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.


  • 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.

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 calculated 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.

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.

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.

Classification of Enzymes

  Enzymes are classified according to the type of reaction they catalyse. All enzymes have formal 'EC' (Enzyme Commission) number and names, and most have trivial names.

International Union of Biochemistry had formulated a committee to look into uniform system of nomenclature and classification of enzymes. It was known as Enzyme Commission. Classification of enzyme is essential for ready reference about the enzyme as well as for the identification of new enzyme. The system of classification accepted by IUB is applied.

According to the International Union of Biochemistry (IUB) enzymes are classified into seven major classes.

IUB - EC numbers

Each enzyme is described by a sequence of four numbers preceded by "EC".

  • First digit represents the Class - Seven classes on the basis of the nature of reaction.
  • Second digit stands for the Subclass - Where, the enzyme classes are further divided on the basis of groups or chemical bond attacked by the enzyme.
  • Third digit is for the Group - indicates Coenzyme/ Cofactor required for enzyme action.
  • Fourth digit gives the number of the particular enzyme in the list indicates - serial number of individual enzyme.

e.g. EC 1:1:1:1 is alcohol NAD Oxidoreductase.

The seven classes as per IUB are as follows:

1]. EC-1 : Oxidoreductase
2]. EC-2 : Transferase
3]. EC-3 : Hydrolase
4]. EC-4 : Lyase
5]. EC-5 : Isomerase
6]. EC-6 : Ligase
7]. EC-7 : Translocases

IUB system of enzyme classification; classes have been formed on the basis of nature of reaction catalyzed.

EC-1 Oxidoreductases

Catalyse removal or addition of Hydrogen atoms, Oxygen atoms or Electrons from one substrate to another.
Oxidoreductases catalyzes oxidation-reduction reactions.

A(red) + B(ox) A(ox) + B(red).

Enzymes in this category include :  

• Dehydrogenases
• Reductases
• Oxidases
• Peroxidases.

Examples of Oxidoreductases

• Lactate dehydrogenase (LDH)
• Glucose 6-phosphate dehydrogenase (G-6-PD)
• Cytochrome oxidase
• Alcohol Dehydrogenase

EC-2 Transferases

Transferase Catalyses the transfer of a group such as, Amino, Carboxyl, Methyl or Phosphate, etc (except hydrogen) from one molecule to another.

A-X + B A + B-X

Enzymes in this category include : 

• Amino transferase or Transaminase

• Kinase: catalyzes the transfer of phosphate groups

Examples of Transferase

• Aspartate amino transaminase(AST)
• Alanine amino transaminase (ALT)
• Hexokinase
• Transmethylase

EC-3 Hydrolases

Hydrolases catalyze the cleavage of Peptide, Glycosidic, Ester and Ether and some other bonds with the addition of water(H₂O).

A-B + H₂O ➞ A-OH + B-H

Enzymes in this category are:

• All digestive enzymes like -
   - α-amylase,
   - Pepsin,
   - Trypsin,
   - Chymotrypsin,
   - Lipase
• Acid phosphatase and alkaline phosphatase.

EC-4 Lyases

Lyases catalyze the cleavage of Carbon-Oxygen(C-O), Carbon-Carbon(C-C) and Carbon-Nitrogen (C-N) bonds by means other than hydrolysis or oxidation, giving rise to compound with double bonds or catalyze the reverse reaction, by the addition of group to a double bond.

X-A-B-Y A=B + X-Y

In cases where reverse reaction is important, then synthase, (not synthetase of group EC-6) is used in the name.

Examples of  Lyases

• Aldolase
• Fumarase
• Argininosuccinase
• Carbonic anhydrase
• HMG CoA Lyases

EC-5 Isomerases

Isomerases catalyze intramolecular structural rearrangement in a molecule. They are called Epimerases, Isomerases or Mutases, depending on the type of isomerism involved.

      ABC CAB

Examples of Isomerases 

 All racemases and epimerases
  - Phosphoglucomutase
  - Triphosphate isomerase
  - Phosphohexose isomerase

EC-6 Ligases (Synthetases)

Ligases catalyze the joining of two molecules coupled with the hydrolysis of ATP.

A + B + ATP ➞ A B+ ADP + Pi

Examples of Ligases

• Glutamine synthetase
• Pyruvate carboxylase
• DNA ligases
• PRPP synthetase

EC-7: Translocases (A new EC Class)

Translocases catalyze the movement of ions or molecules across membranes or their separation within membranes.

Examples of Translocases

  • Enzymes catalyzing the translocation of : Hydrogen ions (H+), Inorganic cations, Inorganic anions, amino acids and Peptides, and carbohydrates and their derivatives.
  • Enzymes of the reaction that provided the driving force for the translocation linked to Oxidoreductase reactions, Hydrolysis of a nucleoside Triphosphate, Hydrolysis of a diphosphate, and Decarboxylation reaction.

Viroids : Characteristics, Structure, Types and Replication

 Viroids are the smallest known infectious agents consisting of a small, circular, RNA molecules.
A VIROIDs are a Virus(VIR) like(OID) particles.
Until 1970, viruses were considered as the smallest infectious agents. However, the discovery of viroids has proved that the infectious entities smaller than virus exist in nature.

Diener & Raymer first time discovered the Potato Spindle Tuber Viroid (PSTV). It was responsible for potato spindle tuber disease.

  Most viroid cause plant discases & most common example is potato spindle tuber viroid. Today, 33 viroids are known.

The human disease known to be caused by viroid is hepatitis D. This viroid is enclosed in a hepatitis B virus capsid. For hepatitis D to occur, there must be simultaneous infection of cell with both the hepatitis B virus & the hepatitis D viroid.

Characteristics of Viroids :

  1. Viroids are obligate intracellular parasite.
  2. They are smaller than viruses. 
  3. Viroids are single stranded covalently closed circular RNA molecules.
  4. They are only 264-400 nucleotides long.
  5. Viroid RNA does not code for any protein.
  6. They use host polymerase for replication.
  7. They do not have capsid (protein coat).

Examples of Viroids

  • Genus Pospiviroids: PSTV (potato spindle tuber viroid)
  • Genus Coleviroids: CbVd 1 (coleus blumei 1)
  • Genus Hostuviroids: HSV (hop stunt viroid)
  • Genus Avsunviroids: ASBV (avocado sunblotch viroid)
  • Genus Cocadviroids CCCV (coconut cadang-cadang viroid)
  • Genus Pelamoviroids:  PLMVD (peach latent mosaic viroid)
  • Genus Apscaviroids: ASSVd (apple scar skin viroid)

Structure of Viroid

  • The viroids are single stranded circular RNA molecules.
  • Most of the nucleotides in the RNA are base paired, producing a double stranded RNA molecules.
  • The single stranded RNA circle has extensive intra-strand base pairing & unpaired loops at intervals.
  • This structure protects the viroid from the action of ribonuclease.
  • Structure of a viroid-circular single-stranded RNA with some pairing between complementary bases and loops where no such pairing occurs.
  • There are two main groups of viroids on the basis of structure, these are self cleaving and non- self cleaving.
  • Non-self cleaving viroids replicate in nucleus and fold into "dog bone" or rod-like structure.

Structure of Viroid 

  • Five domains identifiable in Non-self cleaving viroids
  - Terminal left (TL)
  - Terminal right (TR)
  - Pathogenicity (P)
  - Central (C)
  - Variable (V)

Replication of Viroids

  • The replication of the viroid takes place in the nucleus of the host cell.
  • Viroid RNA is a positive strand RNA and it replicates by the rolling circle mechanism in vivo.
  • All components required for the replication are provided by the host.
  • The rolling circle replication occurs by two mechanisms which are symmetric mode of replication and asymmetric mode of replication.

Symmetric mode of replication

  • Symmetric mode of replication is the most common mode of replication in viroids.
  • According to Symmetric mode of replication, RNA directed RNA polymerase catalyses synthesis of new concatemeric negative(—) strand using the viroid position (+) RNA as a template.
  • Viroids are ribozymes and therefore they catalyze their self cleavage.
  • Self cleavage of concatemeric negative strand produce a monomeric subunits
  • This is followed by the Ligation by host RNA ligase enzyme produce circular molecule.
  • This circular negative strand is copied by the RNA polymerase to produce a concatemeric positive strand.
  • Cleavage of concatemeric positive strand produces monomers.
  • These monomers again circularize and produce positive RNA i.e. viroids.

Replication of Viroids

Asymmetric mode of replication

  • In the Symmetric mode of replication, the concatemeric negative strand is copied directly to a concatemeric positive strand.
  • That means, Here self cleavage of monomers of negative strand is not carried out. Instead of that the intake of negative strand is used to synthesise the complementary concatemeric positive strand.
  • It is then cleaved specifically to monomers.
  • These monomers are ligated to form many viroids. 

Prion Proteins : Characteristics, Types, replication and disease

 Prions are small infectious protein particles responsible for fatal Neurodegenerative diseases in humans and animals. Different from viruses & viroids and they do not contain nucleic acids. Prion is actually misfolded protein.

  All organisms including prokaryotes, eukaryotes & viruses possess nucleic acids. But nucleic acids are absent in some infectious agents and they are called as prions.

In 1982, American Scientist Stanley B. Prusiner isolated and purified such infectious agents. He coined the term 'Prion' by combining first letters of the words proteinaceous and infectious and -on to make analogy to Piron.
  In 1997, Prusiner was awarded the Nobel Prize in medicine and physiology.

Characteristics of Prions :

  • Chemically prions are glycoproteins.
  • Prions do not contain DNA, RNA or capsids.
  • Prions consist of only hydrophobic protein of 33 to 35 kilodalton (253 amino acids). which is often called Prp (Protease resistant protein).
  • Prions are responsible to cause Neurodegenerative discase in which large vacuoles are produccd in the brain tissue.
  • Prions have not been visualised.
  • Prions are highly resistant  nucleases & UV radiations.
  • They are sensitive to chemicals that denature proteins.
  • They are also sensitive to heat.
  • They cause slow infections. The incubation period ranges from one to several years.
  • Prions are very difficult to decompose biologically; they survive in soil many years.
  • Prion discases are transmitted directly from one person to another, indirectly by fomites and by the ingestion of contaminated meat.

Types of Prions :

Prions are mainly 2 types
  1) PrPc (C=cellular)
  2) PrPsc (sc = Scrapie)


  • PrPc is a Cellular prion protein
  • Gene of PRPc is located on chromosome no. 20.
  • PrPc protein consists Alpha helix- 43% and Beta sheets- 03%
  • PrPC present on surface of cell.
  • PrPc Prion proteins involved in communication between neurons, cell death, and controlling sleep patterns.
  • Mutation in PrPc gene which leads to conversion of Aspartate into aspargine was observed in diseased individual.


  • PrPSc is a Scrapie form of Misfolded protein
  • PrPSc prions are Protease resistant
  • PrPSc consists Alpha helix 30% and Beta sheets- 43%.
  • The second type of prion protein, known as PrPSc, is the disease-causing form. Organisms with it develop spongiform disease.
  • These misfolded proteins were observed to be build up in thalamus region.

  The prion diseases are often called transmissible spongiform encephalopathies because of the appearance of holes in the brain tissue.

Replication of Prion :

Scientists are still working out the details, but evidence supports the idea that when PrPc comes into contact with PRPsc it is converted to PrPsc.

The result is a chain reaction that multiplies copy after copy of the infectious prion. Because of their abnormal shape, PrPsc- proteins tend to stick to each other. Over time, the PrP-SC molecules stack up to form long chains called "amyloid fibers".

Prion Disease and Symptoms

  The common symptoms of Transmissible Spongiform Encephalopathies (TSE's) are :

  • loss of muscle co-ordination which leads to difficulty in walking
  • Dementia which is characterized by the loss of memory, diminished intellect & poor judgment
  • Progressive insomnia (inability to sleep)
  • Progressive paralysis and death.

  One of the most common disease known to be caused by prions is the "Scrapie" of Sheep & Goat. It causes the animal to scrap or scratch itself against obstacles such as rocks or trees due to itching sensation.

  High level of kuru appear among the Fore people of Popua New Guniea. Kuru is a one type of Prion disease.

Introduction of the Immune System, Cells and Basic Immunology

  Despite being surrounded by harmful microorganisms, toxins, and the threat of our own cells turning into tumor cells, humans manage to survive, largely thanks to our immune system.

The immune system is made up of organs, tissues, cells and molecules that all work together to generate an immune response that protects us from microorganisms, removes toxins, and destroys tumor cells.

The immune response can identify a threat, mount an attack, eliminate a pathogen, and develop mechanisms to remember the offender in case you encounter it again - all within 10 days.

  In some cases, like if the pathogen is particularly stubborn or if the immune system starts attacking something it shouldn’t like your own tissue, it can last much longer, for months to years, and that leads to chronic inflammation.

Our immune system is of two types
1. Innate immune response
2. Adaptive immune response.

1. Innate Immune Response

  The innate immune response includes cells that are non-specific, meaning that although they distinguish an invader from a human cell, they don’t distinguish one invader from another invader.

  The innate response is also feverishly fast; working within minutes to hours. That’s cause it’s responsible for causing fevers. The trade off for that speed is that there’s no memory associated with innate responses. In other words, the innate response will respond to the same pathogen in the exact same way no matter how many times it sees the pathogen.

The innate immune response includes things that you may not even think of as being part of the immune system. Things like chemical barriers, like lysozymes in the tears and a low pH in the stomach, as well as physical barriers like the epithelium in the skin and gut, and the cilia that line the airways to keep invaders out.

2. Adaptive Immune Response

  In contrast, the adaptive immune response is highly specific for each invader. The cells of the adaptive immune response have receptors that differentiate one pathogen from another by their unique parts called antigens.

These receptors can distinguish between friendly bacteria and potentially deadly ones. The trade off is that the adaptive response relies on cells being primed or activated, so they can fully differentiate into the right kind of fighter to kill that pathogen, and that can take a few weeks. But the great advantage of the adaptive immune response is immunologic memory.

The cells that are activated in the adaptive immune response undergo clonal expansion which means that they massively proliferate. Each time the adaptive cells see that same pathogen, they massively proliferate again, resulting in a stronger and faster response each time that pathogen comes around.

  Once the pathogen is destroyed, most of the clonally expanded cells die off, that’s called clonal deletion. But some of the clonally expanded cells live on as memory cells and they’re ready to expand once more if that pathogen ever resurfaces.

Immune Cells and it's function

White blood cells or leukocytes are the key cell of Immune system. Hematopoiesis is the process of forming white blood cells, as well as red blood cells, and platelets and it takes place in the bone marrow.

Hematopoiesis starts with a multipotent hematopoietic stem cell which can develop into various cell types - it’s future is undecided. Some become myeloid progenitor cells whereas others become lymphoid progenitor cells.

The myeloid progenitor cells develop into myeloid cells which include  neutrophilseosinophilsbasophilsmast cellsdendritic cells, macrophages, and monocytes, all of which are part of the innate immune response and can be found in the blood as well as in the tissues. 

The neutrophils, eosinophils, basophils, and mast cells are considered granulocytes, because they contain granules in their cytoplasm,and the trio of neutrophils, eosinophils, and basophils are also referred to as Polymorphonuclear cells, or PMNs, because they’re nuclei contain multiple lobes instead of being round.
  The mast cells, aren’t considered PMNs because their nucleus is round. During an immune response, the bone marrow produces lots of PMNs, most of which are neutrophils.

Neutrophils use a process called phagocytosis- that’s where they get near a pathogen and reach around it with their cytoplasm to“swallow” it whole, so that it ends up in a phagosome. From there, the neutrophils can destroy the pathogen using two methods - they can use their cytoplasmic granules or oxidative burst. First, the cytoplasmic granules fuse withthe phagosome to form the phagolysosome.

   The granules contain molecules that lower the pH of the phagolysosome, making it very acidic, and that kills about 2% of the pathogens. Now, the neutrophil doesn’t stop there. It keeps swallowing up more and more pathogens until it’s full of pathogens, and at that point, it unleashes the oxidative burst.

  During an oxidative burst, the neutrophil produces lots of highly reactive oxygen molecules like hydrogen peroxide. These molecules start to destroy nearby proteins and nucleic acids - a bit like the neutrophil dumping bleach on itself and then lighting itself on fire. This process kills the neutrophil - a bitof a suicide mission. but each neutrophil takes out a lot of pathogens with it. Now, in comparison to neutrophils, eosinophils and basophils are far less common. They both contain granules that contain histamine and other proinflammatory molecules.

Eosinophils stain pink with the dye eosin- which is where they get their name. Eosinophils are also phagocytic, and they’re best known for fighting large and unwieldy parasites because eosinophils are much larger than neutrophils and have receptors that are specific for parasites.

   Unlike neutrophils and eosinophils, basophils are non-phagocytic. They stain blue with the dye hematoxylin,and like eosinophils they can be helpful at combating large parasites but also cause inflammation in asthma and allergy responses.

  Finally, there are the mast cells which are also non-phagocytic and they’re involved in asthma and allergic responses. Next up are the monocytes, macrophages, and dendritic cells, which are phagocytic cells - they gobble up pathogens, present antigens,and release cytokines a tiny molecules that help attract other immune cells to the area.

Monocytes only circulate in the blood. Some monocytes migrate into tissues and differentiate into macrophages, which remain in tissues and aren’t found in the blood. Other monocytes differentiate into dendritic cells, the prototypical antigen presenting cell, which roam around in the lymph, blood,and tissue.

When dendritic cells are young and immature they’re excellent at phagocytosis, constantly eating large amounts of protein found in the interstitial fluid. But when a dendritic cell phagocytoses a pathogen for the first time. it’s a life-changing, coming of age moment.
  Mature dendritic cells will destroy the pathogenand break up it’s proteins into short amino acid chains. Dendritic cells will then move through the lymph to the nearest lymph node and they will perform antigen presentation which is where they present those amino acid chains  which are antigens to T cells.

Antigen presentation is what connects the innate and adaptive immune systems. Antigen presentation is something that can be done by dendritic cells, macrophages residing in the lymph node, and monocytes which can travel to a lymph node after phagocytosing a bloodborne pathogen which is why all of these cells are referred to as antigen presenting cells.

Now, only T cells with a receptor that can bind to the specific shape of the antigen will get activated - that’s called priming. It’s similar to how a lock will only snap open when a key with a very specific shape goes in. However, T cells can only see their antigen if it is presented to them on a silver platter and on a molecular level that platter is the Major Histocompatibility complex or MHC for short. So the antigen presenting cell will load the antigen onto an MHC molecule and display it to T cells - and when the right T cell comes along it binds.

  Other group the lymphoid progenitor cells  become lymphoid cells which are the B cells. B and T cells make up the adaptive immune system, while NK cells are part of the innate immune system. B cells and NK cells complete their development where they started in the bone marrow, whereas some lymphoid progenitor cells migrate to the thymus where they develop into T cells.

  All of the lymphocytes are able to travel in and out of tissue and the bloodstream. NK cells are large lymphocytes with granules and they target cells infected with intracellular organisms, like viruses, as well as cells that pose a threat like cancer cells.

NK cells kill their target cells by releasing cytotoxic granules in their cytoplasm directly into the target cell. These granules contain some molecules that cause target cells to undergo apoptosis which is a programmed cell death and some that punch holes in the target cell’s membrane by binding directly to the phospholipids and creating pores.

  B-cells, like T-cells, also have a receptor on their surface that allows them to only bind to an antigen that has a very specific shape. The main difference is that B cells don’t need antigen to be presented to them on an MHC molecule, they can simply bind an antigen directly. When a B cell binds to an antigen that’s on the surface of a pathogen, it is capable of phagocytosis and antigen presentation  so technically, they’re also antigen presenting cells as well.

Like other antigen presenting cells, the B-cell will load the antigen onto an MHC molecule called MHC II, and display it to T- cells. When a T-cell gets activated it helps the B-cell mature into a plasma cell, and a plasma cell can secrete lots and lots of antibodies. Typically, it takes a few weeks for antibody levels to peak. The antibodies, or immunoglobulins, have the exact same antigen specificity as the B cell they come from.

  Antibodies, are just the B-cell receptor in a secreted form, so they can circulate in serum, which is the non-cellular part of blood- attaching to pathogens and tagging them for destruction. Because antibodies aren’t bound to cells and float freely in the blood, this is considered humoral immunity a throwback to the term“humors” which refers to body fluids.

  The final type of lymphoid cell is the T-cell and its in charge of cell mediated immunity. T-cells are antigen specific, but they can’t secrete their antigen receptor. A naive T-cell can be activated or primed to allow it to turn into a mature T-cell by any of the antigen presenting cells, but most often it’s done by a dendritic cell. Now, there are two main types of T-cells, CD4 T-cells and CD8 T- cells
“CD” stands for cluster of differentiation.
There are hundreds of CD markers in the immune system, and these CD markers are useful in telling them apart. For example, all T-cells are CD3+, because CD3 is part of the T-cell receptor. So, CD4+ T-cells, are actually CD3+ CD4+, and these cells are called helper cells because they’re like generals on the battlefield, they secrete cytokines that help coordinate the efforts of macrophages, B-cells, and NKcells.

Helper T-cells can only see their antigen if it is presented on an MHC II molecule. CD8+ T-cells are CD3+,CD8+, and they’re called cytotoxic T-cells T-cells because they kill target cells, really similarly to how NK cells doit with one major difference. CD8+ T- cells only kill cells that presenta specific antigen on an MHC I molecule  which is structurally similar to the MHC II molecule,where as NK cells aren’t nearly as specific in who they kill.

  So now let’s go through a complete immune response with a bacterial pathogen in the lungs. To start, the bacteria will have to get breathed in, slip by your nose hairs, past the cilia in the airways, and then will have to penetrate past the epithelium layer of the lungs. Once it’s in the lung tissue, the bacteria will start to divide and might encounter a resident macrophage in the lung tissue which will ingest the bacteria and start releasing cytokines. Those cytokines start the inflammatory process by making blood vessels leaky and attracting nearby eosinophils, basophils, and mast cells, which release their own cytokines and granules amplifying the inflammation.

  Neutrophils from the blood as well as fresh new ones from the bone marrow dive into the tissue and join the battle. If the pathogen was a virus, NK cells would help destroy the infected cells at this point. This is all part of the innate immune response. Around this point in the infection, immature dendritic cells digest the pathogens and move from the lung tissue over to a nearby lymphnode where they present the processed antigen on an MHC II protein to a naive T-cell. The dendritic cell, which is part of the innate immune system, bridges the innate and adaptive immune responses when it presents the antigen to the T-cell part of the adaptive immune system.

Sometimes, if the infection is spreading,bacteria might find its own way to a lymph node without the help of the dendritic cell. In this case, B-cells part of the adaptive immune system might directly phagocytose the bacteria and present it to a naive CD4+ T-cell. Either way, if the antigen is the right “fit” for the T-cell it will begin to differentiate and undergo clonal expansion. Differentiated CD4+ T-cells will release cytokines that will induce B-cells to differentiate into plasma cells which secrete antibodies that will go into the lymph and then the bloodstream. The antibodies will tag pathogens making it easier for phagocytes to eat them. Once again, at this point, if the pathogen was a virus, the CD8+ T- cells would kill any infected cells that express the viral antigen on an MHC I.

Over time, as the invading pathogen dies off, most of the B and T-cells die of neglect, but a few turn into memory B-cells and memory T-cells, which linger for years in case their needed in the future. So, to recap the immune system has innate and adaptive response. The innate immune response is immediate, but non-specific, and lacks memory, where as the adaptive immune response is highly specific and remembers everything, but it takes several days to get started and almost two weeks to peak. 

Steps of rDNA Technology

  Recombinant DNA or r-DNA technology is a technique that is used to refer, identification, isolation and insertion of gene of interest into a vector, Such as plasmid or bacteriophage to form a recombinant DNA molecule and production of large quantities of that gene fragment or product encoded by gene of interest.

Steps in rDNA technology

Step 1 : Identification and isolation of gene of interest or dna fragment to be cloned.

Step 2 : Insertion of this isolated gene into a suitable vector.

Step 3: Introduction of this vector into a suitable host the process is called as transformation.

Step 4 : Selection of the transformed host cell.

Step 5 : Multiplication or expression of the introduced gene of interest inside the host.

Let's discuss this step in detail.

Step 1 : Identification and isolation of gene of interest

  In recombinant DNA technology this gene of interest is isolate from different resources. These include
- Genomic library,
- cDNA library,
- Chemical synthesis of a gene if you know the sequence,
- If the number of copies of a particular gene is very less or not sufficient for gene cloning we can amplify that using polymerase chain reaction.

  For this step we have taken human insulin gene from genomic library. Genomic library is a source from where we get our gene of interest.

Step 2 : Insertion of Gene of interest in a suitable vector

Vector is any DNA molecule that has the ability to replicate inside the host to which the gene of interest has integrated for cloning.

  Vectors include Plasmids, Bacteriophages, Cosmids, BAC, Yeast vectors, Shuttle vectors etc.
most commonly used vector in rDNA technology is plasmid. Plasmid is extra chromosomal DNA that is present in bacteria.

 For the insertion of gene of interest we need to make a cut in the vector using a restriction enzyme. Restriction enzymes are enzymes that are capable of making internal cuts at specific sites in a DNA molecule.

  Now we need to incorporate our insulin gene into this vector. Our gene of interest is settled in between cut and ligated by ligase enzyme. Now we have the recombinant DNA molecule. Recombinant DNA molecule is a DNA molecule from which DNA are from different sources.

Step 3 : Introduction of this vector into a suitable host

  There are different gene transfer methods by which we can introduce our vector into a host. these methods are :
- Electroporation
- Microinjection
- Liposome mediated gene transfer
- Silicon carbide fiber mediated gene transfer
- Gene gun method.
- Poly Ethylene Glycol mediated gene transfer
- Calcium Chloride mediated gene transfer
- DEAD dextran mediated gene transfer.
- Natural transformation by micro-organism

So now our gene of interest is incorporated in the vector, we need to introduce this recombinant DNA into the host(bacterial cell) to manipulate. So now bacterial cell is genetically modified with our gene of interest.

Step 4 : Selection of the transformed host cell

After the transformation we will be getting three types of colonies
- Non-transformed bacterial cell, without any change
- Transform bacterial cell, with unaltered vector (vector is in the cell has transformed but without our gene of interest)
- Transformed bacterial cell with our recombinant vector

 We need Transformed bacterial cell with our gene of interest. Then we need to select this particular colony from the rest majority of the colonies.

There are different ways to select perticular colony which are
- Antibiotic resistance in selective medium
- Visible characters
- Assay for biological activity
- Colony hybridization
- Blotting test.

 Commonly used method for selection is antibiotic resistance selection in recombinant selective medium. In this method our gene of interest contains some antibiotics resistance gene. So in the medium which contain antibiotics only thos bacteria will survive which has our gene of interest.

Step 5 : Multiplication or expression of the introduced gene in the host

 Our intention is to make as many copies of that particular gene or we need to make that gene product to be synthesized inside the bacterium. We have genetically modified bacteria (host) with our recombinant DNA (plasmid). This plasmid will replicate inside the host as well as these bacteria will also replicate making millions of copies of our gene of interest.

 Our first intention is achieved, we have our gene of interest produced in millions of numbers. Our actual intention is to synthesize desired product inside bacteria. This is happening inside the host, gene that will be transcribed and translated and produce our desires protein or product.

 The forst and most common product which is produced by rDNA technology is human insulin. This insulin is called as Humulin.

Major Differences between Viroids and Prions

  Both viroids and prions are Strict intracellular, Subcellular infectious agents, that always requires a living host for replication. Besides there are many differences between Viroids and Prions.

Differences between Viroids and Prions

• Definition :


  • Viroids are infectious RNA particles without protein coat.
  • Viroids are just virus-like.
  • It was discovered by T. Diener.
  • The first viroid discovered was PSTV (Potato spindle tuber viroid) and he called it as virus like or viroid.
  • Viroids are the smallest known Infectious agents.


  • Prions are infectious protein particles that is discovered by Stanley Prisoner in 1982.
  • Prions cause Neurodegenerative diseases.
  • Prions is actually a misfolded protein.

• Chemical Nature and Size


  • Viroids are made up of RNA.
  • It is always small circular single stranded RNA molecule.
  • The size of the genome is 5 to 10 times smaller than the smallest DNA and RNA viral genomes.
  • Generally the size of viroids genome ranges from 246 to 463 nucleotides.


  • Prions are actually misfolded proteins.
  • It is called as PRPc (Prionprotein cellular)
  • Prion protein is made up of 209 amino acids and the function is it is having a neuroprotective function.
  • This prion (PRPc) is converted to PRPsc which is called as Prion Protein Scrapie; the disease that occurs in sheep.
  • The structure of PRPsc is very stable with beta sheets and it accumulates in nerve cells as amyloids and ultimately kill neurons.

• The Replication


  • In viroid replication is done by an RNA-based rolling circle mechanism; using RNA polymerase 2 of the host.
  • Viroid the RNA does not code for any protein but it can act as ribozyme. so it can effectively use the machinery of the host.


  • In the case of prion, The PRPc is converted to PRPsc.
  • The normal protein PRPc is converted to non-abnormal protein or disease causing protein PRPsc.
  • There are two models proposed regarding the replication of Prion,
  • One is heterodimer model and the second is fibrillar model; both suggest that conversion of PRPc to PRPsc is happening during the replication process.

• The Hosts and Examples


  • Viroids infects only plants.
  • Potato spindle tuber viroid (PSTV) is causing potato spindle tuber disease.
  • Other viroids diseases include :
  1. Tomato plant maco viroid (TPMV).
  2. Avocado sunblotch viroid (ASBV)
  3. Peach latent mosaic viroid (PLMV) etc.


  • Prions causes Neurodegenerative diseases in animals.
  • In cattle it causes bovine spongi form encephalopathy (BSE) which is called a "mad cow disease".
  • In humans it causes neurodegenerative diseases like CJD disease and Kuru disease etc.
These are the basic differences between Prions and Viroids. 

Pyruvate Dehydrogenase (PDH) Complex : Machanism, Regulation and Inhibitors

The PDH complex also known as the pyruvate dehydrogenase complex is a multi-enzyme complex.

PDH complex is present on the inner mitochondrial membrane. The molecular weight of the PDH complex is 9×10⁶ kDa.

  Pyruvate is produced in the cytosol as the end product of aerobic glycolysis. It enters mitochondria by a pyruvate protons import which is present in the inner mitochondrial membrane.

  In the mitochondrial matrix pyruvate is oxidatively Decarboxylated to form Acetyl- CoA (acetyl coenzyme A).

The oxidation of pyruvate to acetyl coenzyme A is an irreversible reaction. This is the link reaction between glycolysis and the citric acid cycle.

PDH Complex

The PDH complex consists of 3 Enzymes and 5 coenzymes.
• The 3 enzymes are :
- Pyruvate Dehydrogenase with TPP as a coenzyme
- Dihydrolipoyl Transacetylase
- Dihydrolipoyl Dehydrogenase

• The 5 coenzymes are :
- Thymine pyrophosphate or vitamin B1
- FAD or vitamin B2
- NAD or vitamin B3
- Coenzyme A or vitamin B5
- Lipoic acid.

Reactions of the PDH  complex

Pyruvate Dehydrogenase with TPP as coenzyme,  decarboxylates pyruvate to Hydroxyethyl TPP (thiamine pyrophosphate).

• Then Dihydrolipoyl Transacetylase catalyzes conversion of Hydroxyethyl Thiamine Pyrophosphate into acetyl lipoamide and then transfers the acetyl group to coenzyme A to produce acetyl coenzyme A.

Dihydrolipoyl Dehydrogenase catalyses conversion of reduced Lipamind into Oxidized lipamind by transferring the reduced equivalence of FAD and this completes the cycle.

• FADH2 in turn transfers the reducing equivalents to NAD+ to give NADH and H+.

• This NADH and H+ enters a respiratory chain to give 2.5 ATP by oxidative phosphorylation.

• As a whole five ATP are released in the PDH complex and from two moles of pyruvate which is formed from glucose by glycolysis.

• All these intermediates of PDH catalysed reaction are not free but bound with an enzyme complex together called as the PDH complex.

A comparable enzyme with PDH is alpha ketoglutarate dehydrogenase complex of the citric acid cycle which catalyses the oxidative decarboxylation of alpha-keto glutarate into succinyl CoA.

Regulation of PDH Complex

Regulation of the pyruvate dehydrogenase complex is mainly an example of end product inhibition. It is also regulated by phosphorylation and dephosphorylation.

- PDH is active as Dephosphoenzyme which means in dephosphorylated state.

- While it is inactive as phosphoenzyme that is phosphorylated state.

• PDH phosphatase which is responsible to maintain PDH complex an active state is promoted by Calcium(Ca²+) Magnesium( Mg²+) and mainly insulin. Insulin activates PDH complex.

• PDH kinase which are responsible for the inactive PDH is promoted by ATP, NADH and acetyl coenzyme A.
  while PDH complex is inhibited by NAD+, coenzyme A and pyruvate.

• Net result is that in the presence of high-energy molecules like ATP, NADH.

• The pyruvate dehydrogenase complex is turned off because when enough amount of ATP is already present there is no need for the pyruvate dehydrogenase complex to continue.

Clinical application

Arsenic poisoning :

• Arsenite binds to thiol Which is sulfhydryl-SH group of lipoic acid and makes it unavailable to serve as a cofactor.

• By this way in arsenic poisoning enzymes of the pyruvate dehydrogenase complex and alpha-keto glutarate complex are inhibited by arsenite.

Inherited or Acquired (Alcoholics) deficiency :

• Inherited or acquired alcoholics deficiency of PDH causes lactic acidosis due to rapid conversion of accumulated pyruvate into lactic acid.

• Pyruvate is derived from carbohydrates. An acetyl coenzyme A mainly derived from fats which are triglycerides, fatty acids, cholesterol.

• Carbohydrates in excess can form fats but fats can never be converted to carbohydrates because linked reaction is irreversible.
  An exception to this rule is Glycerol and Propionic acid which are derived from fats, but they can be converted to glucose.

Opsonization : Defination, Opsonins and Machanism

Opsonization is a process which enhances the efficiency of phagocytosis by phagocytic cells. This process involves specific antimicrobial proteins which are termed as Opsonins

  These antimicrobial proteins are present in our body fluids.
Phagocytes such as Macrophages and Neutrophils have receptors for these opsonins on their plasma membrane.  

Opsonins have ability to bind the microbial surface. They coat the microbial surface and this coating increases the number and kind of binding sites on the microbial surface.
In Greek, "opson" means to prepare for eating.

Opsonins coat the microbial surface. Phagocytes have specific receptors for these options.
Optionization helping in the phagocytosis process.

Once opsonins coat the microbial surface, this coating makes the recognition and destruction of the invader more efficient.
This is because the number of binding sites and type of binding sites has increased.

Definition of Opsonization

The process of coating pathogens to promote phagocytosis is called optinization. The proteins which perform this function are called Opsonins.

Types of Opsonins

There are two main options in human body which are
  1). Complement C3b 
  2). Antibody 

Compliment C3b as Opsonin

  Complement or Complement system is a set of plasma proteins. They help in elimination of microbes.

• C3b is a complement proteins which has ability to bind microbial surfaces.
• Phagocytes bear C3b receptors.
• Once the C3b protein coat the microbial surface, The C3b receptors find these C3b proteins and triggers phagocytosis of the microbe.

Antibody as opsonin

Antibody is a part of adaptive immune system. 

When adaptive immune system recognizes a pathogen such as a bacteria, antibody specific to bacteria or that pathogen are produced. 

These antibodies bind to the surface of microbes.

Antibody is a y-shaped molecule and two arms of that Y form the antigen binding site (FB) and stalk is known as FC region.
These antibody binds to the microbial surface with their antigen binding arms.

Phagocytes has a receptor for the FC region of the antibody.
• So first the antibody binds to the antigen on the bacterial cell wall via antigen binding site.
• Then FC receptors of the phagocyte bind the FC region of the antibodies which facilitate phagocytosis.

Opsonins function as a bridge between pathogens and the phagocytes. Opsonization is the process which enhances the phagocytic ability of a phagocyte.