Tuesday, 20 July 2021

Concept of Antigenicity and Immunogenicity

Here we will understand about two more confusing terms in immunology. These are antigenicity and immunogenicity. To understand these terms we should have an idea about how adaptive immune response works.

How adaptive immune system works?

  Main cells of adaptive immune system are known as lymphocytes. The two important lymphocytes are B-cells and T-cells. B-cells deal with extracellular pathogens and the T-cells deal with intracellular pathogens.

Both of these cells are produced in the bone marrow. Although B lymphocytes complete their maturation in the bone marrow itself. T lymphocytes travel to the organ called thymus where they finally mature.

After the maturation stage, These cells are released into the blood they circulate in blood and have ability to enter tissues where they can recognise pathogens, if present. For recognition purpose these cells possess specific receptors. which we know as B-cell receptors and T-cell receptors.

When this happens these lymphocytes are stimulated to respond and mount a specific immune response.
   When B cells recognize an antigen they get activated and get differentiated into plasma cells. These plasma cells produce antibodies specific to that antigen.
  The antibodies bind to those antigens and activate defense mechanisms that lead to the destruction of the pathogen. Some of the activated B-cells remain in circulation as memory B-cells.
This immune response is known as humoral immune response.

Similarly when T cells recognise the antigen. They differentiate into T cell subsets namely T helper cells and T cytotoxic cells. These subsets of T cells further lead to the destruction of the invader and the infected cells.
  Some of these cells also differentiate into memory T cells. This immune response is known as cellular immune response.

Immunogenicity and antigenicity

When pathogen such as bacteria say invaded the body, here bacteria are the antigens. once inside the body B-cells recognise these antigens or more specifically antigenic determinants or epitopes of the bacteria. After recognition of the antigen B cells differentiate into plasma cells which are specific to this antigen.

  These plasma cells produce specific antibodies which bind to the antigens and further lead to the destruction of that pathogen. So Substance and antigen induced an immune response.

The ability of a substance to induce an immune response, that is either humoral or cellular immune response is known as immunogenicity. And such substances are called immunogenic.

Now suppose a related but harmless bacteria with similar epitopes invades the body. This time since antibodies specific to the epitopes of this bacteria are already present inside the body. These antibodies bind to invaded bacteria.

" here note that the bacteria does not induce an immune response it just binds to product of an immune response that is antibodies."

So the ability of a substance to bind specifically to the products of an immune response is known as antigenicity. And such substances are called antigenic.

Definition of immunogenicity and antigenicity

Immunogenicity is defined as the ability of a substance to induce an immune response. Whereas antigenicity is defined as the ability of a substance to bind specifically to the products of the immune response.

"All molecules that are immunogenic are antigenic too."

All molecules which are able to induce an immune response are also able to bind to the final products of the immune response.

Humoral immune response was induced by the antigen (bacteria) and the antibodies produced bind specifically to that antigen. So substances which are immunogenic are also antigenic.

"All antigenic molecules cannot be considered immunogenic."

There are some substances which are able to bind to the final products of an immune response. That means they are antigenic but they cannot induce an immune response by themselves.
  Well-known example of such substances or haptens. Haptens are able to react with the antibodies, but they are unable to stimulate their production.

Thursday, 1 July 2021

Northern Blotting Steps

 Northern Blotting is a blotting technique for the detection of RNA. the overall technique and the steps involved in northern blotting is almost similar to the southern blotting. Northern Blotting is a technique which will be suitable for a study  gene expression and analysis.

Steps of Northern Blotting

1) RNA gel Electrophoresis

• The first step in northern blotting is RNA gel electrophoresis. The RNA molecules isolated from cells are separated according to size by Gel electrophoresis.

• RNA molecules are negatively charged, so they move from negative to the positive electrode during gel electrophoresis.

• RNA is a single-stranded nucleic acid but, still this RNA gel electrophoresis also includes the denaturation step.
- This is because RNA molecules fold onto themselves. Because of intramolecular base pairing they form secondary structures. So if we want to separate them on the basis of their molecular weights we need them to bring in the linear shape.
- Otherwise the secondary structures of RNA molecules will affect their electro phoretic mobility during gel electrophoresis.

• To denature RNA formaldehyde is used as a denaturing agent. Thus denaturring gel electrophoresis is used in this step.


2) Blotting (RNA)

• The second step is blotting.
The separated RNA molecules are now transferred from the gel to the suitable solid support, such as the Nylon membrane.

• There is the traditional way to transfer the RNA to the Nylon membrane. The basis of this transfer is the capillary action.  

System of Northern Blotting

  The tray is filled with the suitable transfer buffer. A support for gel on membrane is kept in this buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. These are placed such that they imbibe the buffer. They act as a wick

-  Next the gel containing RNA is placed on the top of it.
-  An nylon membrane of the same size as that of the gel is placed over it.
- Again a thick stack of blotting papers or absorbent paper is placed over the membrane.
- This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.

  From the tray now Rises through the gel taking with it the RNA molecules. Once the RNA molecules reach the nylon membrane they become adsorbed tightly to the membrane.

  The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. During this transfer the RNA fragments retain the same pattern of separation they had on the gel.  

3) Hybridization and washing

• The third step involves hybridization with probe and washing. Suppose these bands are the RNA molecules on the nylon membrane. For the detection of these RNA molecules first we need a probe that will specifically bind to these target RNA molecules.

• The probe can be a complimentary labeled RNA sequence or labelled complementary DNA sequence.

• When nylon membrane is incubated with these probe molecules probes will bind specifically to their complimentary target RNA molecules.

• Unbound probes are removed by washing.

4). Detection

• In the fourth and final step detection is done. Detection and visualization method depends on the type of labelled molecule we used for hybridization step.

• The probe is detected by autoradiography, fluorescence or a color change depending on what label we have used in the probe.

These were the steps involved in the northern blotting.

Applications of Northern Blotting

The main applications of northern blotting include
• Gene expression studies such as to determine when and where a particular gene is expressed.
• To identify the presence of closely related species
• To determine the size and abundance of RNA.
• For the analysis of RNA processing.

Modern methods such as PCR have replaced the methods of southern and northern blotting. This is because PCR and PCR based techniques are more simple quick and of more precise nature.

Saturday, 26 June 2021

Capsule Staining by Maneval's Method

Capsule Staining by Maneval's Method

Bacterial cell is consisting of various structural components such as  cell wall, cell membrane, capsule, flagella etc.
Capsule is one of the important component but it is not present in all bacteria.

Accordingly bacteria are grouped as capsulated and noncapsulated. Capsule is a slimy, gummy & mucilaginous covering present around the cell wall.

Capsule is made up of 98 % water and 2 % polysaccharides and therefore it is nonionic in nature. However, capsule of some bacteria is made up of polypeptides.

Capsule protects bacterial cell from desiccation, starvation, phagocytosis and phage infection. It also give the property of virulence to the bacterial cell that mean Capsule is increased pathogenicity of an organism.

  Capsule can be demonstrated in microscope by various special staining methods like -India ink method, Anthony's method, Maneval's method & Hiss method.
Maneval's method is most commonly used method for Capsule staining.

Principle of Maneval's capsule staining

It uses the principle of negative staining for demonstration of capsule. Background is stained, cytoplasm is stained and capsule remains colourless.

Requirements

- Clean grease free slide
- Nichrome wireloop
- 24 hrs old culture of capsulated bacteria
- 1% Congo Red Solution
- Maneval's Stain

Composition of Maneval's Stain

• 1% Acid fuchsin - It stains cell cytoplasm
• 5% Phenol - It increases penetration power of stain
• 30 % FeCl3 - It is a Chemical fixative
• 20 % Acetic acid - It Changes the background from red to blue

Procedure (Steps) :

• Prepare the loop full culture smear on the slide by Nichrome wireloop, air dry but do not heat fix
• Put a drop of 1 % congo red at one end of the slide and spread it across the smear with help of another slide
• Dry the film of congo red
• Flood the smear with Maneval's stain and allow to react for 4 minutes.
• Discard the excess stain but do no wash with water.
• Dry the slide and observe under the oil immersion objective.

Microscopic Observation :

In the microscopic field you will see Blue colour background, Pink colour bacterial cell and Colourless Capsule.

Microscopic Observation of Capsule Staining by Maneval's Method

Example of capsulated bacteria :

Klebsiella spp.
Azotobacter spp.
Rhizobium spp.
Bacillus spp
Xantomonas spp.

Friday, 25 June 2021

One Step Growth Curve Experiment of Virus

 One step growth experiment is An experiment by which molecular events that are occurring during reproduction of  virus can be observed.
• It reveals the fundamental nature of virus replication process.

• This process was first performed by Ellis & Delbruck in 1939 by using T2 bacteriophages. 

• They also determined the plague counting method for the enumeration of bacteriophages.

• In this experiment, only a single or one cycle of virus growth is observed.

• Therefore, it is called as one step growth experiment. 

• Excess number of host cells are allowed to infect with phage particles.

• This makes the infection synchronous. That means the simultaneous infection of large number of particles to the host cell is taking place.

• Observation made on such host cell culture is similar to observation made on single host cell infected by a phage. 

• In the experiment, excess host cells are infected with phage particles at a ratio of 1:10. 

• This is done to prevent the adsorption of more than one virus per cell. 

• Mixture is incubated for a short period of time (5 min).

• This incubation allows the adsorption of phage particle on host cell. 

• If the bacteria are in excess, all the phage particles will be adsorbed.

• Such mixture is then diluted to such an extent (1:1000) that the virus particles released after first round of replication cannot adsorb to uninfected cell. 

• Thus, only one step of virus growth can occur. 

• Samples of diluted mixture are then removed at regular time interval & used for plaque count. 

• This gives a measure of infectious centers i.e. the infected bacteria & number of virus particles (i.e. No. of plaques per ml). 

• When a log no, of plague forming units/ml is potted against time, a curve is obtained & it is termed as one step growth curve.

 
• This one step growth curve shows the various events that are occurring during the virus replication cycle.

• This curve gives three distinct phases -
   1]. Latent Period
   2]. Burst or Rise Period
   3]. Plateau Period

One step growth curve of virus

I]. Latent Period:

It is the period from infection to cell lysis. 

• During this, there is no release of new virus particles from infected cells.

• Therefore, the plaque count remains constant. 

• T phage has latent period of 22 to 23 min at 37°C.

• This period can be divided into two phases as 'Eclipse' period & 'Intracellular Accumulation' period. 

• Time from infection until intracellular accumulation of phages is called as 'Eclipse' period. 

• T2 bacteriophage has eclipse period of about 11.5 min at 37°C.

• In this, gene expression, protein synthesis & genome synthesis occurs. 

• The time from initiation to the end of intracellular accumulation of phages is called as  'Intracellular Accumulation' period. 

• During this, phage proteins & genomes assemble into new phage particles. 

• T2 bacteriophage requires the period of about 11 to 12 min. at 37° C for this period.

II]. Burst Period or Rise Period

• The time from initiation of infected host cell lysis to the end is called rise or burst period

• At the end of latent period, each infected cell lyses & liberates a crop of new virus particles. 

• During this phase, there is release of new viral particles from infected cells & therefore, plaque count increases rapidly. 

• T2 bacteriophage has the rise period of about 10 min. at 37°C.

• Due to the asynchrony of infection the rise period is slightly extended.

III] Plateau Period :

• This period represents the end of all infected host cell lysis.

• The newly liberated phage particles fail to meet uninfected host cells due to high dilution. 

• Therefore, during this phase, the plaque count remains constant. 

• T2 phage enters in plateau in about 30 min. at 37°C.

Burst Size - Burst size is defined as the number of virus particles produced from the infection of a single cell. The burst size is caleulated using following formula -

• T2 phage has a burst size of less than 100 phages/cell.

• Burst size varies from 20 to 3000 virions/cell for different viruses.

Thursday, 24 June 2021

Lysosomal Storage Diseases

Lysosomal storage diseases

 Lysosomalstorage disorders (LSDs) arise from the incomplete digestion of macromolecules. Causing the lysosomes to become large and numerous enough to interfere with normal cell functions.

All lysosomal storage disorders are autosomal recessive except
• Fabry's disease and
• Hunters syndrome
These are x-linked recessive diseases.

Affected organs and lysosomal storage diseases depend on the tissue. Where most of the material to be degraded is found and where the degradation normally occurs.

Fabry's disease

Fabry's disease presents with -
• Peripheral Neuropathy of the hands and feet.
• Angiokeratomas, 
• Cardiovascular disease
• Renal disease.
• Patients also have a 20-fold increased risk and stroke.

Fabry's disease is caused by a deficiency in α-galactosidase enzyme.

Gaucher's disease

Gaucher's disease presents with - • Hepatosplenomegaly
• Aseptic necrosis of the femur,
• Bone crisis,
• Pancytopenia or thrombocytopenia.

Gaucher's cells are macrophages that appear like crumpled paper.
Neurological symptoms occur in less frequent subtypes of gaucher's disease.

Gaucher's disease is caused by a deficiency in β-Glucocerebrosidase. This leads to an accumulation of glucocereboside.

Niemann-Pick disease

Niemann-pick disease presents with -
• Progressive Neurodegeneration
• Hepatosplenomegaly
• Cherry red spots on the macula
• Foam cells.
 
Niemann-pick disease is caused by deficiency in enzymes spihingomyelinase. This leads to an accumulation of sphingomyelin with the central nervous system involvement.

Tay-Sachs disease

Tay-sachs disease presents with -
• Progressive Neurodegeneration,
• Developmental delay,
• Cherry red spots on the macula,
• Lysosomes that are appear like onion skins
• There is no hepatosplenomegaly.
 
Tay-sachs disease has a deficiency of Hexosaminidase A. This leads to an accumulation of GM2 Gangliosides.

Krabs disease

Krabs disease presents with -
• Peripheral neuropathy,
• Developmental delay,
• Optic atrophy,
• Fever and
• Globoid cells,
• Often at times it can present with irritability.

Krabs disease has a deficiency in galactocerebrosidase. This leads to an accumulation of galactocerebroside.

Metachromatic leukodystrophy

Metachromatic leukodystrophy presents with -
• Central and peripheral demonization with ataxia and dementia,

Metachromatic leukodystrophy has a deficiency of arylsulfatase A. This leads to an accumulation of cerebroside sulfate.

Hurler's syndrome

Hurler's syndrome presents with -
• Developmental delay,
• Gargoylism,
• Airway obstruction,
• Corneal clouding and
• Hepatosplenomegaly.

  Hurler syndrome has a deficiency in α-L-iduronidase. This leads to an accumulation of heparan sulfate and Dermatan  sulfate. Deposits in coronary arteries leads to ischemic heart disease.

Hunter syndrome

Hunter syndrome presents as a mild form of hurler's syndrome. But also includes aggressive behavior and lacks corneal clouding.

  Hunter syndrome has a deficiency in it iduronate sulfatase. This leads to an accumulation of heparan sulfate and Dermatan sulfate.

Pompe's disease

Pompe's disease presents with -
• Left ventricular hypertrophy which leads to outflow tract obstruction and cardiac failure.

  Pompe's disease has the deficiency in lysosomal α-1,4-glucosidase. This leads to glycogen deposits in the lysosomes.

I-cell disease

I-cell disease presents with -
• Growth and developmental delay,
• Course facial features,
• Gingival hypertrophy and
• Skeletal abnormalities.
  it's called I-cell due to cytoplasmic inclusions in fibroblasts.

It is caused by the inability to properly synthesize the mannose-6-phosphate tag required for targeting enzymes to lysosomes.

Treatments of Lysosomal storage diseases

The treatment of lysosomal storage diseases include
• Enzyme replacement therapy

• Substrate reduction therapy 

•Molecular chaperone therapy. 

Rolling Circle Model of Replication

The rolling circle model of DNA replication was proposed in 1968. This model explains mechanism of DNA replication in circular plasmids and single stranded circular DNA of viruses.

For many plasmids replication is not tied to chromosomal replication. Many circularly closed plasmids replicate autonomously; by a method called rolling circle replication. Rolling circle replication is also called unidirectional replication.

The machanism of Rolling circle replication

  In rolling circle replication a replication initiator protein called Rep A binds to a section of the double-stranded DNA called the origin of replication or Ori.
Rep A is encoded by a plasmid gene.

Rolling circle model of replication

Rep A nicks one strand of the DNA and holds on to the 5' end of the strand.

The 3' end with its free (OH) hydroxyl group serves as a primer for a host DNA polymerase to begin to replicate the intact complementary strand.

The rep A initiator protein recruits a helicase that unwinds the DNA. As the DNA unwinds it becomes coded by single strand DNA binding proteins.

  As replication proceeds the nicked strand which continues to be covered with single strand DNA binding proteins, progressively peels off until replication of the intact strand is complete.

The two ends of the nicked single strand are rejoined by the Rep A protein and released.

DNA ligase seals the nick in the double-stranded molecule.

The single-stranded DNA can now be replicated. A region of the DNA becomes looped allowing RNA polymerase access to the DNA to form a primer.

Host DNA polymerases use the primer as a starting point for the synthesis of DNA.

  Finally DNA ligase seals the remaining nick resulting in a double-stranded plasmid.
Each of these plasmids can undergo replication again by the same method.

Wednesday, 23 June 2021

Detailed Structure of Tobacco Mosaic Virus (TMV)

 TMV (Tobacco Mosaic virus) is a plant virus which infects a wide range of plants. TMV causes disease of Tobacco, Tomato and other members of the family Solanaceae. TMV belongs to Tobamovirus group.

TMV is first virus ever discovered by Iwanowski in 1892, and latter isolated by Stanley in 1935.

The virus infection causes characteristic patterns, such as
"Mosaic"-like mottling(spots) and discoloration on
the leaves (hence the name).

TMV has historical significance as it is

  • a first discovered
  • first crystallized
  • first sequenced plant virus to be shown to contain RNA as genetic material and
  • first virus to be shown to contain RNA and protein.

Symptoms of TMV on plant

  - Mosaic patterns
  - Mottling,
  - Necrosis,
  - Stunting,
  - Leaf curling
  - Yellowing of plant tissue.

Structure of TMV

• TMV is the best studied virus with helical naked capsid

• It is rod shaped virus of 300 nm length and 18 nm diameter. 

• The central opening along the axis has diameter of 4 nm. 

• The capsids are made up of subunits that are called capsomers.

• Capsid consists of 2130 identical capsomers. 

• Each capsomers is made up of 158 amino acids. 

• Capsomers are arranged in a helix around central hole of about 4nm radius. 

Structure of TMV virus

• TMV consist of a single stranded, unsegmented and positive sense RNA as a genetic material. 

• There are about 6395 nucleotide in RNA. 

• RNA has MW of 2.1 x 10⁶ daltans. 

• The RNA is arranged in helix.
• Each turn of RNA helix contains 49 nucleotides and 16.3 capsomeres are attached RNA per turn of helix. That means 3 nucleotide are linked with single capsomer.

• The ratio of nucleoids to capsomers is 3:1

Genome of TMV

The TMV genome contains 4 genes which are -
1]. MTH gene, (Methyl transferase & helicase gene)
  2]. RNP gene,
  3]. MP gene and
  4]. CP gene.

Genome of TMV virus
 
• The MTH gene codes for the enzyme methyl transferase and RNA helicase. The methyl transferase activity plays role in RNA capping and the role of RNA helicase is not clear.

• RNP gene codes for RNA dependent RNA polymerase or RNA replicase. It is useful for making the copies of RNA.

• MP gene codes for movement protein and this proteins mediate the cell to cell movement of the viral RNA in the plant tissues.

• CP gene codes for coat protein or Capsid proteins.