Monoclonal antibodies

Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they were produced by one type of immune cell and are all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications

The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.

In the 1970s the B-cell cancer myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

The process of producing monoclonal antibodies described above was invented by Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody producing B-cells, and be able to select for the successfully fused cells.

In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,removing the reactions that many monoclonal antibodies caused in some patients.

Hybridoma

Monoclonal antibodies can be produced in cell culture or in live animals. If a foreign substance (an antigen) is injected into a vertebrate such as a mouse or a human, some of the immune system's B-cells will turn into plasma cells and start to produce antibodies that recognize that antigen. Each B-cell produces only one kind of antibody, but different B-cells will produce structurally different antibodies that bind to different parts ("epitopes") of the antigen. This natural mixture of antibodies found in serum is known as polyclonal antibodies.

To produce monoclonal antibodies, the B-cells from the spleen or lymph nodes are removed from an animal that has been challenged several times with the antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer or more specifically a plasmacytoma) and that have lost the ability to produce antibodies. This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol (PEG), electroporation or, of historical importance, infection with some virus. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. The hybridomas are sufficiently diluted to ensure clonality (all cells in the culture stem from the same single cell) and grown. The antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot, and the most sensitive one is picked out. When the hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid.

In the above process, myeloma cell lines that have lost their ability to produce their own antibodies or antibody chain are used, so as to not contaminate the target antibody. Furthermore, only myeloma cells that have lost a specific enzyme called hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and therefore cannot grow under certain conditions (e.g. in the presence of a selection medium called HAT medium) are used; these cells are preselected by the use of either 8-azaguanine or 6-thioguanine(8-azaguanine has been shown to produce unreliable results.(van Diggelen et al 1979)) media prior to the fusion since cells that possess the HGPRT will be killed by the 8-azaguanine. During the fusion process many cells can fuse: Myeloma cell with myeloma cell, spleen cell with spleen cell, spleen cell with myeloma cell, etc. The desired fusions for making hybridomas are between a healthy B-cell, which produces antibodies against the antigen of interest, and a myeloma cell. In these relatively rare fusions, the healthy B cell will make the HGPRT enzyme that will allow the fused cell to survive in HAT medium so that only the successfully fused cells will grow in culture. The medium must be enriched during selection to favour hybridoma growth. This can be achieved by the use of a layer of feeder cells or supplement media such as briclone. Production in cell culture is usually preferred as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical.

Recombinant

The production of Recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.These techniques can be used to enhance: the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.

Applications

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence and quantity of this substance, for instance in a Western blot test (to detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a cell). They are also very useful in immunohistochemistry which detect antigen in fixed tissue sections. Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

Complement System Animation

The complement system is a biochemical cascade which helps clear pathogens from an organism. It is one part of the larger immune system.

The complement system consists of a number of small proteins found in the blood, which work together to kill target cells by disrupting the target cell's plasma membrane. Over 20 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors. These proteins are synthesized mainly in the liver, and they account for about 5% of the globulin fraction of blood serum.

The complement system is not adaptable and does not change over the course of an individual's lifetime; as such it belongs to the innate immune system. However, it can be recruited and brought into action by the adaptive immune system.

Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the mannose-binding lectin pathway

Overview
The three pathways all generate homologous variants of the protease C3-convertase. The classical complement pathway typically requires antibodies for activation (specific immune response), while the alternate pathway can be activated by C3 hydrolysis or antigens without the presence of antibodies (non-specific immune response). Mannose-binding lectin pathway belongs to the non-specific immune response as well. C3-convertase cleaves and activates component C3, creating C3a and C3b and causing a cascade of further cleavage and activation events. C3b binds to the surface of pathogens leading to greater internalization by phagocytic cells by opsonization. C5a is an important chemotactic protein, helping recruit inflammatory cells. Both C3a and C5a have anaphylatoxin activity (mast cell degranulation, increased vascular permeability, smooth muscle contraction). C5b initiates the membrane attack pathway, which results in the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and polymeric C9.[2] MAC is the cytolytic endproduct of the complement cascade; it forms a transmembrane channel, which causes osmotic lysis of the target cell. Kupffer cells and other macrophage cell types help clear complement-coated pathogens. As part of the innate immune system, elements of the complement cascade can be found in species earlier than vertebrates; most recently in the protostome horseshoe crab species, putting the origins of the system back further than was previously thought.

Classical pathway

The classical pathway is triggered by activation of the C1-complex (which consists of one molecule C1q and two molecules C1r and C1s), either by C1q's binding to antibodies from classes M and G, complexed with antigens, or by its binding C1q to the surface of the pathogen. This binding leads to conformational changes in C1q molecule, which leads to the activation of two C1r (serine protease) molecules. Then they cleave C1s (another serine protease). The C1-complex now binds to and splits C2 and C4, producing C2a and C4b. The inhibition of C1r and C1s is controlled by C1-inhibitor. C4b and C2a bind to form C3-convertase (C4b2a complex: NB the 2a is actually the larger fragment of the two, contrary to conventional nomenclature designating 'b' fragments as the larger). Production of C3-convertase signals the end of the Classical Pathway, but cleavage of C3 by this enzyme brings us to the start of the Alternative Pathway.


Alternative pathway

The alternative pathway is triggered by C3 hydrolysis directly on the surface of a pathogen. It does not rely on a pathogen-binding protein like the other pathways.[1] In the alternative pathway, the protein C3 is produced in the liver, and is then cleaved into C3a and C3b by enzymes in the blood. If there is no pathogen in the blood, the C3a and C3b protein fragments will be deactivated. However, if there is a nearby pathogen, some of the C3b is bound to the plasma membrane of the pathogen. Then, it will bind to factor B. This complex will then be cleaved by factor D into Ba and the alternative pathway C3-convertase, Bb.

The C3bBb complex, which is "hooked" onto the surface of the pathogen, will then act like a "chain saw", catalyzing the hydrolysis of C3 in the blood into C3a and C3b, which positively effects the number of C3bBb hooked onto a pathogen.

After hydrolysis of C3, C3b complexes to become C3bBbC3b, which cleaves C5 into C5a and C5b. C5a and C3a are known to trigger mast cell degranulation. C5b with C6, C7, C8, and C9 (C5b6789) complex to form the membrane attack complex, also known as MAC, which is inserted into the cell membrane, "punches a hole", and initiates cells lysis.

Lectin pathway (MBL - MASP)
The lectin pathway is homologous to the classical pathway, but with the opsonin, mannan-binding lectin (MBL) and ficolins, instead of C1q. This pathway is activated by binding mannan-binding lectin to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases, MASP-1, MASP-2, MASP-3, which can then split C4 into C4a and C4b and C2 into C2a and C2b. C4b and C2a then bind together to form C3-convertase, as in the classical pathway. Ficolins are homologous to MBL and function via MASP in a similar way. In invertebrates without an adaptive immune system, ficolins are expanded and their binding specificities diversified to compensate for the lack of pathogen-specific recognition molecules.


Regulation of the Complement System

The complement system has the potential to be extremely damaging to host tissues meaning its activation must be tightly regulated. The complement system is regulated by complement control proteins, which are present at a higher concentration in the blood plasma than the complement proteins themselves. Some complement control proteins are present on the membranes of self-cells preventing them from being targeted by complement. One example is CD59, which inhibits C9 polymerisation during the formation of the membrane attack complex.

Role in disease

It is thought that the complement system might play a role in many diseases with an immune component, such as Barraquer-Simons Syndrome, asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries. The complement system is also becoming increasingly implicated in diseases of the central nervous system such as Alzheimer's disease, and other neurodegenerative conditions.

Deficiencies of the terminal pathway predispose to both autoimmune disease and infections (particularly meningitis, due to the role that the C56789 complex plays in attacking Gram negative bacteria).

Modulation by infections

Recent research has suggested that the complement system is manipulated during HIV/AIDS to further damage the body

Genetic Network Lecture

Individual genes have a function (e.g. transforming a substance or binding to a substance)Sets of functions when sequenced can produce pathways (e.g. output of one transformation is the input to another) Sets of pathways, as they interact with other pathways, create a genetic network of interactions.The emergent properties of these networks constitute the “observables” when we study cells.

At one level, biological cells can be thought of as "partially-mixed bags" of biological chemicals -- for the purposes gene regulatory networks, these chemicals are mostly the mRNAs and proteins that arise from gene expression. These mRNA and proteins interact with each other with various degrees of specificity. Some diffuse around the cell. Others are bound to cell membranes, interacting with molecules in the environment. Still others pass through cell membranes and mediate long range signals to other cells in a multi-cellular organism. These molecules and their interactions comprise a gene regulatory network. A typical gene regulatory network looks something like this:

The nodes of this network are proteins, their corresponding mRNAs, and protein/protein complexes. Nodes that are depicted as lying along vertical lines are associated with the cell/environment interfaces, while the others are free-floating and diffusible. Implied are genes, the DNA sequences which are transcribed into the mRNAs that translate into proteins. Edges between nodes represent individual molecular reactions, the protein/protein and protein/mRNA interactions through which the products of one gene affect those of another. These interactions can be inductive (the arrowheads), with an increase in the concentration of one leading to an increase in the other, or inhibitory (the filled circles), with an increase in one leading to a decrease in the other. A series of edges indicates a chain of such dependences, with cycles corresponding to feedback loops. The network structure is an abstraction of the system's chemical dynamics, describing the manifold ways in which one substance affects all the others to which it is connected. In practice, such GRNs are inferred from the biological literature on a given system and represent a distillation of the collective knowledge about a set of related biochemical reactions.

Genes can be viewed as nodes this network, with input being proteins such as transcription factors, and outputs being the level of gene expression. The node itself can also be viewed as a function which can be obtained by combining basic functions upon the inputs (in the Boolean network described below these are Boolean functions or gates computed using the basic AND, OR and NOT gates in electronics). These functions have been interpreted as performing a kind of information processing within the cell, which determines cellular behaviour. The basic drivers within cells are levels of some proteins, which determine both spatial (tissue related) and temporal (developmental stage) co-ordinates of the cell, as a kind of "cellular memory". The gene networks are only beginning to be understood, and it is a next step for biology to attempt to deduce the functions for each gene "node", to assist in modeling behaviour of a cell (see systems biology).

Mathematical models of GRNs have been developed to allow predictions of the models to be tested. The most common modeling technique involves the use of coupled ordinary differential equations (ODEs). Several other promising modeling techniques have been used, including Boolean networks, Petri nets, Bayesian networks, graphical Gaussian models, Stochastic, and Process Calculi. Conversely, techniques have been proposed for generating models of GRNs that best explain a set of time series observations.

MALARIAL INFECTION ANIMATION

Malaria is a vector-borne infectious disease caused by protozoan parasites. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa. Each year, it causes disease in approximately 650 million people and kills between one and three million, most of them young children in Sub-Saharan Africa. Malaria is commonly-associated with poverty, but is also a cause of poverty and a major hindrance to economic development.

Malaria is one of the most common infectious diseases and an enormous public-health problem. The disease is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae, and sometimes Plasmodium knowlesi) can also infect humans. This group of human-pathogenic Plasmodium species is usually referred to as malaria parasites.

Malaria parasites are transmitted by female Anopheles mosquitoes. The parasites multiply within red blood cells, causing symptoms that include symptoms of anemia (light headedness, shortness of breath, tachycardia etc.), as well as other general symptoms such as fever, chills, nausea, flu-like illness, and in severe cases, coma and death. Malaria transmission can be reduced by preventing mosquito bites with mosquito nets and insect repellents, or by mosquito control by spraying insecticides inside houses and draining standing water where mosquitoes lay their eggs.

Pathogenesis

Malaria in humans develops via two phases: an exoerythrocytic (hepatic) and an erythrocytic phase. When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Within 30 minutes of being introduced into the human host, they infect hepatocytes, multiplying asexually and asymptomatically for a period of 6–15 days. During this so-called dormant time in the liver, the sporozoites are often referred to as hypnozoites. Once in the liver these organisms differentiate to yield thousands of merozoites which, following rupture of their host cells, escape into the blood and infect red blood cells, thus beginning the erythrocytic stage of the life cycle.The parasite escapes from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell.

Within the red blood cells the parasites multiply further, again asexually, periodically breaking out of their hosts to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cells.

Some P. vivax and P. ovale sporozoites do not immediately develop into exoerythrocytic-phase merozoites, but instead produce hypnozoites that remain dormant for periods ranging from several months (6–12 months is typical) to as long as three years. After a period of dormancy, they reactivate and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in these two species of malaria.

The parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance. However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels, thereby sequestering the parasite from passage through the general circulation and the spleen. This "stickiness" is the main factor giving rise to hemorrhagic complications of malaria. High endothelial venules (the smallest branches of the circulatory system) can be blocked by the attachment of masses of these infected red blood cells. The blockage of these vessels causes symptoms such as in placental and cerebral malaria. In cerebral malaria the sequestrated red blood cells can breach the blood brain barrier possibly leading to coma.

Although the red blood cell surface adhesive proteins (called PfEMP1, for Plasmodium falciparum erythrocyte membrane protein 1) are exposed to the immune system they do not serve as good immune targets because of their extreme diversity; there are at least 60 variations of the protein within a single parasite and perhaps limitless versions within parasite populations. Like a thief changing disguises or a spy with multiple passports, the parasite switches between a broad repertoire of PfEMP1 surface proteins, thus staying one step ahead of the pursuing immune system.

Some merozoites turn into male and female gametocytes. If a mosquito pierces the skin of an infected person, it potentially picks up gametocytes within the blood. Fertilization and sexual recombination of the parasite occurs in the mosquito's gut, thereby defining the mosquito as the definitive host of the disease. New sporozoites develop and travel to the mosquito's salivary gland, completing the cycle. Pregnant women are especially attractive to the mosquitoes, and malaria in pregnant women is an important cause of stillbirths, infant mortality and low birth weight

Four-Gene DNA Vaccine

DNA mismatch repair Lecture

Any mutational event that disrupts the superhelical structure of DNA carries with it the potential to compromise the genetic stability of a cell. Mismatch repair is a system for recognising and repairing the erroneous insertion, deletion and mis-incorporation of bases that can arise during DNA replication and recombination, as-well as repairing some forms of DNA damage . The fact that the damage detection and repair systems are as complex as the replication machinery itself highlights the importance evolution has attached to DNA fidelity.

Examples of mismatched bases include a G/T or A/C pairing (see DNA repair). The damage is repaired by excising the wrongly incorporated base and replacing it with the correct nucleotide. Usually, this involves more than just the mismatched nucleotide itself, and can lead to the removal of significant tracts of DNA.

Mismatch repair

There are two types of mismatch repair; long patch and short patch. Long patch can repair all types of mismatches (although it is primarily replication associated) and can excise tracts up-to a few kilobases long. Short patch repair handles only specific mismatches caused by damage to the genome, and removes lengths of around 10 nucleotides. Successful mismatch repair requires the error-free execution of three events:

1. Detection of a single mismatch, of which there are eight kinds, in the newly synthesised DNA. 2. Determining which of the two base pairs is incorrect. 3. Correcting the error by excision repair.

Mismatch repair is strand-specific. During DNA synthesis only the newly synthesised (daughter) strand will include errors, and replacing a base in the parental strand would actually introduce an error. The mismatch repair machinery has a number of cues which distinguish the newly synthesised strand from the template (parental). In gram-negative bacteria transient hemimethylation distinguishes the strands (the parental is methylated and daughter is not). In other prokaryotes and eukaryotes the exact mechanism is not clear.

Mismatch repair proteins

Mismatch Repair is a highly conserved process from prokaryotes to eukaryotes. The first evidence for mismatch repair was obtained from S. pneumoniae (the hexA and hexB genes). Subsequent work on E. coli has identified a number of genes that, when mutationally inactivated, cause hypermutable strains. The gene products are therefore called the "Mut" proteins, and are the major active components of the mismatch repair system. Three of these proteins are essential in detecting the mismatch and directing repair machinery to it; MutS, MutH and MutL (MutS is a homologue of HexA and MutL of HexB).

MutS forms a dimer (MutS2) that recognises the mismatched base on the daughter strand and binds the mutated DNA. MutH binds at hemimethylated sites along the daughter DNA, but its action is latent, being activated only upon contact by a MutL dimer (MutL2) which binds the MutS-DNA complex and acts as a mediator between MutS2 and MutH, activating the latter. The DNA is looped out to search for the nearest d(GATC) methylation site nearest the mismatch, which could be up to 1kb away. Upon activation by the MutS-DNA complex, MutH nicks the daughter strand near the mismatch and recruits a UvrD helicase (DNA Helicase II) to separate the two strands with a specific 3' to 5' polarity. The entire MutSHL complex then slides along the DNA in the direction of the mismatch, liberating the strand to be excised as it goes. An exonuclease trails the complex and digests the ss-DNA tail. The exonuclease recruited is dependent on which side of the mismatch MutH incises the strand – 5’ or 3’. If the nick made by MutH is on the 5’ end of the mismatch, either RecJ or ExoVIII (both 5’ to 3’ exonucleases) is used. If however the nick is on the 3’ end of the mismatch, ExoI (a 3' to 5' enzyme) is used.

The entire process ends past the mismatch site - i.e. both the site itself and its surrounding nucleotides are fully excised. The single-stranded gap created by the exonuclease can then be repaired by DNA Polymerase III (assisted by single-strand binding protein), which uses the other strand as a template, and finally sealed by DNA ligase. Dam methylase then rapidly methylates the daughter strand.

MutS

When bound, the MutS2 dimer bends the DNA helix and shields approximately 20 base pairs. It has weak ATPase activity, and binding of ATP leads to the formation of tertiary structures on the surface of the molecule. The crystalline structure of MutS reveals that it is exceptionally asymmetric, and while it's active conformation is a dimer, only one of the two halves interact with the mismatch site.

In Eukaryotes, MutS homologs form two major heterodimers: Msh2/Msh6 and Msh2/Msh3. The Msh2/Msh6 pathway is involved primarily in base substitution and small loop mismatch repair. The Msh2/Msh3 pathway is also involved in small loop repair, in addtion to large loop (~10 nucleotide loops) repair. However, Msh2/Msh3 does not repair base substitutions.

MutL

MutL also has weak ATPase activity (it uses ATP for purposes of movement). It forms a complex with MutS and MutH, increasing the MutS footprint on the DNA.

However, the processivity (the distance the enzyme can move along the DNA before dissociating) of UvrD is only ~40–50bp. Because the distance between the nick created by MutH and the mismatch can average ~600 bp, if there isn't another UvrD loaded the unwound section is then free to reanneal to its complementary strand, forcing the process to start over. However, when assisted by MutL, the rate of UvrD loading is greatly increased. While the processivity (and ATP utilisation) of the individual UvrD molecules remains the same, the total effect on the DNA is boosted considerably; the DNA has no chance to reanneal, as each UvrD unwinds 40-50 bp of DNA, dissociates, and then is immediately replaced by another UvrD, repeating the process. This exposes large sections of DNA to exonuclease digestion, allowing for quick excision(and later replacement) of the incorrect DNA.

Eukaryotes have MutL homologs designated Mlh1 and Pms1. They form a heterodimer which mimics MutL in E. coli. The human homologue of prokaryotic MutL has three forms designated as MutLα, MutLβ and MutLγ. The MutLα complex is made of two subunits MLH1 and PMS2, the MutLβ heterodimer is made of MLH1 and PMS1, while MutLγ is made of MLH1 and MLH3. MutLα acts as the matchmaker or facilitator, coordinating events in mismatch repair. It has recently been shown to be a DNA endonuclease that introduces strand breaks in DNA upon activation by mismatch and other required proteins, MutSa and PCNA. These strand interruptions serve as entry points for an exonuclease activity that removes mismatched DNA. Roles played by MutLβ and MutLγ in mismatch repair are less well understood.

MutH

MutH is a very weak endonuclease that is activated once bound to MutL (which itself is bound to MutS). It nicks unmethylated DNA and the unmethylated strand of hemimethylated DNA but does not nick fully methylated DNA. It has been experimentally shown that mismatch repair is random if neither strand is methylated. These behaviours led to the proposal that MutH determines which strand contains the mismatch. MutH has no Eukaryotic homolog. It's endonuclease function is taken up by MutL homologs, which have some specialized 5'-3' exonuclease activity. The strand bias for removing mismatches from the newly synthesized daughter strand in eukaryotes may be provided by the free 3’ ends of Okazaki fragments in the new strand created during replication

Defects in mismatch repair

Mutations in the human homologues of the Mut proteins affect genomic stability, which can result in microsatellite instability (MI). MI is implicated in most human cancers. Specifically the overwhelming majority of hereditary nonpolyposis colorectal cancers (HNPCC) are attributed to mutations in the genes encoding the MutS and MutL homologues, which allows them to be classified as tumour suppressor genes. A subtype of HNPCC is known as Muir-Torre Syndrome (MTS) which is associated with skin tumors.

Other Animations BioSolutions: Dna Helicase Animation BioSolutions: Peroxisome Proliferator-Activated Receptors BioSolutions: Grid-based Integrated Bioinformatics Systems For High-Throughput Evolutionary Analysis of Genomes and Metabolic Networks BioSolutions: Recombinant DNA Technology BioSolutions: Lecture on Stem Cells and the End of Aging BioSolutions: Eukaryotic translation Animation BioSolutions: Protein Structure Prediction Lecture BioSolutions: DNA Fingerprinting Animation BioSolutions: Embryonic Stem Cells and Disease Lecture BioSolutions: Central Dogma Animation BioSolutions: Immunity and Cancer Lecture

Dna Helicase

DNA helicase is an enzyme that aids in DNA synthesis by 'unzipping' the two strands of a DNA helix so that DNA polymerase can access the DNA to add nucleotides and effect copying.

Many cellular processes (DNA replication, RNA transcription, DNA recombination, DNA repair, Ribosome biogenesis) involve the separation of nucleic acid strands. Helicases are often utilized to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP or GTP hydrolysis. They move incrementally along one nucleic acid strand of the duplex with a directionality specific to each particular enzyme. There are many helicases (14 confirmed in E. coli, 24 in human cells) resulting from the great variety of processes in which strand separation must be catalyzed.

Function

Helicases adopt different structures and oligomerization states. Whereas DnaB-like helicases unwind DNA as donut shaped hexamers, other enzymes have been shown to be active as monomers or dimers. Recent studies showed that helicases do not merely wait passively for the fork to widen, but play an active role in forcing the fork to open, thus "it is an active unwinding motor". However, the unwinding is much faster in cells than in the test tube, so "accessory proteins are helping the helicase out by destabilizing the fork junction".

Structural features

The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology; they all possess common sequence motifs located in the interior of their primary sequence. These are thought to be specifically involved in ATP binding, ATP hydrolysis and translocation on the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.

Based on the presence of defined helicase motifs, it is possible to attribute a putative helicase activity to a given protein, though the presence of a motif does not confirm the protein as a helicase. Conserved motifs do, however, support an evolutionary homology among enzymes. Based on the presence and the form of helicase motifs, helicases have been separated in 4 superfamilies and 2 smaller families. Some members of these families are indicated, with the organism from which they are extracted, and their function.

Superfamilies

• Superfamily I: UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA (Staphylococcus aureus, Bacillus anthracis and Bacillus cereus, regulation of recombination by displacing RecA from DNA and inhibiting RecA-mediated DNA strand exchange), Dda (bacteriophage T4, replication initiation).
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• Superfamily II: RecQ (E. coli, DNA repair), eIF4A (Baker's Yeast, RNA translation), WRN (human, DNA repair), NS3 (Hepatitis C virus, replication). TRCF (Mfd) (E.coli, transcription-repair coupling factor).
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• Superfamily III: LTag (Simian Virus 40, replication), E1 (human papillomavirus, replication), Rep (Adeno-Associated Virus, replication, site-specific integration, virion packaging).
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• DnaB-like family: DnaB (E. coli, replication), gp41 (bacteriophage T4, DNA replication),T7gp4 (bacteriophage T7, DNA replication).
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• Rho-like family: Rho (E. coli, Transcription termination factor ).

PPAR gamma

Peroxisome proliferator-activated receptor delta

Peroxisome Proliferator-Activated Receptors

In cell biology, peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor isoforms that exist across biology. They are intimately connected to cellular metabolism (carbohydrate, lipid and protein) and cell differentiation. They are transcription factors.

Genomics Lecture

Tata Box Inhibitors - Ribosomal

Recombinant DNA Technology

Recombinant DNA is a form of artificial DNA which is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences which would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.

The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

Introduction

Because of the importance in DNA in the replication of new structures and characteristics of living organisms, it has widespread importance in recapitulating via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that have effects upon cellular or organismal processes. Through the use of recombinant DNA, genes that are identified as important can be amplified and isolated for use in other species or applications, where there may be some form of genetic illness or discrepancy, and provides a different approach to complex biological problem solving.

Applications and methods
Cloning and relation to plasmids

The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell which remain identical. In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine which contain recombinant DNA inserted into a structure known as a plasmid.

Plasmids are extrachromosomal self replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), contain genes related to catabolism and metabolic activity, and allow the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria is then used as a viral vector, which is encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects and increase the amount of cells with the recombinant DNA present within them.

The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism. As a general definition of plasmids, the definition is that they contain three common features -- a replicator, selectable marker and a cloning site.The replicator or "ori" refers to the origin of replication with regards to location and bacteria where replication begins. The marker refers to a gene which usually contains resistance to an antibiotic, but may also refer to a gene which is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA. The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs. Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception. In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.

Chimeric plasmids

When recombinant DNA is then further altered or changed to host additional strands of DNA, the molecule formed is referred to as "chimeric" DNA molecule, with reference to the mythological chimera which consisted as a composite of several animals. The presence of chimeric plasmid molecules is somewhat regular in occurrence as throughout the lifetime of an organism the propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells which all contain copies of the original chimeric DNA.

In the production of chimeric plasmids, the processes involved can be somewhat uncertainas the intended outcome of the addition of foreign DNA may not always be achieved and may result in the formation of unusable plasmids. Initially, the plasmid structure is linearised to allow the addition by bonding of complimentary foreign DNA strands to single-stranded "overhangs" or "sticky ends" present at the ends of the DNA molecule from staggered, or "S shaped" cleavages produced by restriction endonucleases.

A common vector used for the donation of plasmids originally was the bacterium Escherichia coli and later, the EcoRI derivative which was used for it's versatility with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol and spectinomycin; later being replaced by the pBR322 plasmid.In the case of EcoRI, the plasmid can anneal with the presence of foreign DNA via the route of sticky-end ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase , which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on blunt ends. Both sticky-end, or overhang ligation and blunt-end ligation can occur between foreign DNA segments, and cleaved ends of the original plasmid depending upon the restriction endonuclease used for cleavage

Lecture on Stem Cells and the End of Aging

Human tissues vary in their ability to heal and regenerate. The nervous system has weak powers of regeneration, while the skin is quick to make new cells for repair. Mammalian muscle cells are intermediate in their ability to regenerate. Human muscle can regenerate in response to minor wounds and normal wear and tear, but humans will not grow a new bicep, for example, in response to amputation. The heart is the most important muscle in the body and yet has feeble regenerative capabilities. Research into the wholesale production of new replacement organs and limbs is in its infancy, but research into enhancing normal levels of regeneration is progressing rapidly. Recent discoveries concerning the location and characteristics of adult stem cells and the signals that wounded tissue produces to activate stem cells have increased our understanding of regeneration. Insulin-like growth factor 1 (IGF1) is an example of an important stem cell communication molecule. If the activity of the growth factor is experimentally enhanced, muscle regeneration improves.

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Protein Structure Prediction Lecture

AIDS Infecting a Lymphocite

HIV can infect a variety of immune cells such as CD4+ T cells, macrophages, and microglial cells. HIV-1 entry to macrophages and CD4+ T cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells and also with chemokine coreceptors.

HIV enters macrophages and CD4+ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell.

Entry to the cell begins through interaction of the trimeric envelope complex (gp160 spike, discussed above) and both CD4 and a chemokine receptor (generally either CCR5 or CXCR4, but others are known to interact) on the cell surface. The gp160 spike contains binding domains for both CD4 and chemokine receptors. The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4 protein, the envelope complex undergoes a structural change, exposing the chemokine binding domains of gp120 and allowing them to interact with the target chemokine receptor. This allows for a more stable two-pronged attachment, which allows the N-terminal fusion peptide gp41 to penetrate the cell membrane. Repeat sequences in gp41, HR1 and HR2 then interact, causing the collapse of the extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid.

Once HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase, ribonuclease and protease, are injected into the cell.

HIV can infect dendritic cells (DCs) by this CD4-CCR5 route, but another route using mannose-specific C-type lectin receptors such as DC-SIGN can also be used. DCs are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T cells once the virus has been captured in the mucosa by DCs

Embryonic Stem Cells and Disease Lecture

Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro fertilization clinic—and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocyst; the blastocoel, which is the hollow cavity inside the blastocyst; and the inner cell mass, which is a group of approximately 30 cells at one end of the blastocoel.

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Immunity and Cancer Lecture

The immune system provides one of the body's main defenses against cancer. When normal cells turn into cancer cells, some of the antigens on their surface change. These new or altered antigens flag immune defenders, including cytotoxic T cells, natural killer cells, and macrophages.

According to one theory, patrolling cells of the immune system provide continuing bodywide surveillance, spying out and eliminating cells that undergo malignant transformation. Tumors develop when the surveillance system breaks down or is overwhelmed. Some tumors may elude the immune defenses by hiding or disguising their tumor antigens. Alternatively, tumors may survive by encouraging the production of suppressor T cells; these T cells act as the tumor's allies, blocking cytotoxic T cells that would normally attack it.

Blood tests show that people can develop antibodies to many types of tumor antigens (although the antibodies may not actually be effective in fighting the tumor). Skin testing (similar to skin testing for tuberculosis) has demonstrated that tumors provoke cellular immunity as well. Furthermore, studies indicated that cancer patients have a better prognosis when their tumors are infiltrated with many immune cells. Immune responses may underlie the spontaneous disappearance of some cancers.

Tests using antibodies derived from batches of human serum can detect various tumor-associated antigens-including carcinoembryonic antigen (CEA) and alphafetoprotein (AFP)-in blood samples. Because such antigens develop not only in cancer but in other diseases as well, the antibody tests are not useful for cancer screening in the general population. They are however, valuable in monitoring the course of disease and the effectiveness of treatment in patients known to have cancer.

Scientists have developed monoclonal antibodies (Hybridoma Technology) that are targeted specifically at tumor antigens. Linked to radioactive substances, these antibodies can be used to track down and reveal hidden cancer metastases within the body. Monoclonal antitumor antibodies are also being used experimentally to treat cancer-either in their native form or as immunotoxins, linked to natural toxins, anticancer drugs, or radioactive substances.

Other efforts to attack cancer through the immune system center on stimulating or replenishing the patient's immune responses with substances known as biological response modifiers. Among these are interferons (now obtained through genetic engineering) and interleukins. In some cases biological response modifiers are injected directly into the patient; in other cases they are used in the laboratory to transform some of the patient's own lymphocytes into tumor-hungry cells known as lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TILS), which are then injected back into the patient. Researchers are even using structures from the tumor cells themselves to construct custom-made anticancer "vaccines."

Animation on Influenza Infection(FLU)

Influenza, commonly known as flu, is an infectious disease of birds and mammals caused by a RNA virus of the family Orthomyxoviridae (the influenza viruses). In humans, common symptoms of influenza infection are fever, sore throat, muscle pains, severe headache, coughing, and weakness and fatigue. In more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. Sometimes confused with the common cold, influenza is a much more severe disease and is caused by a different type of virus.Although nausea and vomiting can be produced, especially in children,these symptoms are more characteristic of the unrelated gastroenteritis, which is sometimes called "stomach flu" or "24-hour flu."

Typically, influenza is transmitted from infected mammals through the air by coughs or sneezes, creating aerosols containing the virus, and from infected birds through their droppings. Influenza can also be transmitted by saliva, nasal secretions, feces and blood. Infections occur through contact with these bodily fluids or with contaminated surfaces. Flu viruses can remain infectious for about one week at human body temperature, over 30 days at 0 °C (32 °F), and indefinitely at very low temperatures (such as lakes in northeast Siberia). Most influenza strains can be inactivated easily by disinfectants and detergents.

Flu spreads around the world in seasonal epidemics, killing millions of people in pandemic years and hundreds of thousands in non-pandemic years. Three influenza pandemics occurred in the 20th century and killed tens of millions of people, with each of these pandemics being caused by the appearance of a new strain of the virus in humans. Often, these new strains result from the spread of an existing flu virus to humans from other animal species. Since it first killed humans in Asia in the 1990s, a deadly avian strain of H5N1 has posed the greatest risk for a new influenza pandemic; however, this virus has not mutated to spread easily between people

Infection and Replication

Influenza viruses bind through hemagglutinin onto sialic acid sugars on the surfaces of epithelial cells; typically in the nose, throat and lungs of mammals and intestines of birds .The cell imports the virus by endocytosis. In the acidic endosome, part of the haemagglutinin protein fuses the viral envelope with the vacuole's membrane, releasing the viral RNA (vRNA) molecules, accessory proteins and RNA-dependent RNA transcriptase into the cytoplasm .These proteins and vRNA form a complex that is transported into the cell nucleus, where the RNA-dependent RNA transcriptase begins transcribing complementary positive-sense vRNA . The vRNA is either exported into the cytoplasm and translated , or remains in the nucleus. Newly-synthesised viral proteins are either secreted through the Golgi apparatus onto the cell surface (in the case of neuraminidase and hemagglutinin, or transported back into the nucleus to bind vRNA and form new viral genome particles . Other viral proteins have multiple actions in the host cell, including degrading cellular mRNA and using the released nucleotides for vRNA synthesis and also inhibiting translation of host-cell mRNAs.

Negative-sense vRNAs that form the genomes of future viruses, RNA-dependent RNA transcriptase, and other viral proteins are assembled into a virion. Hemagglutinin and neuraminidase molecules cluster into a bulge in the cell membrane. The vRNA and viral core proteins leave the nucleus and enter this membrane protrusion . The mature virus buds off from the cell in a sphere of host phospholipid membrane, acquiring hemagglutinin and neuraminidase with this membrane coat . As before, the viruses adhere to the cell through hemagglutinin; the mature viruses detach once their neuraminidase has cleaved sialic acid residues from the host cell.After the release of new influenza virus, the host cell dies.

Because of the absence of RNA proofreading enzymes, the RNA-dependent RNA transcriptase makes a single nucleotide insertion error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, nearly every newly-manufactured influenza virus is a mutant. The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one viral line has infected a single cell. The resulting rapid change in viral genetics produces antigenic shifts and allow the virus to infect new host species and quickly overcome protective immunity

Lecture on Adult Stem Cells and Regeneration

Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues. Also known as somatic (from Greek Σωματικóς, of the body) stem cells, they can be found in children, as well as adults.
Research into adult stem cells has been fueled by their abilities to divide or self-renew indefinitely and generate all the cell types of the organ from which they originate — potentially regenerating the entire organ from a few cells. Unlike embryonic stem cells, the use of adult stem cells in research and therapy is not controversial because the production of adult stem cells does not require the destruction of an embryo. Adult stem cells can be isolated from a tissue sample obtained from an adult. They have mainly been studied in humans and model organisms such as mice and rats.

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Adult stem cells are being developed for use in treatments for a variety of human conditions, ranging from blindness to spinal cord injury.Since adult stem cells can be harvested from the patient, potential ethical issues and immunogenic rejection are averted. Adult stem cells, like embryonic stem cells, have pluripotent potential and can differentiate into cells derived from all three germ layers. Research has demonstrated that pluripotent stem cells can be directly generated from adult fibroblast cultures.



Adult stem cells are available in high quantities in cord blood, which can be collected at birth and are not difficult to isolate and purify. Other adult stem cell types can be multiplied in-vitro to therapeutic numbers, if needed.
While embryonic stem cell potential remains theoretical, adult stem cell treatments are already being used to successfully treat many diseases. The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. Adult stem cells also pose no medical dangers to the patient. Among the most stunning advancements in adult stem cell therapy are treatments for Parkinson's disease, juvenile diabetes, and spinal cord injuries

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DNA Microarray Animation

A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to chemically suitable matrices. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization.

Arrays of DNA can either be spatially arranged, as in the commonly known gene or genome chip, DNA chip, or gene array, or can be specific DNA sequences tagged or labelled such that they can be independently identified in solution. The traditional solid-phase array is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip. The affixed DNA segments are known as probes (although some sources will use different nomenclature such as reporters), thousands of which can be placed in known locations on a single DNA microarray. Microarray technology evolved from Southern blotting, whereby fragmented DNA is attached to a substrate and then probed with a known gene or fragment.

Applications of these arrays include:

• mRNA or gene expression profiling - Monitoring expression levels for thousands of genes simultaneously is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells .
  

• comparative genomic hybridization (Array CGH) - Assessing large genomic rearrangements.
  

• SNP detection arrays - Looking for Single nucleotide polymorphism in the genome of populations.
  

• Chromatin immunoprecipitation (chIP) studies - Determining protein binding site occupancy throughout the genome, employing ChIP-on-chip technology.
  

Fabrication

Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.

DNA microarrays can be used to detect RNAs that may or may not be translated into active proteins. Scientists refer to this kind of analysis as "expression analysis" or expression profiling. Since there can be tens of thousands of distinct probes on an array, each microarray experiment can accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations.

Spotted microarrays
In spotted microarrays (or two-channel or two-colour microarrays), the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs and are spotted onto the microarray surface. This type of array is typically hybridized with cDNA from two samples to be compared (e.g. diseased tissue versus healthy tissue) that are labeled with two different fluorophores (e.g. Rhodamine (Cyanine 5, red) and Fluorescein (Cyanine 3, green)). The two samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores. Relative intensities of each fluorophore are then used to identify up-regulated and down-regulated genes in ratio-based analysis. Absolute levels of gene expression cannot be determined in the two-colour array, but relative differences in expression among different spots (=genes) can be estimated with some oligonucleotide array.


Oligonucleotide microarrays

Two Affymetrix chips

In oligonucleotide microarrays (or single-channel microarrays), the probes are designed to match parts of the sequence of known or predicted mRNAs. T. These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays.

Oligonucleotide Arrays can be either produced by piezoelectric deposition with full length oligonucleotides or in-situ synthesis.

Long Oligonucleotide Arrays are composed of 60-mers, or 50-mers and are produced by ink-jet printing on a silica substrate. Short Oligonucleotide Arrays are composed of 25-mer or 30-mer and are produced by photolithographic synthesis on a silica substrate or piezoelectric deposition on an acrylamide matrix. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes. Arrays can contain up to 390,000 spots, from a custom array design. New array formats are being developed to study specific pathways or disease states for a systems biology approach.

Oligonucleotide microarrays often contain control probes designed to hybridize with RNA spike-ins. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes.


Genotyping microarrays

DNA microarrays can also be used to read the sequence of a genome in particular positions.

SNP microarrays are a particular type of DNA microarrays that are used to identify genetic variation in individuals and across populations. Short oligonucleotide arrays can be used to identify the single nucleotide polymorphisms (SNPs) that are thought to be responsible for genetic variation and the source of susceptibility to genetically caused diseases. Generally termed genotyping applications, DNA microarrays may be used in this fashion for forensic applications, rapidly discovering or measuring genetic predisposition to disease, or identifying DNA-based drug candidates.

These SNP microarrays are also being used to profile somatic mutations in cancer, specifically loss of heterozygosity events and amplifications and deletions of regions of DNA. Amplifications and deletions can also be detected using comparative genomic hybridization, or aCGH, in conjunction with microarrays, but may be limited in detecting novel Copy Number Polymorphisms, or CNPs, by probe coverage.

Resequencing arrays have also been developed to sequence portions of the genome in individuals. These arrays may be used to evaluate germline mutations in individuals, or somatic mutations in cancers.

Genome tiling arrays include overlapping oligonucleotides designed to blanket an entire genomic region of interest. Many companies have successfully designed tiling arrays that cover whole human chromosomes.

COMPARATIVE GENOMICS LECTURE

Comparative genomics is the study of relationships between the genomes of different species or strains. Comparative genomics is an attempt to take advantage of the information provided by the signatures of selection to understand the function and evolutionary processes that act on genomes. While it is still a young field, it holds great promise to yield insights into many aspects of the evolution of modern species. The sheer amount of information contained in modern genomes (several gigabytes in the case of humans) necessitates that the methods of comparative genomics are mostly computational in nature. Gene finding is an important application of comparative genomics, as is discovery of new, non-coding functional elements of the genome.

Comparative genomics exploits both similarities and differences in the proteins, RNA, and regulatory regions of different organisms to infer how selection has acted upon these elements. Those elements that are responsible for similarities between different species should be conserved through time (stabilizing selection), while those elements responsible for differences among species should be divergent (positive selection). Finally, those elements that are unimportant to the evolutionary success of the organism will be unconserved (selection is neutral).


Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is one of the important goals of the field. It is however often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. For this reason comparative genomics studies of small model organisms (for example yeast) are of great importance to advance our understanding of general mechanisms of evolution.

Having come a long way from its initial use of finding functional proteins, comparative genomics is now concentrating on finding regulatory regions and siRNA molecules. Recently, it has been discovered that distantly related species often share long conserved stretches of DNA that do not appear to code for any protein. It is unknown at this time what function such ultra-conserved regions serve.

Anti-Bacterial Defenses Animation

HIV Replication and life cycle

ATP SYNTHASE ANIMATION

An ATP synthase (EC 3.6.3.14) is a general term for an enzyme that can synthesize adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate by utilizing some form of energy.

The overall reaction sequence is:
ADP + Pi → ATP

Molecular model of ATP synthase by X-ray diffraction method

ATP synthase in E. coli

These enzymes are of crucial importance in almost all organisms, because ATP is the common "energy currency" of cells.

In mitochondria, the F1FO ATP synthase has a long history of scientific study. The F1 portion of the ATP synthase is above the membrane, the FO portion is within the membrane. It's easy to visualize the FOF1 particle as resembling the fruiting body of a common mushroom, with the head being the F1 particle, the stalk being the gamma subunit of F1, and the base and "roots" being the FO particle embedded in the membrane. The F1 particle was first isolated by Ephraim Racker in 1961. The nomenclature of the enzyme suffers from a long history. The F1 fraction derives it name from the term "Fraction 1" and FO (written as a subscript "O", not "zero") derives it name from being the oligomycine binding fraction. Taking as an example the nomenclature of subunits in the bovine enzyme, many subunits have Greek and Roman alphabet names (alpha, beta, gamma, delta, epsilon and a, b, c, d, e, f, g, h), while others have more complex names such as F6 (from "Fraction 6"), OSCP (the oligomycin sensitivity conferral protein), A6L (named for the gene that codes for it in the mitochondrial genome) and IF1 (inhibitory factor 1).

The F1 particle is large and can be seen in the transmission electron microscope by negative staining (1962, Fernandez-Moran et al., Journal of Molecular Biology, Vol 22, p 63). These are particles of 9 nm diameter that pepper the inner mitochondrial membrane. They were originally called elementary particles and were thought to contain the entire respiratory apparatus of the mitochondrion, but through a long series of experiments, Ephraim Racker and his colleagues were able to show that this particle is correlated with ATPase activity in uncoupled mitochondria and with the ATPase activity in submitochondrial particles created by exposing mitochondria to ultrasound. This ATPase activity was further associated with the creation of ATP by yet another long series of experiments in many laboratories.


Binding change mechanism

In the 1960s through the 1970s, Paul Boyer developed his binding change, or flip-flop, mechanism, which postulated that ATP synthesis is coupled with a conformational change in the ATP synthase generated by rotation of the gamma subunit. The research group of John E. Walker, then at the MRC Laboratory of Molecular Biology in Cambridge but now at the MRC Dunn Human Nutrition Unit (also in Cambridge) crystallized the F1 catalytic-domain of ATP synthase. The structure, at the time the largest asymmetric protein structure known, indicated that Boyer's rotary-catalysis model was essentially correct. For elucidating this Boyer and Walker shared half of the 1997 Nobel Prize in Chemistry. Jens Christian Skou received the other half of the Chemistry prize that year "for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase"

The crystal structure of the F1 showed alternating alpha and beta subunits (3 of each), arranged like segments of an orange around an asymmetrical gamma subunit. According to the current model of ATP synthesis (known as the alternating catalytic model), the proton-motive force across the inner mitochondrial membrane, generated by the electron transport chain, drives the passage of protons through the membrane via the FO region of ATP synthase. A portion of the FO (the ring of c-subunits) rotates as the protons pass through the membrane. The c-ring is tightly attached to the asymmetric central stalk (consisting primarily of the gamma subunit) which rotates within the alpha3beta3 of F1 causing the 3 catalytic nucleotide binding sites to go through a series of conformational changes that leads to ATP synthesis. The major F1 subunits are prevented from rotating in sympathy with the central stalk rotor by a peripheral stalk that joins the alpha3beta3 to the non-rotating portion of FO. The structure of the intact ATP synthase is currently known at low-resolution from electron cryo-microscopy (cryo-EM) studies of the complex. The cryo-EM model of ATP synthase shows that the peripheral stalk is a flexible rope-like structure that wraps around the complex as it joins F1 to FO. Under the right conditions, the enzyme reaction can also be carried out in reverse, with ATP hydrolysis driving proton pumping across the membrane.


Physiological role

Like other enzymes, the activity of F1FO ATP synthase is reversible. Large enough quantities of ATP cause it to create a transmembrane proton gradient, this is used by fermenting bacteria which do not have an electron transport chain, and hydrolyze ATP to make a proton gradient, which they use for flagella and transport of nutrients into the cell.

In respiring bacteria under physiological conditions, ATP synthase generally runs in the opposite direction, creating ATP while using the protonmotive force created by the electron transport chain as a source of energy. The overall process of creating energy in this fashion is termed oxidative phosphorylation. The same process takes place in mitochondria, where ATP synthase is located in the inner mitochondrial membrane

Lecture on Understanding Embryonic Stem Cells

Embryonic stem cells (ES cells) are stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst. Human embryos reach the blastocyst stage 4-5 days post fertilization, at which time they consist of 50-150 cells.



ES cells are pluripotent. This means they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult body. Pluripotency distinguishes ES cells from multipotent progenitor cells found in the adult; these only form a limited number of cell types. When given no stimuli for differentiation, (i.e. when grown in vitro), ES cells maintain pluripotency through multiple cell divisions

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Immune system Animation

An immune system is a collection of mechanisms within an organism that protects against infection by identifying and killing pathogens. It detects pathogens ranging from viruses to parasitic worms and distinguishes them from the organism's normal cells and tissues. Detection is complicated as pathogens adapt and evolve new ways to successfully infect the host organism.

To survive this challenge, several mechanisms have evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants, fish, reptiles, and insects. These mechanisms include antimicrobial peptides called defensins, pattern recognition receptors, and the complement system. More sophisticated mechanisms, however, developed relatively recently, with the evolution of vertebrates.The immune systems of vertebrates such as humans consist of many types of proteins, cells, organs, and tissues, which interact in an elaborate and dynamic network. As part of this more complex immune response, the vertebrate system adapts over time to recognize particular pathogens more efficiently. The adaptation process creates immunological memories and allows even more effective protection during future encounters with these pathogens. This process of acquired immunity is the basis of vaccination.

Disorders in the immune system can cause disease. Immunodeficiency diseases occur when the immune system is less active than normal, resulting in recurring and life-threatening infections. Immunodeficiency can either be the result of a genetic disease, such as severe combined immunodeficiency, or be produced by pharmaceuticals or an infection, such as the acquired immune deficiency syndrome (AIDS) that is caused by the retrovirus HIV. In contrast, autoimmune diseases result from a hyperactive immune system attacking normal tissues as if they were foreign organisms. Common autoimmune diseases include rheumatoid arthritis, diabetes mellitus type 1 and lupus erythematosus. These critical roles of immunology in health and disease are areas of intense scientific study.



Layered defense in immunity

The immune system protects organisms from infection with layered defenses of increasing specificity. Most simply, physical barriers prevent pathogens such as bacteria and viruses from entering the body. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. Innate immune systems are found in all plants and animals. However, if pathogens successfully evade the innate response, vertebrates possess a third layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.

Surface barriers

Several barriers protect organisms from infection, including mechanical, chemical and biological barriers. The waxy cuticle of many leaves, the exoskeleton of insects, the shells and membranes of externally deposited eggs, and skin are examples of the mechanical barriers that are the first line of defense against infection.
However, as organisms cannot be completely sealed against their environments, other systems act to protect body openings such as the lungs, intestines, and the genitourinary tract. In the lungs, coughing and sneezing mechanically eject pathogens and other irritants from the respiratory tract. The flushing action of tears and urine also mechanically expels pathogens, while mucus secreted by the respiratory and gastrointestinal tract serves to trap and entangle microorganisms.

Chemical barriers also protect against infection. The skin and respiratory tract secrete antimicrobial peptides such as the β-defensins. Enzymes such as lysozyme and phospholipase A in saliva, tears, and breast milk are also antibacterials. Vaginal secretions serve as a chemical barrier following menarche, when they become slightly acidic, while semen contains defensins and zinc to kill pathogens. In the stomach, gastric acid and proteases serve as powerful chemical defenses against ingested pathogens.

Within the genitourinary and gastrointestinal tracts, commensal flora serve as biological barriers by competing with pathogenic bacteria for food and space and, in some cases, by changing the conditions in their environment, such as pH or available iron.This reduces the probability that pathogens will be able to reach sufficient numbers to cause illness. However, since most antibiotics non-specifically target bacteria and do not affect fungi, oral antibiotics can lead to an “overgrowth” of fungi and cause conditions such as a vaginal candidiasis (yeast infection). There is good evidence that re-introduction of probiotic flora, such as pure cultures of the lactobacilli normally found in yoghurt, helps restore a healthy balance of microbial populations in intestinal infections in children and encouraging preliminary data in studies on bacterial gastroenteritis, inflammatory bowel diseases, urinary tract infection and post-surgical infections.

Innate immunity

Microorganisms that successfully enter an organism will encounter the cells and mechanisms of the innate immune system. Innate immune defenses are non-specific, meaning these systems recognize and respond to pathogens in a generic way. This system does not confer long-lasting immunity against a pathogen. The innate immune system is the dominant system of host defense in most organisms

Humoral and chemical barriers

Inflammation

Inflammation is one of the first responses of the immune system to infection. The symptoms of inflammation are redness and swelling, which are caused by increased blood flow into a tissue. Inflammation is produced by eicosanoids and cytokines, which are released by injured or infected cells. Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation, and leukotrienes that attract certain white blood cells (leukocytes). Common cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis; and interferons that have anti-viral effects, such as shutting down protein synthesis in the host cell. Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens.

Complement system

he complement system is a biochemical cascade that attacks the surfaces of foreign cells. It contains over 20 different proteins and is named for its ability to “complement” the killing of pathogens by antibodies. Complement is the major humoral component of the innate immune response. Many species have complement systems, including non-mammals like plants, fish, and some invertebrates.

In humans, this response is activated by the binding of complement proteins to carbohydrates on the surfaces of microbes or by complement binding to antibodies that have attached to these microbes. This recognition signal triggers a rapid killing response. The speed of the response is a result of signal amplification that occurs following sequential proteolytic activation of complement molecules, which are also proteases. After complement proteins initially bind to the microbe, they activate their protease activity, which in turn activates other complement proteases, and so on. This produces a catalytic cascade that amplifies the initial signal by controlled positive feedback. The cascade results in the production of peptides that attract immune cells, increase vascular permeability, and opsonize (coat) the surface of a pathogen, marking it for destruction. This deposition of complement can also kill cells directly by disrupting their plasma membrane.

Adaptive immunity
The adaptive immune system evolved in early vertebrates and allows for a stronger immune response as well as immunological memory, where each pathogen is "remembered" by a signature antigen.The adaptive immune response is antigen-specific and requires the recognition of specific “non-self” antigens during a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.

Lymphocytes

The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow.B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response.

Association of a T cell with MHC class I or MHC class II, and antigen (in red)

Both B cells and T cells carry receptor molecules that recognize specific targets. T cells recognize a “non-self” target, such as a pathogen, only after antigens (small fragments of the pathogen) have been processed and presented in combination with a “self” receptor called a major histocompatibility complex (MHC) molecule. There are two major subtypes of T cells: the killer T cell and the helper T cell. Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell. A third, minor subtype are the γδ T cells that recognize intact antigens that are not bound to MHC receptors.

In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface, and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.


Killer T cells

Killer T cell are a sub-group of T cells that kill cells infected with viruses (and other pathogens), or are otherwise damaged or dysfunctional. As with B cells, each type of T cell recognises a different antigen. Killer T cells are activated when their T cell receptor (TCR) binds to this specific antigen in a complex with the MHC Class I receptor of another cell. Recognition of this MHC:antigen complex is aided by a co-receptor on the T cell, called CD8. The T cell then travels throughout the body in search of cells where the MHC I receptors bear this antigen. When an activated T cell contacts such cells, it releases cytotoxins that form pores in the target cell's plasma membrane, allowing ions, water and toxins to enter. This causes the target cell to undergo apoptosis. T cell killing of host cells is particularly important in preventing the replication of viruses. T cell activation is tightly controlled and generally requires a very strong MHC/antigen activation signal, or additional activation signals provided by "helper" T cells.

Helper T cells

Helper T cells regulate both the innate and adaptive immune responses and help determine which types of immune responses the body will make to a particular pathogen. These cells have no cytotoxic activity and do not kill infected cells or clear pathogens directly. They instead control the immune response by directing other cells to perform these tasks.

Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC molecules. The MHC:antigen complex is also recognized by the helper cell's CD4 co-receptor, which recruits molecules inside the T cell (e.g. Lck) that are responsible for T cell's activation. Helper T cells have a weaker association with the MHC:antigen complex than observed for killer T cells, meaning many receptors (around 200–300) on the helper T cell must be bound by an MHC:antigen in order to activate the helper cell, while killer T cells can be activated by engagement of a single MHC:antigen molecule. Helper T cell activation also requires longer duration of engagement with an antigen-presenting cell. The activation of a resting helper T cell causes it to release cytokines that influence the activity of many cell types. Cytokine signals produced by helper T cells enhance the microbicidal function of macrophages and the activity of killer T cells. In addition, helper T cell activation causes an upregulation of molecules expressed on the T cell's surface, such as CD40 ligand (also called CD154), which provide extra stimulatory signals typically required to activate antibody-producing B cells.