Wednesday, 30 December 2020

DNA Polymerase vs RNA Polymerase

* DNA polymerase

• DNA polymerase is an enzyme that synthesizes the double-stranded DNA molecule.

• Molecular weight of DNA polymerase is 400 k Dalton.

• DNA polymerase's mechanism is during DNA replication where by it synthesizes new DNA strands

• DNA polymerase contains 10 subunits.

• The DNA polymerase  replication mechanism is initiated by a short DNA/RNA primer

• DNA polymerase inserts nucleotide bases after finding the free 3' OH group end by the assistance of the primer-synthesizer, primase enzyme

• DNA polymerase adds deoxy Nucleotide triphosphate (dNTP) molecules like dATP(Adenine), dGTP(Guanine), dCTP(Cytosine) and dTTP(Thiamine) to the growing new DNA strand

• DNA polymerase has polymerization and exonuclease proofreading activity
- Polymerization from 5' to 3'
- Proofreading from 5' to 3' as  
   well as 3' to 5'

• The rate of polymerization by DNA polymerase is about 1000 nucleotide bases per second in prokaryotes

• DNA polymerase enzyme is  efficient, and more accurate considering its proofreading activity.

DNA polymerase has three different subtypes in prokaryotes: Type I, II, and III. and five different subtypes in eukaryotes: α, β, γ, δ, ε.

• The DNA synthesis continues until the end when the strand ends, that is when polymerization stops, thus the entire chromosomal DNA is synthesized.

DNA vs RNA : Comparison and Differences

* RNA polymerase

• RNA polymerase is an enzyme that synthesises a single-stranded RNA molecule

• Molecular weight of RNA polymerase is 500 k Dalton.

• RNA polymerase functions during transcription, which is the synthesis of RNA

• RNA polymerase contains 6 subunits.

• RNA polymerase does not need a primer to initiate transcription

• RNA polymerase adds nucleotides directly. It inserts Nitrogenous bases like Adenine, Guanine, Cytosine and Uracil to the growing RNA strand.

• RNA polymerase only has a polymerization activity.
- Polymerization from 5' to 3'

• The rate of RNA polymerase for adding nucleotide bases is 40 to 80 nucleotide base per second.

• RNA polymerase is, inefficient, and inaccurate many times.

• RNA polymerase has five different subtypes in eukaryotes which are Type I, II, III, IV and V

• The polymerization is terminated when RNA polymerase finds the stop codon or termination codon(UAA, UGA and UAG) on the DNA strand.

Tuesday, 29 December 2020

Inflammation: Vascular Events and Migration of Leukocyte

 Inflammation is fundamentally, a protective response of the body against any offending agent or tissue damage. It involves responses of the vascular tissues and leukocytes which help in getting rid of the primary cause of insult and necrotic cells or tissues resulting as a consequence of the insult. Inflammation could be acute or chronic depending on the duration of the response.

  Acute inflammation is a rapid response, developing within minutes and could last up to a few hours to days. If the insult or damage is not repaired by the acute response, the inflammatory response could become prolonged thus becoming Chronic. Chronic inflammation could go on for few weeks to many months.

Inflammation could be caused by multiple factors ranging from external agents like microbes, drugs and allergens, physical agents like temperature, any form of mechanical injury and other factors like genetic and immunologic disorders. Tissue necrosis due to ischemia, mechanical or chemical injury could also induce inflammation.

Cardinal signs of Inflamatin
There are 5 cardinal signs of inflammation, which actually are a macro-level or an external manifestation of the microscopic events happening during inflammation.

- Inflammation kick starts with vasodilation of arterioles and capillaries near the site of tissue insult and this leads to “RUBOR”or redness.
-  Vasodilation results in increased blood flow causing “CALOR” or heat.
-  Leakage of plasma fluids from the vessels and accumulation in the extra-vascular space causes “TUMOR” or swelling.
-  Chemical mediators of inflammation like histamine and prostaglandins may cause “DOLOR” or pain.
-  Swelling and pain may cause loss of function, of the tissue involved and is called “FUNCTIOLAESA” or loss of function.

  An inflammatory response begins with an insult which could be exogenous or endogenous. Any form of insult causes cell damage and damaged cells release certain molecules are called “Damage Associated Molecular Patterns (DAMPs)”.

These molecules are recognised by receptors on leukocytes like macrophages or dendritic cells. On recognition leukocytes release pro-inflammatory cytokines.

Similarly microbes have specific molecules called “Pathogen Associated Molecular Patterns (PAMPs)” which could be recognised by receptors on leukocytes. On recognition leukocytes release pro-inflammatory cytokines and also other cytokines that help lymphocyte activation.

Now, pro-inflammatory cytokines could cause degranulation of nearby mast cells. Degranulation results in release of inflammatory mediators like histamine, eicosanoids like prostaglandins, leukotrienes and more pro-inflammatory cytokines.

  This cascade of events kick starts an acute inflammatory response. Acute inflammation begins with vasodilation of arterioles and capillaries. Vasodilation could be caused by histamine, leukotrienes and prostaglandins.

This is followed by an increased vascular permeability. So Histamine and leukotrienes can cause retraction of the endothelial cells in the vessels creating small gaps. and Plasma proteins leak out of the blood vessel through these gaps and accumulate in the extra-vascular space causing oedema.

This plasma rich fluid leaking out of the vessel and accumulating in the extravascular space is called an exudate.

Leakage of plasma proteins disturbs the axial flow of blood. It increases blood viscosity thereby slowing down or causing stasis of blood flow. This results in red blood cells getting concentrated at the centre with leukocytes being pushed to the periphery near the vessel wall.

  Acute inflammation serves to recruit leukocytes to the site of infection or injury in order to eliminate the offending agents and damaged tissue. Neutrophils and monocytes are key leukocytes that are recruited and are capable of phagocytosis.

However in acute inflammation, neutrophils are the first batch of leukocytes to be recruited to the site of infection. Migration of monocytes follows many hours later. Leukocyte recruitment to the site of infection involves a series of steps.

Once the blood flow slows down, leukocytes are pushed to the periphery and move along in close association to the endothelium. This process is called margination.

Pro-inflammatory cytokines like IL-1 and TNF-α released by macrophages and mast cells cause endothelial cells and leukocytes to express what are called adhesion molecules.

Adhesion molecules on the endothelium are called selections and those on leukocytes are called sialyl Lewis X proteins. Sialyl Lewis X proteins bind with selections causing leukocytes to slow down and tumble along the endothelium, a process called 'rolling'. Rolling and slowing down, helps leukocytes firmly adhere to the endothelium. This is accomplished by binding of surface proteins on leukocytes called integrins to adhesion molecules on the endothelium called, VCAM – vascular cell adhesion molecule.

  Expression of VCAMs on the endothelium are induced by pro-inflammatory cytokines like IL-1 and TNF-α.

The next step involves the transmigration of adherent leukocytes through the endothelial gaps to the extravascular space. This process called diapedesis is induced by chemokines. Once in the extravascular space, leukocytes migrate towards the site of infection along a concentration gradient, a process called chemotaxis.

Chemotaxis is induced by a variety of chemokines produced by mast cells, complement proteins and the microbes themselves. Examples of chemokines are IL-8, LTB4 and complement proteins like C5a.

Neutrophils predominate in the site of infection for the first 6-24 hours followed by monocytes getting into action post 24-48 hours. After recruitment to the site of infection leukocytes recognise microbes or dead cells and kill them by a process called phagocytosis

Monoclonal Antibodies - Production by Hybridoma Technology

Antibodies are produced against antigen. whenever an antigen enters our body our immune system produces antibodies against that antigen. In normal condition as antigen enters it triggers or stimulates our immune system and our immune system produce antibodies.

  These antibodies are of different specificities and they recognise different epitops on the same antigen. The antibodies derived from the multiple cell are known as polyclonal antibodies. Polyclonal antibodies represent a collection of antibodies from different B-cells, that  recognise multiple epitops on the same antigen.

Monoclonal antibodies

preparations contain only one type of antibody derived from a single cloned B-cell. So these antibodies will recognise and bind only one particular epitop on an antigen. Monoclonal antibodies with same specificity are identical antibodies.

  Cesar Milsten and Grorges J.F. Kohlar described the first technique developed for stable Monoclonal antibodies production in 1975. This technique is known as Hybridoma Technology. They were jointly awarded Nobal Prise in physiology and medicine in 1984.

Hybridoma technology is used for the production of large number of monoclonal antibodies against a particular antigen and these monoclonal antibodies are used for the different purpose. i.e. in the treatment of cancer, used as anti-venoms.

Production of Monoclonal Antibodies by Hybridoma Technology

Production of Monoclonal Antibodies by Hybridoma Technology

1). Animal Immunization :

   First step in production of Monoclonal antibodies is the immunization of an animal, usually the animal is used as the mouse. Mouse is immunized with the antigen against which we need antibodies.

   Mouse is injected with the antigen (i.e. antigen which has three epitops) several time, as a result mouse B-lymphocytes are stimulated against the epitops or antigenic determinants of the injected antigen.

  After several weeks when these B-lymphocytes reach to and optimal amount the mouse is sacrificed, spleen of mouse is removed aseptically. Spleen is secondary lymphoid organ and we can easily harvest activated B-lymphocytes from spleen.

  Then we have activated B-cells which are capable of producing antibodies of against the specific epitops present on the antigen. Activated B-cells produce the antibodies of interest but can not replicate in culture.

2). Cell Fusion :

   In this step activated B-lymphocytes are fused with myeloma cells. The rational behind fusing these two cell type togather is that spleen cells produce the antibodies of interest but can not replicate in culture. Thus it is difficult to harvest antibodies from them. Myeloma cells on the other hand do not produce antibodies but to replicate in culture quite easily.

  Hybridomas take advantages of the properties of both cell types to mass produce is antibodies of interest.

As a result of cell fusion, we will have five type of cells:
-  Unfused B-cells,
-  Fused B-cells,
-  Unfused Myeloma cells,
-  Fused Myeloma cells and
-  Hybrid cells formed by fussion of an activated B-cell and in myeloma cell.
These hybrid cells are also known as Hybridomas

In our illustration we have five types of activated B-cells each specific to one of the three epitops on the antigen. So hybrid cells will  also be of three types.

3). Selection of Hybridomas 

    Next aim is to select these hybridomas from this mixture of cells. Selection of hybridomas from the mixture can be accomplished through the use of media containing Hipoxanthine, Aminopterin and Thymidine (HAT).

  In order to understand the rational behind this approch it is important to know that mammalian cells can synthesize nucleotide using two different pathways that are -
  • De novo pathway and
  • Salvage pathway.

  Under normal conditions mammalian cells will use the de novo pathway to replicate. but de novo pathway is blocked by Aminopterin, when the de novo pathway is blocked cells will then utilize the salvage pathway as an alternative means to replicate. but this will only happen if Hypoxanthanine and Thymidine are present. 

  The kye to this approch is to use myeloma cells that are deficient in an enzyme called HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) which is required for the salvage pathway in this scenario unfused myeloma cells that are deficient in an enzyme called HGPRT.

Myeloma cells are unable to replicate because the de novo pathway is blocked by Aminopterin and the salvage pathway is blocked by deficiency and HGPRT.

  Fused and unfused B-cell died within few days because of their short lifespan, They are not able to devide indefinitely in cell culture. Fused and unfused myeloma cells also die. This is also because of de novo pathway is blocked by Aminopterin and the salvage pathway is blocked by deficiency and HGPRT.

  Hybrid cells almost survived in HAT medium these hybrid cells are able to synthesise nucleotide by the salvage pathway. The functional HGPRT enzyme is contributed by the activated B-cell partner and these cells are abe to divide indefinitely.

4). Isolation of Hybridomas :

    Our aim is to select legate single antibody producing hybrid cell. we need to isolate these hybridomas and grow them individually. Therefore next step is the isolation of hybridomas of single specificity, this is done by a method known as limiting dilution. In this method the cells are hybridomas are distributed in multi well culture plates at very density. This is done such that on an everage each will contain a single cell.

5). screening :  

  In this step these hybridomas are screened for the secretion of the antibody of desired specificity. This screening  is done mostly by two technique namely ELISA and RIA. Once the hybridoma cells producing the desired antibody are identified they are isolated and cloned.

6). Cloning and Propagation :

   In this step we have seperated clones of activated B-cells each producing antibodies of a single specificity in each case. The antibodies produced are known as monoclonal antibodies.

7). Characterization and storage of Monoclonal antibodies :

  This is the final step in the step these hybridomas and monoclonal antibodies are characterized and stored. Mostly they are stored in liquid nitrogen. Now these monoclonal antibodies  are ready to treating and diagnosing disease.


Sunday, 27 December 2020

Gene Gun: Method, Applications and Limitations

Gene gun is not so widely used method as is always the case. however it has some advantages thet may be useful for your research.In general it is a way of transfecting cells. Suppose you want to deliver a gene or plasmid into the diffrent cell and this particular cell culture is difficult to transfect using another method. This method was invented by John C. Sanford, Ed Wolf, and Nelson Allen.

How does the gene gun works?

    In this method you have a nanoparticles (gold or tungsten), which are coated by a desired gene or plasmid.

    Now you have a equipment capable of accelerating these coated particles just like a gun.
   In this gun Helium gas fuels the chamber and pressure propels micro carrier with modified  nanoparticles into the stopping screen.
   When the micro carrier hits it, DNA coated nanoparticles arr propelled through the screen and into the target cell in petridish.
You can actually shoot DNA coated gold particals any cells without damaging most of these cells. Of course it depend on both nanoparticles size and the impulse. Once with the diameter of about 1micrometer had initially been used but relatively recent study had revealed two orders of megnitude. Smaller nanoparticles are also suitable.

For long period of time tungsten or gold nanoparticles
1micrometer in diameter were widely used by researchers. Insted of gold and tungsten silica nanoparticles are much better due to their biodegradability and non toxicity.

Applications of Gene gun Technique

1) To study of neuronal dendritic branching and dendritic spine morphology- special dye is used as a molecule to deliver into the cell.
2) Plant transformation- reported gene is used to find out whether it's successful or not.
3) Transfection of neurons- some of which are notoriously difficult to transfect.
4)Transfection of cells deep in tissue.
5)DNA vaccination for inducing protective immunity to infection and malingnancy.

Weaknesses of Gene gun method

1) Perticular nanoparticle may contain molecule to deliver or may not.
2) Each particular nanoparticles contain different quantity of that molecule on its surface.
3) Low efficiency of the metal nanoparticles in reaching the entire tissue due to the low penetration of the particles.
4) Surgery is often necessary to use the technique in order to reach any non superficial tissue.

X-linked Inheritance : Morgon's work on fruit fly

 Mendel described factors of inheritance called genes, but he did this before anyone knew about the existence of the chromosomes that contain them, or the DNA they are made of.

  The early 20th century saw incredible progress in contextualising Mendel’s work within new discoveries in molecular biology, as it was then possible to see chromosomes in a microscope, and watch them replicate and redistribute themselves during cell division, which led to the chromosome theory of inheritance.

It was at this point that studies regarding mitosis and meiosis were able to explain Mendel’s laws of segregation and independent assortment, the two alleles for a gene separate during gamete formation, and that alleles for two different genes separate in random combinations, so long as they are on different chromosomes, as the chromosomes themselves, which contain the genes at specific loci, will undergo segregation and independent assortment, during gamete formation.

  But more evidence was still needed to corroborate the chromosome theory of inheritance. Some very compelling evidence for the chromosome theory of inheritance came from the work of Thomas Hunt Morgan.

  Morgan studied fruit flies, which have only four pairs of chromosomes, those being three pairs of autosomes and one pair of sex chromosomes, or an X and Y chromosome, just like humans. Two X’s for a female, and an X and a Y for a male.

  In studying the flies, he noticed a rare characteristic that sometimes appeared in males, and that was white eyes instead of the typical red.
- If considering phenotypes, red eyes would be referred to as the wild type, and
- white eyes would be considered the mutant, or non-wild type.

Morgan did some experiments that were similar to Mendel and the pea plants. He bred a red-eyed female with a white-eyed male.
- The entire F1 generation had red eyes, suggesting that red eyes are dominant, which is consistent with Mendelian genetics.
- Then he bred the F1 generation amongst it self, and he observed the expected 3 : 1 ratio for red to white eye color in the F2 generation, with an additional result.White eyes showed up only in males.
- All the F2 females had red eyes, and half the F2 males had red while half had white.

This was firm evidence that eye color was linked to sex, which makes perfect sense considering the discrepancy in sex chromosomes for males and females. It was suggested that the gene for eye color in fruit flies was an X-linked gene.

The reasoning went as follows.
- The original parental generation was a red-eyed female, with two X chromosomes, each with the red allele, which is represented by the letter W with a plus in superscript, and a white-eyed male, with an X chromsome containing the white allele, represented by the letter W, and a Y chromosome, which does not contain the gene in question.

In producing gametes, all the female eggs would have an X chromosome with the red allele, while for the male, half would have an X chromosome with with the white allele, and half would have the Y chromosome, without this gene.

  This means that all of the F1 generation would have precisely one red allele, and thus red eyes, since this is the dominant allele. But then,
  For the F2 generation,
25% would be homozygous dominant,
50% would be heterozygous, with the presence of the white allele or the Y chromosome being irrelevant to the phenotype, and 25% would have the white allele and the Y chromosome, thus missing the red allele, and possessing white eyes. But all of these flies will necessarily be male, which is indeed what was observed.

This constituted tremendous evidence for the chromosome theory of inheritance, because it demonstrated that information regarding the inheritance of a particular phenotype was associated with a particular chromosome, in this case, the X chromosome.
  Beyond corroborating the chromosome theory of inheritance, it was therefore realised that sex-linked genes have interesting patterns of inheritance. It is worth noting that not all organisms that reproduce sexually have the same X/Y system of sex determination that humans and flies do.

Chromosomal System of Sex Determination
1) X-0 System
The X-0 system is found in certain insects like grasshoppers and cockroaches. Here there is only one sex chromosome, the X chromosome. Females have two, and males have one.
  Their sperm may contain an X chromosome, or no sex chromosome.

2) Z-W System
In various birds, fishes, and certain insects, a Z-W system is utilised. Females are ZW, and males are ZZ.

3) haplo-diploid system
  A bit stranger is the haplo-diploid system, found with bees and ants. Here there is no sex chromosome, it is simply that eggs that are unfertilized develop into males, while eggs that are fertilized develop into females.
   This means that male bees and ants, having no fathers, are haploid organisms. All of their cells have only one set of chromosomes, just like our gametes, while females are diploid, each cell containing two sets of chromosomes, like most other animals.

Color-blindness: an X linked disorder

 With sex-linked genes better understood, we can examine the inheritance of certain traits like color blindness, which is an X-linked disorder. The normal allele is represented by the letter X with a capital N in superscript (XN), while the recessive allele has a lower case n in superscript(Xn).

Homozygous dominant female reproduces with a color blind male, meaning one recessive allele, and a Y chromosome, lacking the gene entirely. Off spring have a 50% probability of being female and heterozygous, and 50% probability of being male, with the dominant allele, a situation we would call hemizygous dominant, where hemizygous means that only one allele is present. Thus, there is a zero probability of passing on color blindness, but a 50% probability of producing a carrier.

  Now if the mother is heterozygous, and the father has the dominant has the dominant allele, there is a
25% probability of offspring being homozygous dominant,
25% heterozygous and therefore a carrier,
25% hemizygous dominant, and 25%hemizygous recessive, and therefore color blind.

if the mother is a carrier and the father is color blind is color blind, there is a
25% percent chance offspring will be a carrier,
25% hemizygous dominant,and  
50% chance of being color blind, with the possibility of producing a color blind female, which is far more rare, given that both parents must possess the rare recessive gene.

There are a large number of X-linked disorders, given that the X chromosome is so much larger than the Y chromosome, containing some 1100 genes, whereas there are far fewer Y-linked disorders, since it has only 78 genes, some of which are duplicates, although these can exist as well.

Thursday, 24 December 2020

Overview of Phagocytosis

 Microbes and dead cells could trigger inflammatory mediators to cause inflammation, which is basically the body’s response of delivering leukocytes and plasma proteins to the site of infection or injury, so that the offending agents could be removed or eliminated.

When the body is inflamed due to microbes or other external agents, leukocytes migrate from the blood vessels to the site of infection through a process called chemotaxis.

  Once in the site of infection, leukocytes have to recognise the offending agents, in order to be activated and to eliminate them. Once activated, the leukocytes perform various functions to eliminate the offending agents or microbes.

  Leukocytes possess numerous receptors for recognising external agents, some of them being Toll like receptors and G-protein coupled receptors. They also possess receptors for other proteins like cytokines, which could activate them to start the cleansing process.

  Once activated, leukocytes perform various functions to amplify the process of inflammation that is it could cause further migration of leukocytes to the site of infection and help in production of various inflammatory mediators like cytokines and arachidonic acid metabolites.

  Apart from amplifying the inflammatory response leukocytes also eliminate offending agents like microbes by ingesting and destroying them, a process called phagocytosis.

Phagocytosis involves 3 steps, them being,
1) Attachment of the offending agent/microbe to the phagocyte receptors,
2) Engulfment of the offending agent/microbe and
3) Intra-cellular killing of the offending agent/microbe.

1) Attachment of the offending agent/microbe to the phagocyte receptors

There are receptors like mannose and scavenger receptors on the phagocyte which can bind to microbes and help in engulfment. Another important receptor is the opsonin receptor which helps in binding to opsonins that have coated cell surface of microbes.   
   For example, immunoglobulins like IgG and complement proteins like C3b act as opsonins that could bind to cell surfaces of microbes, which in turn could be recognised by opsonin receptors on phagocytes to help in engulfment of the microbe.

  After attachment to phagocyte receptors, the plasma membrane of the phagocyte throw extensions called pseudopods around the microbe, engulfing it. This part of the plasma membrane gets pinched off forming a vesicle called phagosome with the microbe inside. The phagosome then fuses with lysosomes inside the cell forming phagolysosomes.

  The killing or elimination of microbes is accomplished by chemicals called free radicals which are essentially of two types - Reactive Oxygen Species (ROS) and Reactive Nitrogen Species.
Reactive Oxygen Species are derived by reduction of oxygen byan enzyme called phagocyte oxidase or NADPH oxidase and Reactive Nitrogen Species are derived from nitric oxide (NO) combining with superoxide (ROS).

  Phagocyte oxidase present in the cytoplasm, translocates to the membrane of the phagolysosome and reduces oxygen to a superoxide anion. The superoxide anion is converted to Hydrogen peroxide (H2O2) which is further converted to hypochlorite, by amyeloperoxidase. Hydrogen peroxide is also converted to hydroxyl (OH) radical.

  Hypochlorite and hydroxyl radical are powerful destructive agents helping in elimination of microbes. Further, nitric oxide derived from arginine by the action of nitric oxide synthase (NOS) combines with superoxide to form a free radical called peroxynitrite, which also participates in microbial killing. The free radicals target the lipids, proteins and nucleic acids of microbes, thus killing them.

  Apart from free radicals, phagocytes also possess numerous enzymes like lysozyme, elastase, cathlecidin, lactoferrin and defensins which are anti-microbial. 

Wednesday, 23 December 2020

Structure and Organisation of Chromosomes

 During nucleotides polymerization two strands of DNA come together to form a long double helix, with millions of base pairs. But to go from this to a more complete understanding of all the chromosomes in every one of your cells is a pretty big leap.

  DNA is not just floating around the nucleus in the double helix form. DNA is typically coiled up to save space, because there is so much of it to store. DNA strands are wrapped around proteins called histones, and these DNA-histone complexes are called nucleosomes. Then, this undergoes further supercoiling until we get chromosomes of the familiar shape.

  In a human diploid cell, which is every cell in your body except for reproductive cells, there are two versions of every chromosome, one maternal and one paternal, which makes two sets of 23 chromosomes, or 46 in total. Each chromosome will duplicate through DNA replication to give two identical sister chromatids.

Homologous pair of chromosomes contains the same genes, but different alleles for that gene, one from each parent, so these are not precisely identical to one another. But each chromosome consists of two identical sister chromatids, which will be pulled apart during mitosis. So that each daughter cell can have a complete set of chromosomes.

  When Mendel spoke of genes, it was an abstract concept, as no one knew about DNA at that time. But later in the century when microscopes became powerful enough to see chromosomes and watch mitosis take place, scientists began to see that Mendel had been exactly right, and they developed the chromosome theory of inheritance.
They realized that the genes we learned about from Mendelian genetics are actually long stretches of DNA that code for various proteins. These genes have specific locations on specific chromosomes, and each chromosome in a homologous pair has the same gene at the same spot. This new understanding explained all of Mendel’s observations.

   In meiosis, homologous pairs of chromosomesare separated, which accounts for the law of segregation. Only one allele for a particular gene will show up in a gamete, not both. And the fact that homologous pairs are arranged only during this process accounts for the law of independent assortment, because if two genes are located on two different chromosomes, the combination of alleles for those two genes that ends up in a particular gamete will be totally random as well.

  So before DNA structure was fully understood, we knew that chromosomes contained genes. Now we understand, each chromosome contains hundreds or even thousands of genes. Even still, genes only comprise around one to one and a half percent of the genome. So rest is, in between all the genes is noncoding DNA. This is the majority of the DNA, which doesnot code for any proteins.

However, this area still serves a varietyof functions, like
• Transcribing RNA’s other than mRNA,
• Serving as origins of replication,
• Regulating gene expression,
• Comprising centromeres as well as telomeres.
  Telomeres, are found at the ends of each chromosome, and they are sections of DNA where, in humans, the sequence TTAGGG is repeated hundreds of times. This is because with every round of DNA replication, the enzymes involved can’t quite copy the last couple bases, so this extra padding is present so that even after many rounds of replication, the ends haven’t shortened so much that the genetic information present within an actual gene starts to get eroded away, which would be harmful to a cell. If this does happen, it is called replicative senescence.

In some cells, an enzyme called telomerase regularly extends the telomeres, which buys a cell a little more time. Beyond telomeres, some are as of noncoding DNA are called transposons. These are sequences that can change position within a genome.

Key difference between the chromosomes of human males and females.

There is a pair of sex chromosomes present in each cell, and for a female these are both X chromosomes, with the familiar shape. But for a male, one of these is X and oneof these is a Y chromosome, which is much smaller.
  During meiosis, all egg cells get X chromosomes, since that’s all there is in the parent cells of a female, but sperm cells can end up with either an X or a Y,
since they are both present in diploid cells for the male.

A cell or zygote that inherits two X chromosomes upon fertilization will become a female, and one that gets an X and a Y will become male. These two chromosomes are partially homologous,but obviously the Y chromosome is missing genes that are present on the X chromosome, as the Y is much smaller. This means that males have only one allele for certain genes where females will have two. These are called X-linked genes, because theyare present only on the X chromosome and not the Y.
  In such a case, if the singular allele is recessive, a male will express the recessive phenotype, as there is no dominant allele present to override this. There are a number of disorders that are attributed to X-linked genes, such as color-blindness and hemophilia.

We should also note that females, with two X chromosomes, typically have one of these largely inactivated in each cell, and the inactive one is chosen at random, so some cells have an active X chromosome that camefrom the mother, and some from the father.

This results in phenotypes like two colors of fur on female cats, because some of the cells have an X chromosome active with anallele for one color of fur, and other cells have the other X chromosome active, which has the other allele, and corresponds to a different color. A replicated chromosome consisting of two sister chromatids, is made of looped domains wrapped around a scaffold. These looped domains can be unwound to reveala fiber of nucleosomes, which result when DNA wraps around histones to form tiny beads. 

Monday, 21 December 2020

Gel-Electrophoresis principle and Application

  Gel electrophoresis is a method of separating large molecules like segments of DNA. When we introduced some concepts in biotechnology, for make recombinant DNA plasmids and insert them in bacteria to produce many copies of a gene, or many copies of the protein produced when a gene is expressed.

We want to cut up the plasmid and analyze it, to make sure it is being copied as desired. Or imagine any other scenario where we have a mixture of DNA fragments that we want to separate and visualise. Gel electrophoresis enables us to do this.

Principle of Electrophoresis :

In electrophoresis there is a tray. In this tray there sits a slab of either polyacrylamide or agarose gel, which is immersed in an aqueous buffer solution. At one end of the gel there are a series of wells, and a number of samples can be loaded into these wells, each of which is a mixture of some DNA molecules of varying length.

This apparatus is also equipped with electrodes at each end, with the cathode, or negatively charged electrode, at the end where the wells sit, and the anode, or positively charged electrode, at the other end. Once everything is loaded and ready to go, the current is turned on.

Phosphate groups line the DNA backbone, and that each phosphate group contains one oxyanion, and thus carries a formal negative charge. We can therefore say that DNA molecules are negatively charged.

This means that the negatively charged cathode will repel the DNA molecules, and they will begin to travel along the gel, towards the positively charged anode, to which they are attracted.

The thing is, this gel is a sticky, porous substance, and the molecules have to migrate through the pores to move along the gel, in a process called sieving.

The larger the DNA molecule, the more difficulty it will have in navigating through the pores, which means that smaller DNA molecules will travel greater distances greater distances through the gel, while larger ones will travel shorter distances through the gel, in the same time interval.

This process is so reliable and quantifiable, that we can plot the approximate number of base pairs in a DNA molecule as a function of the precise distance it travels during gel electrophoresis.

  Once separation is complete, the current is turned off, and a DNA-binding dye is added to the system that glows a fluorescent pink in UV light. This is how the data is gathered, which will show up as thin bands that sometimes resemble a ladder, if many different DNA molecules were present in the sample, and each band contains thousands of identical DNA molecules of that particular length.

Remember, the farther away from the well a band shows up, the shorter the DNA molecule is that has produced that band.


(Note : For a standard agarose gel electrophoresis, a 0.8% gel gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments. 1% gels is often used for a standard electrophoresis.)

1. Take 300mg of agarose and dissolve it in 20 ml of 0.5X TEB buffer to prepare 1.5% Agarose gel. 

2. Boil until the Agarose is completely dissolved and no obvious particles of the Agarose remain in the suspension. 

3. When the gel temperature is around 40° C, add 2ul of ethidium bromide(EtBr) and mix properly. 4. Seal the gel-costing/running tray on two sides; place the comb in the gel-tray in appropriate place, 

5. Pour the Agarose solution into the gel-costing/running tray-containing comb. 

6. Allow the Agarose to solidify in the tray, then remove the seal from the two sides of the tray without disturbing the gel. 

7. Then keep the gel-tray in the tank containing 0.5X TEB buffer with the wells in the cathode (negative side). The buffer level in the tank should be maintained above the gel tray. 

8. Gently lift the comb without damaging the wells; the gel is now ready for loading. 

9. Connect the cords between electrophoresis tank and the power pack before loading the samples. 

10. To prepare samples for electrophoresis, add 5μl of gel loading dye in to the restricted sample and mix well by pipetting. Load 15-20μl of the sample in the respective wells. 

11. In the nearby well load 3μl of DNA marker provided. 

12. After loading, switch on the power pack and adjust the voltage to 50V – 100V. 

13. Continue the electrophoresis until the dye reaches to 1/3rd of the gel or above. 

14. Observe the DNA bands by using UV Transilluminator.

Applications of Gel Electrophoresis

• This technique may be used to chop up plasmids with restriction enzymes and analyse the results. • It can be used to assess the products of gene amplification using the polymerase chain reaction.
• It can be used to isolate a specific DNA molecule of a mixture for sequencing or further characterization via a technique called Southern blotting.
• In addition to separating mixtures of DNA according to length, this technique can also be used to separate mixtures of proteins according to electrical charge, which offers information about the identity of the side chains, or a number of other very useful applications.
• The simplicity and immense utility of gel electrophoresis make it a very important part of any molecular biology laboratory today. 

Saturday, 19 December 2020

Biological Weapon Anthrax: Bacillus anthracis

 Anthrax is a disease that is most recently famous for being sent through the mail as a biological weapon. Anthrax is an ancient disease, and many researchers believe we can trace it all the way back to the year 700 BC in ancient Egypt.

Over the years, some of the greatest microbiologists have studied it, including Robert Koch, who used it to develop his famous Koch Postulates. In fact, Bacillus anthrecis was the first bacterium ever shown to be the cause of a disease in 1876, and Louis Pasteur made the first vaccine for anthrax.

The symptoms of anthrax depend on the type of infection, and the type of infection depends on

How anthrax enters the body?

  The most common entry points are the lungs, the skin, and the gastrointestinal system, but it can also be injected, though this type of infection is very rare.
  All types of anthrax, if left untreated, have the potential to spread through the body and cause severe illness or death. 

Types of infections:

Cutaneous anthrax, or anthrax of the skin, is the most common form of anthrax infection. Infection can begin one to seven days after exposure, appearing as small, painless bumps called papules, that may or may not develop swelling or an ulcer with a black center. Generally, cutaneous anthrax is thought to be the least dangerous of the types.

  Inhalation anthrax is the deadliest form of anthrax, which can take up to two months or more to begin to show symptoms. Symptoms begin as nonspecific fever, shortness of breath, headache, abdominal pain, and chills, and can escalate to a more serious fever and significant swelling of the lymph nodes.
  Even though this form of anthrax is inhaled, symptoms rarely involve the pulmonary system. Almost every case of inhalation anthrax progresses to shock and death within three days of initial symptoms unless swift treatment is delivered.

Gastrointestinal anthrax is more rare in the U.S., and symptoms depend on which portion of the intestinal tract is infected. If the upper gastrointestinal tract is infected, an infected person might develop ulcers in the esophagus or mouth. If the infection occurs in the intestines, an infected person will experience vomiting and nausea. In both cases, disease develops rapidly, and is thought to have a mortality rate close to 100%.

Bacillus anthracis
  Anthrax is caused by the rod-shaped, gram-positive bacterium Bacillus anthracis. This bacterium is found naturally in soil and produces spores, which are round, highly durable cells that can lie dormant for years, even decades.
   These spores can activate when they find themselves in favorable conditions, like, say, the body of an animal or person, which is rich with water, sugar, and other nutrients.
   Once active, these bacteria can multiply and spread throughout the body, causing severe illness and death.

How do they cause damage?  
   Once ingested, the spores become activated and the bacteria begin to reproduce and make proteins. Reproducing bacteria make three different proteins:
1. protective antigen or PA.
2. lethal factor or LF. 
3. edema factor, or EF.
These proteins then combine to form two different toxins.
  When protective antigen(PA) and lethal factor(LF) combine, they form the lethal toxin. When protective antigen(PA) and edema factor(EF) combine,they form edema toxin. Together, these toxins cause a fluid buildup around the lungs that kills infected cells, ultimately causing severe disease and death in the infected animal or human.

It turns out that anthrax is not contagious, meaning it’s not spread from person-to-person. For the most part, anthrax is a disease of livestock that ingest spores in plants, soil, or water.

Common causes of infection in humans:
  Humans most commonly become infected where they handle infected animal products like wool or leather, or inhale anthrax spores from infected animal products. Humans can also get anthrax from eating undercooked meat of infected animals.

Treatment :

Time is of the essence for those with an anthrax infection. Getting proper medical care as quickly as possible can make the difference between life and death. All types of anthrax can be treated with antibiotics, and some cases are treated with antitoxin.
   Livestock vaccines can prevent outbreaks in animals, and there are several types of vaccines for humans. One type protects adults that routinely handle animals or animal products, and another has been approved by the FDA for use after exposure.

  Anthrax is most common in agricultural regions of Central and South America, central and southwestern Asia, sub-Saharan Africa, southern and eastern Europe, and the Caribbean, so readers in those regions take particular caution. 

Friday, 18 December 2020

How Hormons Impact on Your Sexual Behaviour

  Even with what we know about hormones, it still may seem incredible that the secretion of a few molecules from a handful of glands can be responsible for something as intense as the sex drive, but sexual behavior can be understood scientifically just like any other type of human behavior.

hormones will be the key players in this realm, and these hormones orchestrate a wide variety of things.
  From conception through to adulthood, they guide the development of our primary and secondary sexual characteristics, as well as behavioral characteristics, which together distinguish us as either male or female. And once mature, these same hormones regulatethe sex drive and reproductive behavior as well. All of this activity is orchestrated by the neuroendocrine system.

Some of the glands and associated with sexual behaviar are Pineal, Pypothalamus, Pituitary, Thyroid, Parathyroid, Thymus, and Adrenal glands, all of which secrete hormones.

Of course there are several organs that are relevant to this system as well, most notably the gonads, which are the testes for a male and ovaries for a female, as well as the pancreas and other organs that secrete hormones in addition to performing their other functions.

Types of Hormons

We can also recall that hormones are of several classes, those being
• Amino acid derivatives,
• Peptides and proteins, and
• Steroids, are last of these categories is the one that all the sex hormones belong to.

These are released into the bloodstream to reach their targets, often slipping right through the membrane due to their size and structure to find receptors within the cell. They interact with these receptors to elicit a specific physiological response related to body development or behavior, typically in some manner related to gene expression. As we may recall, the sex hormones can be divided into androgens, like testosterone, and estrogens, like estradiol.

It is true that the male gonads release more androgens than estrogens, and the opposite is true for the female gonads, but all men and women have both in their bodies, so it is overly reductive to refer to them as male and female hormones.

Hypothalamic Pituitary Gonad Axis

Release of sex hormones by the gonads is regulated by the anterior pituitary gland, which releases other hormones like gonadotropin, which travels through the bloodstream to the gonads to stimulate them.

  The levels of these hormones are relatively stable for males, whereas in females they fluctuate in a cycle lasting around 28 days,which we call the menstrual cycle.

The anterior pituitary, in turn, is controlled by the hypothalamus, again through the release of other hormones, which in this case travel through the hypothalamo pituitary portal system. For example, gonadotropin-releasing hormone is released from the hypothalamus, which stimulates release of gonadotropin in the anterior pituitary, which then travels to the gonads to stimulate release of sex hormones.

Hormones Can be Released as a response to
•Other hormones,
• Non-hormonal chemicals,
• Psychosocial inputs, or even
• Direct neural regulation. The exception to this last method is the anterior pituitary, which has no neural input.

  In general, hormone release due to neural signals tends to be a response to some experience, such as arousal, or more subtle environmental factors.

How do hormons regulate human developments ?
  Humans exhibit sexual dimorphism, which means that similarly to many other animal species, males and females have very different physical features. This starts at fertilization. When a zygote is produced, it gets 23 chromosomes from each gamete, one set from the egg, and one set from the sperm.

One of the chromosomes in each set is a sex chromosome, which will be either an X or a Y chromosome. The egg always has an X chromosome, and the sperm has either an X or a Y chromosome.

If the resulting zygote has two Xs, the fetus will develop into a female. If an X and a Y, the fetus will begin to develop into a female, but at a certain point this process will be overruled due to the chromosomal difference and it will develop into a male.
  A six week old fetus possesses gonads that are essentially identical regardless of the sex, but they will develop into either ovaries or testes depending on the genetic instructions.

  On the Y chromosome, there is a gene that,when expressed, produces the Sry protein, which influences the development of the gonad such that it will develop into a testis. In the absence of Sry protein, development results in an ovary. All the features of the external genitalia develop in similar ways, from identical precursors that differentiate due to specific interactions involving the differing products of gene expression, as well as the presence or absence of hormones like testosterone.

A similar process occurs again during puberty, when the secondary sexual characteristics develop. Growth hormone, gonadotropic hormone, and adrenocorticotropic hormone cause the gonads and adrenal cortex to release other hormones which initiate the development of these characteristics, as well as the maturation of the genitals.

  In this way, while testosterone is not anexclusively male hormone, it is responsible for masculinization, and while estrogen is not exclusively female, it is responsible for feminization.

Obviously, the differences in the physical characteristics of males and females are very clearly seen, but much more subtle is the sexual differentiation of the brain. Those who place political correctness over scientific fact may not like it, but the male and female brain are quite different in a variety of ways. 

  There are differences in regional sizes, such as with the sexually dimorphic nucleus of the hypothalamus. There are differences in numbers of neural and glial cells, number and types of synapses, and more. Of course there is plenty of overlap, as weare all humans, but it is undeniable that these differences exist, and it seems clear that these differences develop at different times and by different mechanisms.

  When analysing sexual behavior, cultural factors must be taken into account, but we are also beginning to understand mechanisms in the brain that inform our sexuality. These tend to be relegated to the cortex, hypothalamus, amygdala, and ventral striatum.
•  The cortex mediates the more complex and abstract aspects of any sexual experience, including the variety of emotions that can be felt during the act.
•  The hypothalamus is home to structural differences between males and females, given the differing size of the sexually dimorphic nucleus, which influences male and female sexual behavior, and it is also involved in hormone release.
•  The amygdala, in a general sense, is responsible for our experience of emotions and social salience detection, so in this context, it would seem to be involved in mate selection.
•  The ventral striatum is involved in reward,such as the anticipation of sexual activity, and other rewarding stimuli. 

Nature of Genetic Codes

The linear sequence of bases in DNA constitutes alphabet (hereditary lettering of 4 bases - A, T, G, C) which 'codes' for another linear structure, a protein, written in another alphabet of 20 amino acids.

  All properties of protein, including its secondary and tertiary structure, are ultimately determined by chromosomal DNA, and all biological properties are in turn determined by the amino acid sequence of the proteins within an organism, through protein structure and enzyme activity.

  The term 'coding' implies the relationship between DNA and protein. By coding, the hereditary lettering carried in the four alphabet of DNA is ultimately converted into the protein languag composed of twenty letter alphabet of amino acids.

Triplet Nature of the Genetic Code : 

 The genetic message coded in m-RNA molecule is translated into the amino acid sequence of a polypeptide. During this procesa sets of three m-RNA nucleotides are read successively starting from an initiator codon, AUG, which codes for methionine, till a termination codon arrives at the site on the ribosome. 
 Since a termination codon does not code for any amino acid, the polypeptide chain synthesis stops. The termination codons are also known as non-sense codons.

  The whole sequence of m-RNA starting from the initiator codon up to the triplet preceding the termination codon is known as the reading-frame, Since the reading-frame is a continuous sequence of nucleotides , addition or deletion of a single nucleotide results in a change of the triplets from that point downstream. 

  Such an event constitutes a type of mutation known as frame- shift mutation which can be induced artificially by treatment with a mutagenic dye, like acridine. Frame-shift mutations in the coliphage T4 provide a strong evidence in support of the triplet nature of codons. 

Non-Overlapping Nature of the Code : 

 The non-overlapping nature of the genetic code means that the reading frame is read in sets of three consecutive nucleotides and that the same nucleotide is not used for the consecutive triplets. For example, the non-overlapping code reads a frame ABCDABCDA as ABC, DAB and CDA. The code been overlapping involving one nucleotide, it would have been read as ABC, CDA, ABC, CDA. 

  In that case, a single change in the nucleotide sequence would cause change in more than one amino acids because the same changed nucleotide would be used in more than one codon. Experimental determination of amino acid sequences of a normal (wild-type) protel and a mutant protein shows that a single mutational event alway causes a change of only one amino acid. Thus, it is proved that the codons are non-overlapping.

Degenerate Nature of the Code :

 Another important feature of the genetic code is its degeneracy. Hed the genetic code been absolute, then each amino acid would have been coded by a single codon. In that case, the chance of mutation would have been much greater than the rate of mutation observed in practice. 
Since most amino acids have more than one codon, (i.e. degenerate),a mutant codon may be substituted by another without 

  Thus, even if a mutation occurs, the organism may still produce nermal protein. Degeneracey therefore, should be considered as a positive attribute for the stability of the genetic make-up of an organism. It is a strength of the genetic code and not a weakness.

  A notable feature of degeneracy is that, in most codons, the third nucleotide at the 3-end of the triplet appears to be of less importance than the first two. 
For example, α-alanine has four codons, GCA, GCC, GCU and GCG, or threonine, has also four codons, ACA, ACC, ACG and ACU. The first two nucleotides are fixed, while the third position can be filled by any one of the four nucleotides. During protein synthesis, the m-RNA codons form base- pairs with the t-RNA anticodons.

  The degeneracy of the m-RNA codons assumes a special significance in this perspective. Crick proposed the wobble hypothesis to explain the relationship between codons and anticodons. According to this hypothesis, the third base of the degenerate codons can form non-standard base pairing with a base in the anticodon. Standard base-pairing relationship is between A and U, and G and C. But anticodons often contain some unusual bases, like inosine, pseudo-uracil etc. 

Universality of the Genetic  Code :

 Analysis of the sequence of nucleotide s of m-RNA and of amino acids of proteins of different organisms has adduced evidence in favour of universality of the genetic code which means that the same codons stand for the same amino acids in all organisms irrespective of their taxonomic position. Although this is largely true, some exceptions have been discovered which prove that the genetic code is not absolutely universal. 

  The most notable exceptions are the mitochondrial codons Mitechondria have their own DNA which is transcribed and translated to produce proteins the mitochondrial genetic codes which are different from the universal codons are presented in Table. 
Table : Differences in genetic codes of mitochondria and organisms 

  Apart from those mentioned in Table, in the mitochondria of maize (Zea mays) the codon CGG codes for tryptophan, while this codon stands for arginine in the universal code. Also, it should be noted that tryptophan is coded by UGA in the mitochondria of mammals, Drosophila and yeast. 
Thus, mitochondrial codons are not uniform in all organisms. Maize mitochondria use the codons AGA and AGG for arginine, like the yeast mitochondria.

   In more recent times, deviations of the universal code have also been discovered in some organisms. For example, in Mycoplasma capricoleum, tryptophan is coded by the codon UGA, as in mitochondria, whereas in the universal genetic code, it is one of the termination codons. In the eukaryotic protozoan Tetrahymena UAA codes for glutamine and not for termination. Probably, more such discrepancies would be revealed in future, challenging the concept of universality of the genetic code.

Thursday, 17 December 2020

Type 1 Hypersensitivity - Pathogenesis and Clinical Menifestations

 The immune system protects the human body against disease by dispatching a bunch of immune cells, whenever the body encounters foreign material or antigens. These immune cells or effector cells elicit an inflammatory response in order to remove or eliminate these foreign antigens without causing much damage to the host.

However under certain conditions the host may elicit an exaggerated or an inappropriate immune response to a foreign antigen causing much damage to the host tissues. This exaggerated or inappropriate immune response is termed hypersensitivity.

Type I Hypersensitivity

  Type I hypersensitivity is also termed as allergy and immediate hypersensitivity since symptoms manifest rapidly within minutes to hours. Antigens eliciting these allergic reactions are termed allergens and could be anything ranging from dust, food, pollen, drugs, insect products like bee venom, microbes and many different chemicals.

  Certain individuals are genetically prone to develop Type I hypersensitivity reactions because they may inherit certain genes making them susceptible to this exaggerated immune response.
  For example they may inherit certain MHC genes making their T-cells capable of recognising an allergen or they may have abnormally high levels of circulating IgE antibodies making them more prone to develop these reactions.

  When an exogenous antigen or allergen is inhaled or ingested by a susceptible individual, circulating dendritic cells or antigen presenting cells(APC) may pick these allergens swim to a nearby lymphnode and present it over to CD4+ helper T-cells.  

   These T-cells are naive or inactive T-cells and an inactive or a naive T-cell could differentiate into an effector Th1 or a Th2 cell depending on the stimuli and the environment.

The naive T-cell after recognition of the antigen presented by the dendritic cell, in this case differentiates into an active effector Th2 cell. This may happen due to certain a cytokines like IL-4 present locally in the environment. IL-4 drives naive T-cells to differentiate into the Th2 subset.

  On subsequent encounter with the antigen these activated Th2 cells produce a number of cytokines like IL-4, IL-5 and IL-13.

IL-4 besides stimulating differentiation of more Th2 cells also causes B-cells to class switch, meaning the B-cells instead of producing IgM antibodies now would produce antigen specific IgE antibodies.

IL-5 activates and recruits eosinophils, which may release a bunch of enzymes that could damage host tissue. IL-13 enhances IgE production along with IL-4 and also stimulates epithelial cells to secrete mucus.

Mast cells in the connective tissue express certain high affinity receptors called FCε receptors. These FCε receptors are specific to the FC portion of the IgE antibodies. So the antigen specific IgE antibodies bind to these high affinity surface receptors on the mast cells. The individual at this point is said to be sensitised to the antigen.

On subsequent re-exposure the antigen binds to these antibodies sitting on the mast cells cross linking them and causing the granulation of the mast cell contents. The contents in the mast cells are preformed chemical mediators already present within the cells and newly formed mediators as well as cytokines.
  These are powerful mediators responsible for the clinical manifestation of Type I hypersensitivity reaction. Some of the preformed mediators contained in the mast cell granules are histamine, enzymes like chymase, tryptase and eosinophilic chemotactic factors.

  Histamine causes smooth muscle contraction of the airways making it difficult to breathe.

  It also causes vasodilation of blood vessels and increases their permeability. This causes an increased blood supply to the site of action and leakage of fluid through the vessels causing edema and swelling.
  Enzymes like chymase and tryptase cause damage to the adjacent tissues and lead to generation of kinins and complement, leading to further inflammation.
  Eosinophilic chemotactic factors recruit eosinophils to the site of allergy and are implicated in late phase reactions.

Manifestations of these mediators namely histamine and enzymes are called early phase reactions since these manifestations occur within 5 to 30 minutes of the allergen exposure and may subside within 60 minutes.

Newly synthesized mediators are usually arachidonic acid derivatives namely leukotrienes and prostaglandins.
   Leukotrienes C4 and D4 are several thousand times more powerful than histamine in causing smooth muscle contraction and also causes an increased vascular permeability.  
Leukotrienes B4 is a chemotactic agent for neutrophils, monocytes and eosinophils.

Prostaglandin D2 is the most abundant prostaglandin produced and causes vasodilation, increased permeability of the blood vessels and also bronchospasm.

Cytokines like IL-1 and TNF-α are pro-inflammatory cytokines that can cause further inflammation by recruiting leukocytes.
IL-4 causes further differentiation of the Th2 cells and amplifies the response.
IL-5 activates and recruits eosinophils. These mediators namely leukotrienes and cytokines are responsible for a late phase reaction which kicks in 2 to 24 hours later and does not require additional antigen exposure.
  They may last for several days and are due to recruitment of more eosinophils, neutrophils, monocytes as well as CD-4 T-cells and a sustained inflammation in the environment.

Clinical Manifestations of  Type-I Hypersensitivity 

   Some of the clinical manifestations of Type I hypersensitivity could be itching, urticaria and edema where there could be pruritic wheels surrounded by erythema.
 •  Allergic rhinitis and allergic asthma are other manifestations of Type I hypersensitivity.
• One of the most important complications of Type I hypersensitivity is systemic anaphylaxis, which is a widespread systemic manifestation of Type I Hypersensitivity characterized by bronchospasm resulting in difficulty in breathing, laryngeal edema for the compromising breathing, abdominal cramps as well as diarrhea.
• The patient may also develop ischemia in multiple organs due to widespread vascular permeability leading to anaphylactic shock and death.