APOPTOSIS------How is our cells dead ???

This video will give you an inspiration, how our cell will die...Nice creation of the God....!!!



Apoptosis

For every cell, there is a time to live and a time to die.

There are two ways in which cells die:

* They are killed by injurious agents.
* They are induced to commit suicide.

Death by injury
Cells that are damaged by injury, such as by

* mechanical damage
* exposure to toxic chemicals

undergo a characteristic series of changes:

* They (and their organelles like mitochondria) swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted).
* The cell contents leak out, leading to
* inflammation of surrounding tissues.

Death by suicide
Cells that are induced to commit suicide:

* shrink;
* develop bubble-like blebs on their surface;
* have the chromatin (DNA and protein) in their nucleus degraded;
* have their mitochondria break down with the release of cytochrome c;
* break into small, membrane-wrapped, fragments.
* The phospholipid phosphatidylserine, which is normally hidden within the plasma membrane, is exposed on the surface.
* This "eat me" signal is bound by receptors on phagocytic cells like macrophages and dendritic cells which then engulf the cell fragments.
* The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)

The pattern of events in death by suicide is so orderly that the process is often called programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis.

Programmed cell death is also called apoptosis. (There is no consensus yet on how to pronounce it; some say APE oh TOE sis; some say uh POP tuh sis.)
Why should a cell commit suicide?
There are two different reasons.
1. Programmed cell death is as needed for proper development as mitosis is.
Examples:

* The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis.
* The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them.
* The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation occurs by apoptosis.
* The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis

2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism.
Examples:

Cells infected with viruses
One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis [diagram of the mechanism]. (And some viruses mount countermeasures to thwart it — Link)

Cells of the immune system
As cell-mediated immune responses wane, the effector cells must be removed to prevent them from attacking body constituents. CTLs induce apoptosis in each other and even in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases such as lupus erythematosus and rheumatoid arthritis.

Cells with DNA damage
Damage to its genome can cause a cell

* to disrupt proper embryonic development leading to birth defects
* to become cancerous.

Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis. Is it any wonder that mutations in the p53 gene, producing a defective protein, are so often found in cancer cells (that represent a lethal threat to the organism if permitted to live)?

Cancer cells
Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells.

What makes a cell decide to commit suicide?
The balance between:

* the withdrawal of positive signals; that is, signals needed for continued survival, and
* the receipt of negative signals.

Withdrawal of positive signals
The continued survival of most cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals:

* growth factors for neurons
* Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes

Receipt of negative signals

* increased levels of oxidants within the cell
* damage to DNA by these oxidants or other agents like
o ultraviolet light
o x-rays
o chemotherapeutic drugs
* accumulation of proteins that failed to fold properly into their proper tertiary structure
* molecules that bind to specific receptors on the cell surface and signal the cell to begin the apoptosis program. These death activators include:
o Tumor necrosis factor-alpha (TNF-α ) that binds to the TNF receptor;
o Lymphotoxin (also known as TNF-β ) that also binds to the TNF receptor;
o Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called CD95).

The Mechanisms of Apoptosis
There are 3 different mechanisms by which a cell commits suicide by apoptosis.

1. One generated by signals arising within the cell;
2. another triggered by death activators binding to receptors at the cell surface:
* TNF-α
* Lymphotoxin
* Fas ligand (FasL)
3. A third that may be triggered by dangerous reactive oxygen species.

1. Apoptosis triggered by internal signals: the intrinsic or mitochondrial pathway

* In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on their surface. Bcl-2 inhibits apoptosis.
* Internal damage to the cell (e.g., from reactive oxygen species) causes
o related proteins, Bad and Bax, to migrate to the surface of the mitochondrion where they bind to Bcl-2 — blocking its protective effect — and punch holes in the outer mitochondrial membrane, causing
o cytochrome c to leak out.
* The released cytochrome c binds to the protein Apaf-1 ("apoptotic protease activating factor-1").
* Using the energy provided by ATP,
* these complexes aggregate to form apoptosomes.
* The apoptosomes bind to and activate caspase-9.
* Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get their name because they cleave proteins — mostly each other — at aspartic acid (Asp) residues).
* Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7).
* The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity (rather like that in blood clotting and complement activation) which leads to
o digestion of structural proteins in the cytoplasm,
o degradation of chromosomal DNA, and
* phagocytosis of the cell.

2. Apoptosis triggered by external signals: the extrinsic or death receptor pathway

* Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell
* binding of the complementary death activator (FasL and TNF respectively) transmits a signal to the cytoplasm that leads to
* activation of caspase 8
* caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to
* phagocytosis of the cell.

Example (right): When cytotoxic T cells recognize (bind to) their target,

* they produce more FasL at their surface.
* This binds with the Fas on the surface of the target cell leading to its death by apoptosis.

The early steps in apoptosis are reversible — at least in C. elegans. In some cases, final destruction of the cell is guaranteed only with its engulfment by a phagocyte.
3. Apoptosis-Inducing Factor (AIF)

Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases.
Apoptosis-inducing factor (AIF) is a protein that is normally located in the intermembrane space of mitochondria. When the cell receives a signal telling it that it is time to die, AIF

* is released from the mitochondria (like the release of cytochrome c in the first pathway);
* migrates into the nucleus;
* binds to DNA, which
* triggers the destruction of the DNA and cell death.

Apoptosis and Cancer
Some viruses associated with cancers use tricks to prevent apoptosis of the cells they have transformed.

* Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53.
* Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas
o produces a protein similar to Bcl-2
o produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).

Even cancer cells produced without the participation of viruses may have tricks to avoid apoptosis.

* Some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production. [Discussion].
* Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.
* Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells by the mechanism shown above.
* Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).

Apoptosis in the Immune System

The immune response to a foreign invader involves the proliferation of lymphocytes — T and/or B cells [Link]. When their job is done, they must be removed leaving only a small population of memory cells [Link]. This is done by apoptosis.

Very rarely humans are encountered with genetic defects in apoptosis. The most common one is a mutation in the gene for Fas, but mutations in the gene for FasL or even one of the caspases are occasionally seen. In all cases, the genetic problem produces autoimmune lymphoproliferative syndrome or ALPS.
Features:

* an accumulation of lymphocytes in the lymph nodes and spleen greatly enlarging them.
* the appearance of clones that are autoreactive; that is, attack "self" components producing such autoimmune disorders as
o hemolytic anemia
o thrombocytopenia
* the appearance of lymphoma — a cancerous clone of lymphocytes.

In most patients with ALPS, the mutation is present in the germline; that is, every cell in their body carries it. In a few cases, however, the mutation is somatic; that is, has occurred in a precursor cell in the bone marrow. These later patients are genetic mosaics — with some lymphocytes that undergo apoptosis normally and others that do not. The latter tend to out-compete the former and grow to become the major population in the lymph nodes and blood.
Apoptosis and AIDS

The hallmark of AIDS (acquired immunodeficiency syndrome) is the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (µl) of blood). CD4+ T cells are responsible, directly or indirectly (as helper cells), for all immune responses. When their number declines below about 200 per µl, the patient is no longer able to mount effective immune responses and begins to suffer a series of dangerous infections.

What causes the disappearance of CD4+ T cells?
HIV (human immunodeficiency virus) invades CD4+ T cells, and one might assume that it this infection by HIV that causes the great dying-off of these cells. However, that appears not to the main culprit. Fewer than 1 in 100,000 CD4+ T cells in the blood of AIDS patients are actually infected with the virus.

So what kills so many uninfected CD4+ cells?

The answer is clear: apoptosis.
The mechanism is not clear. There are several possibilities. One of them:

* All T cells, both infected and uninfected, express Fas.
* Expression of a HIV gene (called Nef) in a HIV-infected cell causes
o the cell to express high levels of FasL at its surface
o while preventing an interaction with its own Fas from causing it to self-destruct.
* However, when the infected T cell encounters an uninfected one (e.g. in a lymph node), the interaction of FasL with Fas on the uninfected cell kills it by apoptosis.

Apoptosis and Organ Transplants
For many years it has been known that certain parts of the body such as

* the anterior chamber of the eye
* the testes

are "immunologically privileged sites". Antigens within these sites fail to elicit an immune response.

It turns out that cells in these sites differ from the other cells of the body in that they express high levels of FasL at all times. Thus antigen-reactive T cells, which express Fas, would be killed when they enter these sites. (This is the reverse of the mechanism described above.)
This finding raises the possibility of a new way of preventing graft rejection.

If at least some of the cells on a transplanted kidney, liver, heart, etc. could be made to express high levels of FasL, that might protect the graft from attack by the T cells of the host's cell-mediated immune system. If so, then the present need for treatment with immunosuppressive drugs for the rest of the transplant recipient's life would be reduced or eliminated.

So far, the results in animal experiments have been mixed. Allografts engineered to express FasL have shown increased survival for kidneys but not for hearts or islets of Langerhans.
Apoptosis in Plants

Plant, too, can turn on a system of programmed cell death; for example, in an attempt to halt the spread of virus infection.

The mechanism differs from that in animals although it, too, involves a protease that — like caspases — cleaves other proteins at Asp (and Asn) residues.

Activation of this enzyme destroys the central vacuole, which is followed by disintegration of the rest of the cell.

New PCR Machine Microchip

Researchers in Hong Kong have miniaturized technology needed to perform the versatile polymerase chain reaction (PCR) -- widely used in criminal investigations, disease diagnosis, and a range of other key applications. They report development of a long-sought PCR microchip that could permit use of PCR at crime scenes, in doctors' offices, and other out-of-lab locations.

I-Ming Hsing and colleagues note that PCR works like a biological copy machine, transforming a few wisps of DNA into billions of copies. However, existing PCR machines are so big and complex that they can be used only in laboratories. Scientists have searched for years for a portable, PCR technique that can be used outside the lab.

The study describes a new PCR technique that uses electrochemical DNA sensors to provide simultaneous DNA amplification and detection on a silicon-glass microchip. Their performance tests show that the new technique, called electrochemical real-time PCR (ERT-PCR), is about as fast and sensitive as conventional PCR. The new technique shows "tremendous" promise as a portable system for moving DNA analysis out of the lab and into remote locations, the researchers say.

The article "Electrochemistry-Based Real-Time PCR on a Microchip" is published in the Jan. 15 issue of ACS' Analytical Chemistry.

taken from : sciencedaily Jan 4th, 2008

monoclonal antibody industry...next bom industry ..???

Monoclonal antibody drugs offer several advantages over other types of drugs. Technologically, antibodies exhibit amazing specificity that allows for a safe and targeted attack on disease-causing cells or compounds. The antibody itself may be used as a weapon or the antibody may serve as the vehicle to deliver a drug. Along with high specificity, monoclonal antibodies are effective in a broad range of diseases including autoimmune, cardiovascular and infectious diseases, cancer and inflammation. Also of great importance is that antibodies are virtually never toxic.

As of May 2005, there were 18 therapeutic monoclonal antibody products on the U.S. market. Worldwide, there were an estimated 500 monoclonal antibody products in development by more than 200 companies for the treatment of virtually every debilitating disease. Approximately 80 of these were in clinical trials.

This BCC report provides detailed market analyses and industry trends. It quantifies and qualifies the emerging market for monoclonal and polyclonal antibody drugs and imaging products. Forecasts and trends are gleaned from industry sources as well as from considered assessments of available and emerging technologies. The study also examines strategies used by biotechnology companies and pharmaceutical firms to develop and market products in this explosive market sector.

We will be waiting for the next big industry .....!!!!!

Detection fragile X Using a little split out ?

Hello reader,

I just learning about the article about detection of Fragile X syndrome patients using methylation-specific PCR of the FMR1 gene. It has been reported that using this method could detect fragile X patient, especally in man with 100 % accuracy. I also have friend of mine in lab who did many samples with this method. All the samples were blood that was drawn from the samples. I am just wondering...We can develop the test like this USING a little split out..since it can be run in PCR......So in the future we can screen the people in large population, more cheapest than Southern, and in one single day....!!!!.....Hmmmmm...very interesting....!!!
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here is the article :

INTRODUCTION

The fragile X syndrome (FRAXA) is the most common inherited form of mental retardation in man. The molecular pathogenesis of the disease generally involves expansion of a (CGG)_n trinucleotide repeat located on the 5' region of the FMR1 gene, leading to hypermethylation of the promoter and shutdown of gene expression (reviewed by Nelson, 1998; Kaufmann and Reiss, 1999). The molecular diagnosis of FRAXA has depended on simultaneous Southern blot analysis of (CGG)_n length and methylation status. However, this is a complex procedure and, to screen for FRAXA among mentally retarded children, we need simpler and cheaper PCR-based diagnostic tests.

The first PCR-based test that we developed was based on direct PCR amplification of the (CGG)_n trinucleotide repeat with primers flanking the microsatellite, with a product of 557 bp for the (CGG)₂₉ allele (Haddad et al., 1996). Conditions were established so that full mutations failed to amplify. To produce an internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 base pairs upstream the polymorphic region. In blind trials the PCR-based test showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. The test had two main disadvantages. Firstly, a normal (CGG)_n allele was preferentially amplified by PCR due to its smaller size and thus the PCR technique could not be used for the diagnosis of FRAXA in females, because they are heterozygous and would be scored as normal. For the same reason, mosaic patients with a normal sized allele might yield a false negative result. That is why the test was not 100% sensitive. The second drawback resulted from the "failure-to-amplify" characteristic of the test that thus could not provide a definitive diagnosis of fragile X syndrome. Although not quite suitable for medical diagnosis, the PCR test proved to be a useful tool for fragile X syndrome screening in populations of mentally retarded males (Haddad et al., 1999).

Recently Herman et al. (1996) developed an elegant PCR assay for methylation status of CpG islands. DNA samples are first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We wish to report the successful application of this methylation-specific PCR (MSP) for the study of the FMR1 promoter. This led to a much-improved method for PCR diagnosis of the fragile X syndrome in affected males.

MATERIAL AND METHODS

Patients

We used DNA from eight patients with fragile X syndrome, all confirmed by Southern blot analysis: five of these were ascertained in a screening study of mentally retarded boys in Brazil (Haddad et al., 1999), two were patients diagnosed at GENE and the other (NA06852) was obtained from the Coriell Mutant Cell Repository (Camden, NJ, USA). DNA samples from 42 normal controls were obtained from paternity testing cases at GENE.

PCR primers

For development of primers we used the data of Stöger et al. (1997) who described the pattern of cytosine methylation at the CpG island of the FMR1 gene. We designed two primer pairs: the first, 5'-AAATGGGCGTTTTGGTTTTCGC-3' and 5'-GCCAAAAATCATCGCGCATACG-3', produces a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other, 5'-TGTTTTTTATTAAGTTTGTGTAT-3' and 5'-ACCAAAAATCATCACACATACA-3', generates an 84-bp product from the treated non-methylated promoter. The strategy behind the primer development is shown schematically in Figure 1.

Methylation-specific PCR

DNA samples were treated with sodium bisulfite as described by Herman et al. (1996). They were then submitted to PCR amplification in separate tubes with primers specific for the methylated (M) or non-methylated (N) versions of the CpG island of the FMR1 gene. In the reaction with the M primers we also included primers specific for the methylated version of the SNRPN gene (Kubota et al., 1997). PCR was performed in a final volume of 13 µl using 0.65 µ AmpliTaq Gold (Perkin Elmer, Foster City, CA, USA) in the manufacturer's recommended buffer, 200 µM of each dNTP, 0.4 µM of each primer and 100 ng of human genomic DNA. Thermal cycling conditions were: initial denaturation at 95^oC for 5 min, followed by 35 cycles of 1 min of annealing at 53^oC for the M reaction and 43^oC for the N reaction, 1 min of extension at 72^oC and denaturation at 95^oC for 1 min. Afterwards, the PCR reaction products were separated by electrophoresis in a 6% polyacrylamide gel and visualized by silver staining.

RESULTS AND DISCUSSION

In normal males only the 84-bp fragment was seen (Figure 2), while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with visualization of a much weaker 84-bp band (Figure 2). The probable reasons that the 84-bp product did not disappear as could be expected are that methylation is generally not complete (Stöger et al., 1997) and that somatic mosaicism occurs in the length of the (CGG)_n repeat in complete mutations. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15 (Kubota et al., 1997) generating a fragment of 174 bp (Figure 2).

Using the methylation-specific PCR we identified with 100% specificity, sensitivity and accuracy, eight previously diagnosed FRAXA male patients mixed with 42 normal controls. In theory the test should not be prone to producing false positive results and should also be very sensitive, permitting diagnosis even in mosaics with normal-sized alleles. Indeed, we have found that we can still obtain a clear methylated product even when DNA from FRAXA patients is diluted 20-fold with normal male DNA. If needed, sensitivity could be further increased by the use of fluorescently labeled primers and detection in an automatic DNA sequencer.

Apparently the pattern of methylation in the promoter region of the FMR1 is identical in full mutations of FRAXA and in X inactivation in normal females (Stöger et al., 1997). Thus, the MSP test cannot be used to diagnose the fragile X syndrome in affected females, since they already have, in virtue of X inactivation, a methylated FMR1 promoter region.

In summary, methylation-specific PCR emerges as a simple and efficient method for assessing methylation in the FMR1 CpG island. Indeed, it may become the method of choice for diagnosis of the fragile X syndrome in mentally retarded males.