subsequent digestive processes pass into cytosol to be used as food. Phagocytosis required other than nutrition is carried out by specialized cells called professional phagocytes. In mammals two main classes of professional phagocytes are macrophages and neutrophils. Any indigestible particle inside the phagocytes forms residual bodies which will be expelled out of the cell through Exocytosis.

It’s a triggered process which required the activation of receptors that transmit signals to the cell interior and initiate the response. Eg: Antibodies triggers phagocytosis by exposing the tail Fc region to the phagocytic cells.

Localized actin polymerization initiated by Rho family GTPases and their activating Rho-GEFs shapes the pseudopods. An active Rho GTPase switches on the kinase activity of local PI kinases and initial actin polymerization occurs in response to an accumulation of PIP2. To seal off the phagosome and complete its engulfment, actin is depolymerized at its base as PIP2 is subject to a PIP3 kinase which converts it into PIP3 is required for closure of the phagosome and may also contribute to reshaping the actin network to help drive the invagination of the forming phagosome.

Don’t eat me signal: the cell surface protein binds to the inhibitory receptor of the phagocytic cell and recruits tyrosine phosphatase proteins which antagonize the intracellular signals required for the phagocytosis.

Eat me signal: Phosphatidyl serine present inside the cell membrane will get exposed to the extracellular region by the enzyme Flipase. The exposure will lead to the initiation of eat me signal and macrophage will phagocytose the whole cell.

Pinocytosis: Fluid and solutes are ingested via small pinocytic vesicles which are about 100 nm in diameter. This process is a constitutive process where it occurs continuously regardless of the needs of the cell. The pinocytic vesicles are formed from the coated pits of the plasma membrane. The pits are mostly coated by Clathrin and it will be pinched off from the membrane by dynamins.

Not all the pinocytic vesicle is coated with Clathrin, but there are other vesicles which are not understood very well and they are caveolae. They are recognized by their ability to transport molecules across endothelial cells which form the inner lining of blood vessels. They are thought to form from membrane microdomains or lipid rafts. The major structural proteins in caveolae are caveolins which are integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane. The caveolins don’t not dissociate like Clathrin do because these proteins are integral proteins hence they stick with the vesicle surface even after they are pinched off. Instead they are delivered to the target compartments where they are maintained as discrete membrane domains.

Receptor mediated endocytosis:

The macromolecules bind to complementary transmembrane receptor proteins, accumulate in coated pits and then enter the cell as receptor macromolecules complexes in clathrin coated vesicles. This process is called receptor mediated endocytosis. This happens basically because the ligands are selectively captured by receptors, receptor mediated endocytosis provides a selective concentrating mechanism that increases the efficacy of internalization of particular ligands more than a hundred fold. The LDL receptor goes to the early endosome and gives off the cholestrol molecules. Then retrives back to the plasma membrane without any coating.

Short Note on Diabetes Mellitus

noreply@blogger.com (R Santhosh Kalash) — Sun, 08 Sep 2013 14:34:00 +0000

Diabetes Mellitus is a metabolic disease in which the patient blood glucose level goes higher due to two possible reasons:

Pancreatic cells called beta cells stops producing enough Insulin,
Cells are not responding to the hormone insulin.

Insulin is a peptide hormone produced by the beta cells of the pancreases, which regulates the metabolism of carbohydrate and fat in the body. It helps the cells except brain cells to uptake the blood glucose through the glucose transporter. Insulin activates the insulin receptor, which in turn actives the glucose transporter.

Based on the insulin failure, there are three main types of Diabetes mellitus:

Type I Diabetes Mellitus (Insulin Dependent Diabetes Mellitus)

The patient’s body fails to produce insulin because the beta cells of the islets of langerhans are destroyed by the self immune cells like T cells or genetic defects in beta cells function. Therefore it is considered as autoimmunity disease.

Type II Diabetes Mellitus (Non Insulin Dependent Diabetes Mellitus)

The patient’s body fails to accept the insulin produced by the beta cells of the islets of langerhans, because the target cells lose their sensitivity, which often referred as insulin resistance.

Blood Glucose Profile:

The normal blood glucose level before and after the meal is 70 -110 mg/dL and 140 mg/dL.

In Type I diabetes: after the meal the level increases to 162 mg/dL.

In Type II diabetes: after the meal the level increases to 153 mg/dL.

Apart from increase in blood glucose level, there is also increase utilization of fats for energy and for formation of cholesterol by the liver and also depletion in body’s proteins.

Ketone bodies à Ketone bodies produced from acetyl CoA, which is excessively formed by the breakdown of fatty acids by β oxidation for energy production. Since NADH levels increases, Citric acid cycle shuts down and therefore acetyl CoA cant enter the cycle. But they take another route and form three endogenous ketone bodies: acetone, acetoacetic acid, and beta-hydroxypentanoate. In normal condition, their level in blood is maintained at 1 mg/dL. If it increase beyond this, then it leads to ketosis (Ketonemia and Ketouria).

Ketosis or ketoacidosis leads to damage of organs as well as lowers the pH in blood.

The lipid profile of a normal person is

Total Cholesterol à 150-250 mg/dL

LDL: 130 mg/dL

HDL: For men: 30-60 mg/dL; For women: 40-70 mg/dL

TG: 10-190 mg/dL

LDL (bad cholesterol) is elevated in the blood of diabetic patient, but HDL (good cholesterol) is found less compared to the normal. Elevated LDL causes atherosclerosis, and leads to blockage of blood vessels to heart. Therefore, diabetic patients are more prone to heart attack.

High level of glucose in the bloods shows three basic symptoms in the diabetic patient:

Polyuria à Frequent urination

Polydipsia à Increased thirst

Polyphagia à Increased hunger

What is the reason behind the occurrence of these symptoms in diabetic patient?

Polyuria and polydipsia: Glucose does not diffuse easily through the pores of the cell membrane and the increase osmotic pressure in the extracellular fluids causes osmotic transfer of water out of the cells. And also due to excess of glucose in blood, it leads to lose of glucose in the urine. Therefore even the presence of glucose in the renal tubules greatly decreases the tubular readsorption of fluid.

So overall there is excessive loss of fluid in urine causing frequent urination and dehydration which eventually increases the thirst also.

Polyphagia: Due to the failure of glucose utilization, the body starts to increase the utilization of protein and fats for energy. Therefore rapid weight loss and increase in apatite leads to polyphagia.

Effect of Environment on Growth Kinetics- Temperature

noreply@blogger.com (R Santhosh Kalash) — Wed, 04 Sep 2013 18:44:00 +0000

Growth kinetics can be affected by three main environmental factors:

Temperature
pH
Dissolved oxygen concentration

Temperature

According to temperature optima (T_opt), organisms are classified into three types:

Psychrophiles (T_opt< 20°C)
Mesophiles (T_opt50°C-80°C)
Thermophiles (T_opt> 50°C)

Figure 1: Schematic representation of effect of Temperature in growth

Now, As the temperature increase towards this optimal growth temperature, the growth rate actually doubles for every 10 degree Celsius.

Please look into the figure above. Consider the Car is the cell and as it moves towards the optimal growth temperature, its growth rate i.e µg doubles for every 10 degree Celsius increase in temperature.

but, beyond this optimal temperature, µg drops slowly and finally thermal death occurs. So What could be the net replication rate for temperature above optimal level?

First the net replication rate in the absence of cell death i.e kd is said to be

N (µ’R) = dN/dt

Now thermal death occurs above the optimal growth temperature and that could be explained by another equation. i.e

N (-k'd) =dN/dt

Combining both these equation, we get

dN/dt = (µ’R - k'd) N

So, at higher temperature the thermal death rate exceeds the growth rate, which causes a net decrease in the concentration of viable cells.

Both the net replication rate constant and death rate constant vary with temperature which is according to Arrhenius Equation.

µ’R = Ae^(-Ea/RT)
k’d = A'e^(-Ed/RT)

Ea and Ed are the activation energies for growth and thermal death.
For growth Ea is 10-20 kcal/mol and thermal death Ed is 60-80 kcal/mol. So, Thermal death is more sensitive to temperature changes than microbial growth.

Temperature affects four things:

Why maintenance coefficient increase with increase in temperature?

As the temperature increases, the proteins and cell organelles starts to ware out and in order to replace the losses, the cell utilizes more of the substrate.

Maintenance Coefficient is the specific rate of substrate uptake for cellular maintenance. And increase in 'm' happens with activation energy range between 15-20 kcal/mol, which results in decrease in the yield coefficient.

Complement system

noreply@blogger.com (R Santhosh Kalash) — Wed, 04 Sep 2013 17:49:00 +0000

Activities of complement system

Lysis, opsonisation, activation of inflammatory response, clearance of immune complexes. They are produced by liver hepatocytes and small amount by blood monocytes, macrophage, and epithelial cells. Zymogens are inactive form of complement proteins and they are activated by proteolytic cleavage which removes the inhibitory fragment and exposes the active site of molecule.

There are three pathways of complement activity

Classical pathway
Alternative pathway
Lectin pathway

The common among all these pathways is the formation of MAC (membrane attacking complex).

1. Classical pathway

The activation starts with formation of soluble antigen antibody complexes or with the binding of antibody to antigen on a suitable target. IgM and IgG activate the complement pathway and initial stage of activation involves components C1, C2, C3, and C4 which are present in plasma in functionally inactive forms.

Fc portion of IgM conformational change on binding of antigen. Exposure site on IgM for C1 complement protein to bind. C1 consists of C1qr₂s₂ held together in a complex stabilized by calcium ions.

C1q molecule à 18 polypeptide chains that associate to form six collagen like triple helical arms, tips of which bind to exposed C1q binding sites in C_H2 domain of the antibody molecule.

Each C1r and C1s monomer contains a catalytic domain and an interaction domain, the latter facilitates interaction with C1q or with each other.

After the C1q is bound to Fc binding sites, a conformational changes induce C1r to become a active serine protease enzyme C1r* which then cleaves C1s to a similar active enzyme C1s*.

C1s* à two substrates C4 and C2. The C4 is glycoprotein contains three polypeptide chains α, β, and γ.

C4 is activated when C1s* hydrolyzes a small fragment C4a from the amino terminus of α chain, exposing a binding site on the larger fragment (C4b).

The C4b fragment attaches to the target surface in the vicinity of C1 and the C2 proenzyme then attaches to the exposed binding site on C4b, where the C2 is then cleaved by the neighbouring C1s*, the small fragment C2b diffuses away.

The resulting C4b2a* complex is called C3 convertase.

C3 convertase converts C3 into active form. C4a is an anaphylatoxin or mediator of inflammation which does not participate directly in the lytic function of the complement cascade. Similarly C3a and C5a is also an anaphylatoxin.

The C3 component consists of two polypeptide chains α and β. Hydrolysis of a short fragment C3a from the amino terminus of α chain by the C3 convertase generates C3b.

Some C3b component binds to C4b2a* complex to form a trimolecular complex called C5 convertase (C4b2a3b*).

C3b component of this complex binds C5 and alters its conformation allowing C4b2a* component to cleave C5 into C5a, which diffuses away and C5b which attaches to C6 and initiates formation of the membrane attack complex.

C3b sometimes function as opsonin and promote phagocytosis. C3b may also bind directly to cell membranes.

2. Alternative pathway

It happens similar to the classical pathway, but it does without the antigen antibody complexes for initiation. Since, it is a component of innate immunity.

It involves four serum proteins C3, factor B, factor D and properdin.

Serum C3 gets cleaved into C3a and C3b on the microbial cell surface.

The C3b bind to either foreign surface antigens or to the host cell. But on the host cell is not affected by C3b binding because host cell surface is having high sialic acid which contributes to rapid inactivation of bound C3b molecules on host cells.

Then another serum protein called factor B forms complex with C3b and it is stabilized by Mg²⁺.

Now this complex serve as a substrate for factor D, which convert them into C3bBb* complex.

The complex is having C3 convertase activity which is limited half life unless the serum protein properdin binds to it.

Now the C3 convertase cleaves the C3 into C3b and C3a. C5 convertase is formed by the binding of C3b to the C3 convertase (C3bBbC3b). C5 convertase generates C5a and C5b. C5b is used for MAC.

3. Lectin pathway

Lectins are proteins that recognize and bind to specific carbohydrate targets. Lectin activates complements binds to mannose residues.

Lectin pathway is activated by the binding of mannose binding lectin to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms, including bacteria such as salmonella, listeria and neisseria strains.

MBL is a collectin family acute phase proteins and its concentration increases during inflammatory responses. Its function in the complement pathway is similar to that of C1q, which it resembles in structure.

After MBL binds to the carbohydrates residues on the surface of cell or pathogen, MBL associated serine proteases, MASP-1 and MASP-2 bind to MBL.

Now the active complex formed by this association causes cleavage and activation of C4 and C2. MASP-1 and MASP-2 are structurally similar to C1r and C1s and mimic their activity.

MAC formation

C5b, C6, C7, C8, and C9 which interact sequentially to form a macromolecular structure called the membrane attack complex (MAC). It forms a large channel on the membrane of the target cell enabling ions and small molecules to diffuse freely across the membrane.

C3b and C4b binding facilitates opsonisation.

Complement system neutralize viral infectivity.

Regulation of complement system

C1 inhibitor à form complex with C1r2s2 causing it to dissociate from C1q and preventing further activation of C4 or C2.

C3b is hydrolysed once it moves away at 40 nm distance from the C4b2a* or C3bBb* convertase enzymes.

C3 convertase activity is regulated by proteins which contain repeating amino sequences of about 60 residues termed short consensus repeats (SCRs). All this proteins are encoded in single location on chromosome 1 in humans, known as the regulators of complement activation (RCA) gene cluster.

RCA proteins prevent C3 convertase assembly and it includes proteins like C4b binding protein (C4bBP), and two membrane bound proteins, complement receptor type 1 (CR1) and membrane cofactor protein (MCP).

Each of these regulatory proteins binds to C4b and prevents its association with C2a. Once the regulatory proteins binds to C4b, another regulatory protein factor I cleaves the C4b into bound C4d and soluble C4c.

In other case, MCP, CR1 and Factor H binds to C3b and prevents it association with factor B. After this, Factor I cleaves C3b into a bound iC3b and release C3c fragment.

Decay accelerating factor (DAF or CD55) which is a glycoprotein covalently anchored to a glycophospholipid membrane protein has the ability to dissociate C3 convertase.

Homologous restriction factor (HRF) or membrane inhibitor of reactive lysis (MIRL or CD59). CD59 protects cells from nonspecific complement mediated lysis by binding to C8 preventing assembly of poly C9 and its insertion into plasma membrane.

Immune organs: Primary and Secondary Lymphoid organs

noreply@blogger.com (R Santhosh Kalash) — Wed, 04 Sep 2013 17:35:00 +0000

Primary lymphoid organs à maturation of lymphocytes takes places.

Thymus

It is the site for T cell development and maturation.

Bilobed organ situated above the heart. Each lobe is surrounded by a capsule and divided into lobules, which are separated from each other by strands of connective tissue called trabeculae. Each lobule is organized into two compartments: cortex which is densely packed with immature T cells called Thymocytes whereas the inner compartment or medulla is sparsely populated with thymocytes.

Both the compartments consist of epithelial cells, dendritic cells, and macrophages which make up the framework of the organ and contribute to the growth and maturation of thymocytes.

Function of thymus: to generate and select a repertoire of T cells that will protect the body from infection. As thymocytes develop, enormous diversity of T cell receptors is generated by gene rearrangement.

It produces some T cells with receptors capable of recognizing antigen MHC complexes. But only small portion of cells survive and rest will undergo apoptosis.

Bone marrow

It is a complex tissue that is the site of haematopoiesis and a fat depot. Hematopoietic cells are generated here move through the walls of blood vessels and enter the bloodstream, which carries them out of the marrow and distributes these various cell types to the rest of the body.

It is site of B cell origin and development. Immature B cells proliferate and differentiated within the Bone marrow and stromal cells within the bone marrow interact directly with the B cells and secrete various cytokines that are required for development.

Secondary lymphoid organs à site for mature lymphocytes to interact with antigen.

Lymphatic system

lymphoid follicles: primary follicle à network of follicular dendritic cells and small resting B cells. After antigenic challenge, a primary follicle becomes secondary follicle à a ring of concentrically packed B lymphocytes surrounding a center (the germinal center) in which one finds a focus of proliferating B lymphocytes and an area that contains nondividing B cells and some helper T cells interspersed with macrophages and follicular dendritic cells.

Lymph nodes and spleen à surrounded by fibrous capsules.

MALT à less organized lymphoid tissue found in various body sites.

Lymph node:

Encapsulated bean shaped structures containing a reticular network packed with lymphocytes, macrophages and dendritic cells. They are organised in such a way to encounter antigens that enter the tissue spaces.

Cortex à outermost layer contains B cells mostly, macrophages and follicular dendritic cells arranged in primary follicles. After antigenic challenge, the primary follicles enlarge into secondary follicles, each containing germinal center.

Paracortex à it is beneath cortex which is populated largely by T cells and also contains dendritic cells that migrated from tissue to the node.

Medulla à more sparsely populated with lymphoid lineage cells, and of those present, many are plasma cells actively secreting antibody molecules.

Lymph nodes traps the antigens from local tissues.

Spleen:

Present in the high left abdominal cavity. It is large ovoid, plays a major role in mounting immune responses to antigens in the blood stream. Spleen filters the blood and traps the blood borne antigens. So it responds to systemic infections. It is not supplied by lymphatic vessels, instead antigens and cells are carried into spleen through splenic artery. It is surrounded by capsules from which a number of projections extend into the interior to form a compartmentalized structure.

Two compartments:

Red pulp: network of sinusoids populated by macrophages, numerous red blood cells, and few lymphocytes. Defective and old RBCs are destroyed and removed.

White pulp: it surrounds a branches of splenic artery forming a periarteriolar lymphoid sheath (PALS) populated mainly by T cells. Primary lymphoid follicles are attached to PALS.

Marginal zone contains lymphocytes and macrophages. Antigens are trapped in marginal zone by dendritic cells, which then carry it to the PALS. And also lymphocytes enter through the same route as of the antigen.

MALT (mucosa associated lymphoid tissue)

The vulnerable membrane surface of digestive, respiratory and urogenital systems are defended by a group of organized lymphoid tissues called MALT.

Respiratory epithelium à bronchus associated lymphoid tissue (BALT)

Epithelium of the digestive tract à Gut- associated lymphoid tissue (GALT)

MALT includes tonsils, and appendix.

M cells carry the antigen from lumina of the tracts to the underlaying MALT. They are flattened epithelial cells lacking the microvilli that characterize the rest of the mucous epithelium. They have deep invagination, or pocket in the basolateral, plasma membrane which is filled with a cluster of B cells, T cells and Macrophages.

NOD and Toll Like Receptor Protein

noreply@blogger.com (R Santhosh Kalash) — Wed, 04 Sep 2013 17:25:00 +0000

NOD proteins

Cytosolic and has two members in this family i.e. NOD1 and NOD2, which recognize products derived from bacterial peptidoglycans.

NOD1 binds to the tripeptide products of peptidoglycans (NAM and NAG) breakdown and NOD2 recognizes muramyl dipeptide, derived from the degradation of peptidoglycan from gram positive bacterial cell walls.

NOD1 recognizes meso-diaminopimellic acid (meso-DAP) in Gram –ve bacteria and NOD2 senses intracellular muramyl dipeptide (MDP) in S. Pneumoniae and M.tuberculosis.

Mutation of NOD2 is associated with crohn’s disease or Blau syndrome.

Toll Like Receptor proteins (TLR)

Membrane spanning proteins that share a common structural element in their extracellular region, repeating segments of 24 to 29 amino acids containing the sequence xLxxLxLxx. These structural motifs are called leucine rich repeats. The intracellular domain of TLR is called TIR domain.

TLR1 à ligand is Triacyl lipopeptides and a target microbe is mycobacteria.

TLR2 à ligand is peptidoglycans, GPI linked proteins, lipoproteins and zymosan. Target molecules are gram positive bacteria, trypanosomes, mycobacteria, yeasts and other fungi.

TLR3 à double stranded RNA (dsRNA) and target microbes are viruses.

TLR4 àLPS and F-protein and target microbes are Gram –ve and respiratory syncytial virus (RSV)

TLR5 à flagellin and target is bacteria.

TLR6 à diacyl lipopeptides and zymosan. Mycobacteria, yeast and fungi.

TLR7 à single stranded RNA (ssRNA) and viruses.

TLR8 à single stranded RNA (ssRNA) and viruses.

TLR9 à CpG unmethylated dinucleotides, dinucleotides, herpesvirus infection. Bacterial DNA and some herpesvirus

TLR10 and TLR11 à unknown

Innate immunity

noreply@blogger.com (R Santhosh Kalash) — Wed, 04 Sep 2013 17:18:00 +0000

Its a non specific immunity.

It’s not long lasting defense system like adaptive immunity, but provides immediate defense against infection.

First line of defense against foreign organisms

Cells involved in innate immunity are mast cells, phagocytic cells like macrophages, neutrophiles, dendritic cells, basophiles and eosinophiles, natural killer cells, γδ T cells.

Barriers which prevent the entry or growth of microorganisms inside the body: Skin, Phagocytic cells, Antibacterial proteins such as α defensin, Psoriasin.

Psoriasin is a small protein with potent antibacterial activity against E.Coli.

Anatomical barriers are skin which defense against the microorganism through the sweat, desquamation, flushing and organic acids. GI tract, which has defensin, gut flora, thiocynate, bile acids, gastric juice, and peristalsis. Airway and lungs has surfactant, mucociliary elevator. Eyes have tears. Nasopharynx has mucus, saliva, lysozyme.

C- Reactive protein belongs to family of pentameric proteins called pentraxins, which bind ligands in a calcium dependent reaction.

BPI (bactericidal/permeability-increasing protein) is 55 kDa proteins that bind with high affinity to LPS in the walls of gram negative bacteria and cause damage to the bacterium’s inner membrane.

iNOS (inducible nitric oxide synthetase), an enzyme that oxidizes L- arginine to yield L- citrulline and Nitric oxide (NO).

ROS species production

Oxygen O2 à .O₂^- à H₂O₂ à HClO^-

Oxygen is converted to superoxide anion by NADPH phagosome oxidase, and then Superoxide anion is converted to hydrogen peroxide by superoxide dismutase. Then with addition of chlorine ion, hydrogen peroxide is converted to hypochlorite by myeloperoxidase.

Natural killer cells produce interferon gamma and TNF alpha. These cytokines stimulate the maturation of dendritic cells. INFG is a mediator of macrophage activation and regulator of T helper cells development.

How Selenocysteine is incorporated into polypeptide chain during translation?

noreply@blogger.com (R Santhosh Kalash) — Tue, 03 Sep 2013 18:53:00 +0000

No free pool of Selenocysteine exists in the cell.

Its high reactivity would incur damage to cells. Instead, cells store selenium in the less reactive selenide form (H₂Se).

Selenocysteine synthesis occurs on a specialized tRNA, which also functions to incorporate it into nascent polypeptides.

The primary and secondary structure of Selenocysteine tRNA, tRNA(Sec), differ from those of standard tRNAs in several respects, most notably in having an 8-base (bacteria) or 10-base (eukaryotes) pair acceptor stem, a long variable region arm, and substitutions at several well-conserved base positions.

The Selenocysteine tRNAs are initially charged with serine by seryl-tRNA ligase, but the resulting Ser-tRNA(Sec) is not used for translation because it is not recognized by the normal translation factor (EF-Tu in bacteria, eEF1A in eukaryotes). Rather, the tRNA-bound seryl residue is converted to a selenocysteine-residue by the pyridoxal phosphate-containing enzyme selenocysteine synthase.

Finally, the resulting Sec-tRNA(Sec) is specifically bound to an alternative translational elongation factor (SelB or mSelB (a.k.a. eEFSec)), which delivers it in a targeted manner to the ribosomes translating mRNAs for selenoproteins.

The specificity of this delivery mechanism is brought about by the presence of an extra protein domain (in bacteria, SelB) or an extra subunit (SBP2 for eukaryotic mSelB/eEFSec) which bind to the corresponding RNA secondary structures formed by the SECIS elements in selenoprotein mRNAs.

There are more than 20 human proteins that contain Selenocysteine.

where does Selenocysteine comes from?

Selenocysteine is not coded for directly in the genetic code. Instead, it is encoded in a special way by a UGA codon, which is normally a stop codon. When cells are grown in the absence of selenium, translation of selenoproteins terminates at the UGA codon, resulting in a truncated, nonfunctional enzyme.

The UGA codon is made to encode Selenocysteine by the presence of a SECIS element (Selenocysteine Insertion Sequence) in the mRNA. The SECIS element is defined by characteristic nucleotide sequences and secondary structure base-pairing patterns. In bacteria, the SECIS element is located immediately following the UGA codon within the reading frame for the selenoproteins. In archaea and in eukaryotes, the SECIS element is in the 3' untranslated region (3' UTR) of the mRNA, and can direct multiple UGA codons to encode Selenocysteine residues.

Inhibitors of DNA Replication in Eukaryotic cells

noreply@blogger.com (R Santhosh Kalash) — Tue, 03 Sep 2013 18:44:00 +0000

DOXORUBICIN

The exact mechanism of action of doxorubicin is complex and still somewhat unclear, though it is thought to interact with DNA by intercalation. Doxorubicin is known to interact with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.

The planar aromatic chromophore portion of the molecule intercalates between two base pairs of the DNA, while the six-membered daunosamine sugar sits in the minor groove and interacts with flanking base pairs immediately adjacent to the intercalation site, as evidenced by several crystal structures.

Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA.

ETOPOSIDE

Etoposide phosphate (brand names: Eposin, Etopophos, Vepesid, VP-16) is an anti-cancer agent. It inhibits the enzyme topoisomerase II, which unwinds DNA, and by doing so causes DNA strands to break.

Etoposide forms a ternary complex with DNA and the topoisomerase II enzyme, preventing re-ligation of the DNA strands. This causes errors in DNA synthesis and promotes apoptosis of the cancer cell.

CAMPTOTHECIN

Camptothecin (CPT)cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I. CPT binds to the topo I and DNA complex (the covalent complex) resulting in a ternary complex, and thereby stabilizing it. This prevents DNA re-ligation and therefore causes DNA damage which results in apoptosis. CPT binds both to the enzyme and DNA with hydrogen bonds.

Toxicity of CPT is primarily a result of conversion of single-strand breaks into double-strand breaks during the S-phase when the replication fork collides with the cleavage complexes formed by DNA and CPT

RIFAMPICIN

Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, thus preventing transcription to RNA and subsequent translation to proteins. Its lipophilic nature makes it a good candidate to treat the meningitis form of tuberculosis, which requires distribution to the central nervous system and penetration through the blood-brain barrier.

Rifampicin acts directly on messenger RNA synthesis. It inhibits only prokaryotic DNA-primed RNA polymerase, especially those that are Gram-stain-positive and Mycobacterium tuberculosis. Much of this acid-fast positive bacteria's membrane is mycolic acid complexed with peptidoglycan, which allows easy movement of the drug into the cell. Evidence shows that in vitro DNA treated with concentrations 5000 times higher than normal dosage remained unaffected; in vivo eukaryotic specimens' RNA and DNA polymerases suffered few problems as well. Rifampicin interacts with the β subunit of RNA polymerase when it is in an α2β trimer. This halts mRNA transcription, therefore preventing translation of polypeptides. It should be made clear, however, that it cannot stop the elongation of mRNA once binding to the template-strand of DNA has been initiated. The Rifampicin-RNA polymerase complex is extremely stable and yet experiments have shown that this is not due to any form of covalent linkage. It is hypothesized that hydrogen bonds and π-π bond interactions between naphthoquinone and the aromatic amino acids are the major stabilizers, though this requires the oxidation of naphthohydroquinone which is found most commonly in Rifampicin. It is this last hypothesis that explains the explosion of multi-drug-resistant bacteria: mutations in therpoB gene that replace phenylalanine, tryptophan, and tyrosine with non-aromatic amino acids result in poor bonding between Rifampicin and the RNA polymerase.

Well due to blocking in RNA transcription, DNA initiation is also not possible since the primer synthesis is also blocked by the drug.

APHIDICOLIN

Aphidicolin is defined as a tetracyclic diterpene antibiotic with antiviral and antimitotical properties. Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNA replication. It blocks the cell cycle at early S phase. It is a specific inhibitor of DNA polymerase A, D in eukaryotic cells and in some viruses and an apoptosis inducer in HeLa cells.

NOVOBIOCIN

The molecular basis of action of novobiocin, and other related drugs clorobiocin and coumermycin A1 has been examined. Aminocoumarins are very potent inhibitors of bacterial DNA gyrase and work by targeting the GyrB subunit of the enzyme involved in energy transduction. Novobiocin as well as the other aminocoumarin antibiotics act as competitive inhibitors of the ATPase reaction catalysed by GyrB. The potency of novobiocin is considerably higher than that of the fluoroquinolones that also target DNA gyrase, but at a different site on the enzyme. The GyrA subunit is involved in the DNA nicking and ligation activity.

CIPROFLOXACIN

Ciprofloxacin is a broad-spectrum antibiotic active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby inhibiting cell division.

Actinomycin -binding between adjacent G-C bases in DNA (intercalation
chloramphenicol- inhibits peptidyltransferase of the 70S ribosome
erythromycin - binds to the 50S particle and arrests synthesis of the 70S ribosome
neomycin- binds to the 30S ribosomal subunits and inhibits binding of a tRNA
puromycin- premature chain termination
Rifamycin- inhibits RNA synthesis by binding to the β subunit of the RNA polymerase holoenzyme
streptomycin as erythromycin
tetracyclin -inhibits binding of tRNA to the 30S ribosomal subunit in eukaryotes
α-amanitin - inhibits polymerase II
chloramphenicol - inhibits peptidyltransferase of the mitochondrial ribosome
cycloheximide -inhibits peptidyltransferase
diptheria toxin - inhibits factor 2 and translocation

Antimicrobial agents and mode of action

noreply@blogger.com (R Santhosh Kalash) — Tue, 03 Sep 2013 18:37:00 +0000

Antimicrobial agents are chemical substance that kills or inhibit the growth of microorganism such as bacteria, fungus and yeast. Most of this agents are found in the nature and some are artificial synthesized. They are used for sterilization of instruments, cultures or even wounds.

Alexander Fleming is the first person to discover a natural antimicrobial agent i.e penicillin. After that vast number of agents are discovered or synthesized in the lab.

Chemical class	Examples	Biological source	Spectrum (effective against)	Mode of action
Beta-lactams (penicillins and cephalosporins)	Penicillin G, Cephalothin	Penicillium notatum and Cephalosporium species	Gram-positive bacteria	Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly
Semisynthetic beta-lactams	Ampicillin, Amoxicillin		Gram-positive and Gram-negative bacteria	Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly
Clavulanic Acid	Augmentin is clavulanic acid plus Amoxicillin	Streptomyces clavuligerus	Gram-positive and Gram-negative bacteria	Inhibitor of bacterial beta-lactamases
Monobactams	Aztreonam	Chromobacterium violaceum	Gram-positive and Gram-negative bacteria	Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly
Carboxypenems	Imipenem	Streptomyces cattleya	Gram-positive and Gram-negative bacteria	Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly
Aminoglycosides	Streptomycin	Streptomyces griseus	Gram-positive and Gram-negative bacteria	Inhibits translation (protein synthesis)
	Gentamicin	Micromonosporaspecies	Gram-positive and Gram-negative bacteria esp.Pseudomonas	Inhibits translation (protein synthesis)
Glycopeptides	Vancomycin	Amycolatopsis orientalisNocardia orientalis(formerly designated)	Gram-positive bacteria, esp.Staphylococcus aureus	Inhibits steps in murein (peptidoglycan) biosynthesis and assembly
Lincomycins	Clindamycin	Streptomyces lincolnensis	Gram-positive and Gram-negative bacteria esp. anaerobicBacteroides	Inhibits translation (protein synthesis)
Macrolides	Erythromycin Azithromycin	Streptomyces erythreus	Gram-positive bacteria, Gram-negative bacteria not enterics, Neisseria, Legionella, Mycoplasma	Inhibit translation (protein synthesis)
Polypeptides	Polymyxin	Bacillus polymyxa	Gram-negative bacteria	Damages cytoplasmic membranes
	Bacitracin	Bacillus subtilis	Gram-positive bacteria	Inhibits steps in murein (peptidoglycan) biosynthesis and assembly
Polyenes	Amphotericin	Streptomyces nodosus	Fungi (Histoplasma)	Inactivate membranes containing sterols
	Nystatin	Streptomyces noursei	Fungi (Candida)	Inactivate membranes containing sterols
Rifamycins	Rifampicin	Streptomyces mediterranei	Gram-positive and Gram-negative bacteria,Mycobacterium tuberculosis	Inhibits transcription (bacterial RNA polymerase)
Tetracyclines	Tetracycline	Streptomycesspecies	Gram-positive and Gram-negative bacteria, Rickettsias	Inhibit translation (protein synthesis)
Semisynthetic tetracycline	Doxycycline		Gram-positive and Gram-negative bacteria, RickettsiasEhrlichia,Borrelia	Inhibit translation (protein synthesis)
Chloramphenicol	Chloramphenicol	Streptomyces venezuelae	Gram-positive and Gram-negative bacteria	Inhibits translation (protein synthesis)
Quinolones	Nalidixic acid	synthetic	Mainly Gram-negative bacteria	Inhibits DNA replication
Fluoroquinolones	Ciprofloxacin	synthetic	Gram-negative and some Gram-positive bacteria (Bacillus anthracis)	Inhibits DNA replication
Growth factor analogs	Sulfanilamide, Gantrisin, Trimethoprim	synthetic	Gram-positive and Gram-negative bacteria	Inhibits folic acid metabolism (anti-folate)
	Isoniazid (INH)	synthetic	Mycobacterium tuberculosis	Inhibits mycolic acid synthesis; analog of pyridoxine (Vit B6)
	para-aminosalicylic acid (PAS)	synthetic	Mycobacterium tuberculosis	Anti-folate

Growth Kinetics - Introduction

noreply@blogger.com (R Santhosh Kalash) — Tue, 03 Sep 2013 17:18:00 +0000

Growth kinetics of Bacteria is studied by culturing a bacteria of our interest in a liquid medium with limited amount of nutrients. By using Spectrometer, the Optical density of the culture is measured and a curve is drawn by keeping O.D in Y axis and time (hrs) in X axis. Growth kinetics provide insight about the conditions in which the bacteria grows and also at what stage the bacteria secrets primary or secondary metabolite in the media or within the cell. Growth kinetics is depend on the substrate concentration, product concentration, Environmental factors such as temperature, pH and oxygen uptake.

Industrial use of microorganism

noreply@blogger.com (R Santhosh Kalash) — Sat, 31 Aug 2013 19:07:00 +0000

Microorganisms play a vital role in the production of many food stuffs, cosmetics, pharmaceutical and construction materials etc. In order to increase the growth efficiency and product yield, some of the microorganisms are genetically modified. The use of microorganism reduce the slaughtering of animals for the production of enzymes, hormones and other biological fluids.

Below is the list of microorganism that revolutionize the industrial world.

S.No	Microorganism	Uses in industries
1.	E.Coli	Ethanol, creatininase, 1, 3-propanediol Production.
2.	Penicillium chrysogenum	Cephalosporin precursor, penicillin
3.	Saccharomyces cerevisiae	Lactic acid, xylitol production,
		pediocin, recombinant albumin,
		heterologous protein, ethanol
4.	Clostridium acetobutylicum	Acetone and butanol
5.	Streptomyces coelicolor	Actinorhodin
6.	Clostridium gene in bacillus	Cellulose
7.	Kluyveromyces fragilis	Ethanol from lactose
8.	Enterobacter, Serratia	2,3 butanediol
9.	Gibberella fujikuroi	Gibberellins
10.	Corynebacterium glutamicum	Amino acids eg., lysine, nucleotides
11.	Aspergillus niger	Organic acids, citric acid, gluconic acid
12.	Ashbya, Eremothecium, Blakeslae	Vitamins
13.	Xanthomonas	Polysaccharides
14.	Claviceps purpurea	Alkaloids
15.	Rhizopus, arthrobacter	Steroid transformation
16.	Methanobacterium	Methane
17.	Rhizopus nigricans	Fumaric acid
18.	Aspergillus terreus	Itaconic acid, statinslovastatin
19.	Aspergillus flavus- oryzae	Kojic acid
20.	Lactobacillus delbrueckii	Lactic acid
21.	Streptomyces cinnamonensis	PolyetherMonensin
22.	S. lasaliensis	PolyetherLasalocid
23.	S. albus	PolyetherSalinomycin
24.	S. avermilitis	Avermectins
25.	Penicillium citrinum	Pravastatin
26.	S. clavaligerus	Clavulanic acid
27.	Actinoplanes sp.	Acarbose
28.	S. hygroscopicus	Bialaphos
29.	Tolypocladium inflatum	Cyclosporin A
30.	S. tsukabaensis	FK-506
31.	S. hygroscopicus	Rapamycin
32.	Gibberella zeae	Zearalenone
33.	Claviceps purpurea	Ergot alkaloids
34.	S. peuceticus subsp. caesius	Doxorubicin
35.	S. peuceticus	Daunorubicin
36.	S. caespitosus	Mitomycin
37.	S. verticillus	Bialaphos
38.	Rhizopus nigricans.	Hydroxylation of progesterone
39.	Thiobacillus thiooxidans	Bioleaching
40.	Phanerochaete chrysosporium	Biodegradation
41.	Bacillus thuringiensis, Bacillus popillia, Pseudomonas fluorescens, nuclear polyhedrosis virus (NPV), granulosis virus (GV), and cytoplasmic polyhedrosis virus (CPV), Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii,	Bioinsecticide
42.	Entomophthora spp	Single cell protein
	Methylococcus capsulatus
	Methylophilus methylotrophus
	Fusarium venenatum,
	Kluyveromyces marxianus,
	Paecilomyces variotii,
	Saccharomyces cerevisiae
	Methylococcus capsulatus,
	Candida utilis
43.	Xanthomonas campestris	Xanthan
44.	Azotobacter vinelandii	Alginates
45.	Acetobacter xylinum	Cellulose
46.	Leuconostoc mesenteroides	Dextran
47.	Sphingomonas paucimobilis	Gellan
48.	Ralstonia eutropha	Polyhydroxybutyrate
49.	Acetobacter suboxydans	Ascorbic acid Vit C
50.	Pseudomonas denitrificans	Vitamin B12 cyanocobalamine
51.	Blakeslea trispora	β-carotene Vit A
52.	Ashbya gossypii	Riboflavin
53.	Acremonium chrysogenum	Cephalosporins
54.	Saccharapolyopora erythraea	Erythromycin
55.	Bacillus licheniformis	Bacitracin
56.	Bacillus brevis	Gramicidin

Mutation and Its types

noreply@blogger.com (R Santhosh Kalash) — Sat, 31 Aug 2013 17:53:00 +0000

Mutation

Mutation is defined in simply words as "Stable Genetic alteration" occurs due to change in the nucleotide sequences of a short region of a genome, caused by either error during DNA replication or by the damaging effects of mutagens such chemical and radiation.

Chemical Mutagens are Reactive oxygen species, nitrous acid, ethidium bromide,sodium azide etc.

Radiations are ultraviolet, gamma rays, X-rays etc.

List of Mutation types and their examples.

Mutation type	Definition	Example	Notes
Transitions	base pair substitution (purine to another purine and pyrimidine to another pyrimidine )	A --> G or G --> A (Purine), C --> T or T --> C (Pyrimidine)
Transversion	base pair substitution (purine to pyrimidine or pyrimidine to purine)	A --> C or C --> A, G --> T or T --> G
Frame shift	Indels of number of nucleotides in a DNA sequence (not divisible by three) can change the reading frame resulting in a different translational.	Original mRNA sequence: 5′ GTC GTT TTA CAA 3′ --> functional protein. Frame shift mutation sequence : GTC GTT T TTA CAA or GTC GTT C TTA CAA	Indels means insertion or deletion in molecular biology.
Point	Single nucleotide is replace by a different nucleotide	examples similar to transition and transversion mutation	It’s a type of base substitution mutation.
Silent	mutation is a result of substitution occurring at the third position of the codon, but it has no effect in the protein activity	GAA is mutated to GAG, but both the codons code for small amino acid i.e. glutamate.	It’s a type of point mutation.
Missense	This results in change in protein activity, since the substitution of nucleotide in the codon codes for a different amino acid.	CAT which codes for histidine are changed to CCT that code for proline.	It’s a type of point mutation.
Nonsense	The mutation is similar to Missense expect that the resulting codon will be a STOP codon.	CAG --> TAG i.e. Glutamine to STOP codon.	It’s a type of point mutation.
Deamination	Conversion of a primary amino group to a ketone.	Deamination of cytosine to uracil results in a base substitution.	ung gene product in E.Coli is an enzyme that specifically removes uracil from DNA.
Base analogs	A compound similar to that of the nitrogenous base is incorporated in the growing polynucleotide chain during replication. It causes a stable mutation.	5-Bromouracil is an analog for Thymine. So, AT --> A5BU. Now during replication, Since 5BU is ionized, its complementary binds to G rather than A. So A5BU results in AT and G5BU. Now during next replication, G5BU is again gives rise to G5BU and GC.	It’s a stable mutation and also it only can be induced artificially.
Leaky mutation	Amino acid substitution mutation causes only partial disruption of the protein function.	A mutation makes the bacteria not to survive in a particular medium, But the bacteria has a leaky mutation at the mutation site, then it slowly grow on the restricted media.
Transposition mutation	Insertion of transposons at the site causes this mutation.	It basically gives rise to antibiotic resistance and most Multi antibiotic resistant bacteria have this mutation.

Cell Communication- Introduction

noreply@blogger.com (R Santhosh Kalash) — Fri, 30 Aug 2013 08:24:00 +0000

A cell always needs a command from its surrounding inorder to maintain its structure and function. So, either cells or proteins present in the extracellular region that can bind specifically to a receptor are called ligand. A ligand can either be a small peptide or large globular protein molecule. Hormones which are produced from endocrine gland act as ligand and it is also termed to be first messengers, because they basically trigger the signaling cascade inside the cell through a receiver protein molecule called receptor.

Receptor is basically found attached to the cell membrane and it gets activated only when a matched ligand binds to it. First messengers cannot enter the cell membrane; hence they have to bind to the receptor molecule Inorder to activate the signaling cascade. Certain ligands like steroid hormones need not be requiring receptor for mediating the signaling cascade; instead they directly pass through the cell membrane and trigger the target molecules such as transcription factors, or nuclear receptor.

So, the ligand binds to the receptor of the cell and then what happens?

The activated receptor will activate the downstream molecules like protein kinase enzyme and then thereby leads to the release of second messengers inside the cell. The second messenger will further activate other enzyme, which modulates the ion channels and transcriptional activities.

Cell signaling versus signal transduction

Cell signalling is regarding the generation and transmission of a signal, the reception of the signal and the propagation of that signal within the receiving cell.

Signal transduction is the process whereby an extracellular signal is converted into a different form of intracellular signal.

Secretions by glands

Glands can be divided into two types: exocrine gland and endocrine gland. Exocrine glands secretes either in the gastrointestinal tract or into the surface of the body. Its secretion is not involved in the signalling, but only play general role in digestive or innate immune system.

Endocrine glands secretes informational molecules into the blood stream and not all first messenger travel through blood, instead some can act locally or even self- stimulate via autocrine secretion.

The four crines

Autocrine

Cell produces a hormone for which it has a specific receptor on itself. Cell becomes self stimulating. For example breast cancer cells produce TGFα which causes uncontrolled growth via interaction with the EGF receptor.

Paracrine

Hormones are secreted locally and the producing cells have no receptors. They will diffuse through the interstitial fluid to nearby target cells. For example in breast cancer cells the PDGF produced will stimulate the nearby fibroblasts which possess the PDGF receptors.

Juxtacrine

Membrane bound hormone precursor interacts with receptor on the adjacent cell.

Intracrine

Hormone receptor complex forms inside cell from newly synthesised proteins.

Endocrine

The signaling molecule act on target cells distant from their site of synthesis by cells of the various endocrine organs. Blood carries the hormone from the glands to the target cell.

“Cell signalling means cell to cell or within cell communication”

In this communication, the following steps are involved à

Synthesis
Release of the signalling molecule by the signalling cell
Transport of the signal to the target cell
Binding of the signal by a specific receptor protein leading to its activation
Initiation of one or more intracellular signal-transduction pathways by the activated receptor
Specific changes in cellular function, metabolism, or development
Removal of the signal – termination of the cellular response

Telomere and Its enzyme

noreply@blogger.com (R Santhosh Kalash) — Sun, 12 May 2013 09:59:00 +0000

In eukaryotes, the linear chromosomes ends with a specific nucleoprotein structure called telomere.

In human, telomere consists of thousands of repeats of six base pair motif (5’-TTAGGG-3’) ending in a G rich 3’ overhang 30-300 nt long, associated with a complex of six proteins named shelterin.

The structure prevents chromosome ends from being recognized as DNA double strand breaks and therefore being processed by the DNA repair machinery.

End replication problem is the inability of DNA polymerase to completely replicate the linear genomes, in which cells undergo telomere shortening at every replication round.

The loss of terminal DNA is counteracted by the activity of telomerase enzyme, which adds de novo telomeric repeats to the G rich 30- overhang.

Telomerase is active only in germ line and in stem cells, but not in somatic cells where telomeres shorten till they reach a critical length that activates a DNA damage response (DDR) leading to replicative senescence or to apoptosis.

Thus, limiting the number of cell division this constitutes an important barrier against cancer proliferation, because it reduces the risk of accumulating harmful mutations that could lead to malignant transformation.

Telomere erosion could result in high chromosomal instability and facilitates the generation of tumor promoting mutations, if cells escape p53 and Rb-dependent DNA damage checkpoints.

Telomeres are extremely dynamic structure and their organisation switch between a protected and a deprotected state throughout the cell cycle and cell differentiation, inorder to accomplish their multiple tasks.

During cell cycle, telomere structure has to change from a closed conformation concealing chromosome ends from repairing enzymes, to an open one in S phase inorder to allow controlled access to DNA replication factors.

In the telomerase negative cells, after reaching the critical short length of telomere, the uncap region will trigger the ATM/ATR signalling cascade, that eventually leads to a p53 dependent cell cycle arrest or apoptosis.

Capping of telomere requires the binding of specific proteins that recognize telomeric DNA and shields single stranded G overhangs by hiding them into specific structures.

T loops (Telomeric loop) are lasso like structures were the 3’ overhang folds back and invade the upstream telomeric region, generating a displacement loop (D-loop).

G – quadruplex are four stranded DNA structure, derived from folding of single stranded DNA containing runs of three to four consecutive guanines to form stacked tetrad of Gs, stabilized by Hoogsteen hydrogen bonding and cation coordination.

Both these structures need RTEL1 helicase inorder to be resolved and allow an efficient telomere replication.