Tuesday, August 31, 2010

Antibody-Mediated Effector Functions

Opsonization
  • promotion of phagocytosis of  the antigen by macrophages, neutrophiles
  • FcR present on the surfaces of macrophages, neutrophiles bind the constant region of Ig
                                                                            ↓
                                     secures pathogen to the phagocyte membrane
                                                                            ↓
                                           initiates signal transduction pathways
                                                                            ↓
                                     phagocytosis of Ag═Ab complex takes place

Complement Activation
  • IgM, most subclasses of IgG activate collection of serum glycoproteins (complement activators) that perforate cell membranes
  • Binding of the Ag═Ab complex to C3b receptors of red blood corpuscles (RBCs)
                                                                           ↓
                                                     RBC delivers to liver/spleen
                                                                           ↓
                       resident macrophages remove complex without destroying the RBC


Antibody-dependent cell-mediated cytotoxicity (ADCC)

                         Infected cell═Ab complex
                                                ║
                                             FcR of NK cells
                                                     ↓
                     activates cytotoxic activity of the effector cells

Antibodies can also cross epithelial layers by a process called transcytosis; for example, transfer of IgG from mother to fetus ("passive immunization")

Saturday, August 28, 2010

Structure and Function of Antibodies

Recall:
  • B-cells can recognize an antigen via their surface immunoglobulin (Ig) molecules
  • T-cells can only recognize an antigen that is associated with a major histocompatibility complex (MHC) molecule

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

Antigens and Antibodies

Immunogenicity → the ability to induce humoral/cell-mediated immune response

B-cell + Antigen → Effector B-cell + memory B-cell
                                           ↓
                                  plasma cell → Antibody


T-cell + Antigen → Effector T-cell + memory T-cell
                                            ↓
                                 CTLs + THELPER, etc → cytokines, cytotoxic factors

Antigenicity → the ability to combine specifically with antibodies and surface receptors on T-cells

Friday, August 27, 2010

Analytical Aptitude - Problem Set I

Analytical questions are intended to assess a student's knowledge of the subject matter emphasized in the CSIR-NET examination as preparation for doctoral study. As of 2011, the CSIR-NET committee is introducing a new pattern of examination which will contain multiple choice questions, a number of which may be grouped in sets and based on descriptions of laboratory situations, diagrams, or experimental results. Throughout the test, there will be an emphasis on questions requiring problem-solving skills (including mathematical calculations that do not require the use of a calculator) as well as content knowledge.

The CSIR-NET examination is designed to evaluate the following abilities and background of the student:
  • Knowledge of basic vocabulary and facts in several life science fields at the equivalent of MSc degree
  • Conceptual understanding of ideas, relationships, and processes
  • Understanding of basic scientific research, procedures, and tools
  • Capacity to read, evaluate, and draw conclusions from unfamiliar laboratory and field studies
  • Understanding of the connections among the life science as well as between life and allied sciences

Thursday, August 26, 2010

Membrane Transport

  • Cell membranes are permeable to large uncharged polar molecules (amino acids, glucose and nucleotides) and charged molecules (ions). Transfer of these water-soluble molecules depends on membrane transport proteins
  •  If a cell is not to be torn apart by electrical forces, then the quantity of positive charge inside or outside the cell must be balanced by an almost exactly equal quantity of negative charge (outside or inside respectively)
  • Nature has therefore employed mechanisms by which small molecules cross lipid bilayers
    • passive diffusion → a molecule simply dissolves in the lipid bilayer, diffuses across it, and then dissolves in the aqueous solution at the other side of the membrane
      • gases (O2, CO2), hydrophobic molecules (benzene) and small polar uncharged molecules (H2O, ethanol) are able to dissolve in the lipid bilayer
      • no membrane proteins are involved
      • no external source of energy is required
      • the net flow of molecules is always down their concentration gradient →  the direction of transport is determined only by the relative concentrations of the molecule inside and outside of the cell

The Cell Membrane

Cell Membrane
  • All cells are enclosed by a thin, film-like membrane called the plasmalemma or more popularly as the plasma membrane
  • Danielli and Davson (1935) proposed a “trilaminar model” according to which, the plasma membrane is formed of a bimolecular layer of phospholipids (35 Å thick) sandwitched between two layers of proteins (each 20 Å thick). The model was proposed even before the plasma membrane was seen under the electron microscope
  • J.D. Robertson (1959) proposed a “unit membrane concept” according to which, all biological membranes shared the same basic structure:
    • thickness of about 75 Å
    • a characteristic trilaminar appearance when viewed with electron microscope
    • the three layers are a result of the same arrangement of proteins and lipids as proposed by Danielli and Davson
  • S. J. Singer and G. Nicolson (1972) put forward the “fluid mosaic model” of membrane structure which is presently the most widely accepted model.
Components of the Plasma Membrane
    The Plasma membrane structure
  • According to the fluid mosaic model, the cell membrane consists of a highly viscous fluid matrix of two layers of phospholipid molecules which serve as a relatively impermeable barrier to the passage of most water soluble molecules
  • The plasma membrane contains lipids (32%), proteins (42%), carbohydrates (6%) and water (20%) although variations are always there
  • Protein molecules or their complexes occur in the membrane, but not in continuous layer; instead, these occur as separate particles asymmetrically arranged in a mosaic pattern

Monday, August 23, 2010

Fundamental Processes - Overview of the Cell Cycle

Control of Cell Division
  • Most cells have two major phases: mitosis and interphase often together referred to as the cell cycle
  • For most tissues at any given time, only a few cells are in mitosis while the rest remain in interphase which is the period between divisions of the cytoplasm and is where a typical eukaryotic cell spends most of its life
  • Some cells lose the capacity to divide altogether and stay in interphase indefinitely (for example in humans: nerve cells and muscle cells), while some divide regularly and others only occasionally

The Cell Cycle
Interphase consists of three sub-phases:
  • G1 is Gap 1 → the period just after mitosis and before the beginning of DNA synthesis
  • S (synthesis) → which is the time when the cell’s DNA is replicated
  • G2 is Gap 2 → the time after S and prior to mitosis
Mitosis and cytokinesis are referred to as M-phase:
  • the G1→S transition commits the cell to enter another cell cycle


Saturday, August 21, 2010

X-chromosome Inactivation & Dosage Compensation

Dosage Compensation

  • A phenomenon of neutralization of differences in sex-linked gene doses, i.e. males with one X-chromosome and females with two X-chromosomes have identical X-linked gene products
  • First report of this phenomenon → Muller (1931) while working with the wa allele observed that there is a mechanism compensating for the difference in the dosage of X-linked gene between the sexes

Tuesday, August 17, 2010

Proteomics

Mixed-peptide sequencing → enables internal peptide sequence information to be derived from proteins electroblotted onto hydrophobic membranes

Proteomics
  • Coined in 1995
  • Defined as the large-scale characterization of the entire protein complement of a cell line, tissue or organism
  • 2 types:
    • Classical → large-scale analysis of gene products to studies involving only proteins
    • Inclusive → combines protein studies with analyses that have a genetic readout such as mRNA analysis, genomics and yeast 2-hybrid analysis

Analytical Techniques - Pulsed Field Gel Electrophoresis (PFGE)

Pulsed-Field Gel Electrophoresis - PFGE
  • Introduced in 1982 by Schwartz et al.
  • Addresses the inability of conventional gel electrophoresis to separate fragments above 50 Kb due to loss of sieving action of the gel because of the large size of molecules
  • Uses 2 alternating electric fields
  • Provides means for routine separation of fragments exceeding 6000 Kb
  • Ability to separate small, natural linear chromosomal DNAs from 50 Kb parasite minichromosomes to multimillion-bp long yeast chromosomes
  • Basically separates DNAs from few Kb to more than 10 Mb

Types of PFGE

DNA Ligases

Ligation → a process that involves the formation of 4 phosphodiester (PD) bonds -- 2 at each end of the molecule between neighbouring 3'-OH and 5'-PO4


DNA Ligases
  • Exquisite specificity for nicked DNA
  • In E. coli:
    • homologous and monomeric
    • polypeptide chain with molecular weight of 75 KDa
    • require NAD+ as a co-factor
  • In T4 phages, mammalian and plant cells:
    • polypeptide chain with molecular weight of 68 KDa
    • requires ATP as a co-factor and energy source

Restriction Endonucleases

Discovery
  • In his 1969 paper, Werner Arber proposed that host-controlled restriction in bacteriophages occurs as result of enzymatic cleavage of infecting DNA molecules by what he termed as endonuclease while refering to endonuclease R (later to be renamed as EcoB)
  • Arber, along with Struat Linn received the Nobel Prize in Physiology and Medicine in 1978 for the discovery of restriction "enzymes" or "endonucleases"
  • They also proposed that DNA modification is the result of DNA methylation by DNA "methylases"
  • Shortly after the discovery, another restriction enzyme was found in E. coli (EcoK)
Nomenclature

Monday, August 16, 2010

Biology of DNA Restriction

Restriction-Modification
  • Phenomenon first described 40 yrs ago in viruses
  • Restriction occurs by an endonucleolytic cleavage of foreign DNA
  • 2 different types:
    • one where only DNA that carries specific modifications is cleaved (host DNA avoids restriction because it is not modified)
    • other where cellular DNA is protected from restriction by modification i.e. by methylation of adenosyl or cytosyl residues within the sequences recognized by Restriction Enzymes -- The classical R-M systems
Restriction-Modification (R-M) systems

Hallmarks of a cancer cell

Hanahan and Weinberg (who first discovered the oncogene RAS from a bladder cancer cell line) proposed six characteristics for a cancer cell that define its patho-physiological and cellular properties:
Functional capabilities of Cancer cells

1) Limitless replicative potential

2) Self sufficiency in growth signals

3) Insensitivity to anti-growth signals

4) Evasion of apoptosis

5) Sustained angiogenesis

6) Tissue invasion and metastasis