BioSolutions

The Power of Basic Science Applied to Medical Progress: Past Examples and Hope for Schizophrenia and Bipolar Illness

2009-11-19T18:10:00.003+05:30

An exemplar of the purpose-driven life in medical science, Ed Scolnick details research milestones from a remarkably varied career, revealing how scientific insight and collaborative effort translate into life-saving solutions for millions.

This physician turned biochemist has held distinguished positions at the National Institutes of Health, Merck, and now at MIT, but common themes unite his pursuits: “I’m always excited by the inherent beauty of molecular and biochemical insights into how biology works. Making scientific discoveries for me is tremendously emotionally satisfying and in fact addicting.”

In his talk, Scolnick touches on such research breakthroughs as identifying virus oncogenes, and developing treatments for cardiovascular disease, Hepatitis B, and osteoporosis, among others. He emphasizes that teasing out the biochemistry of diseases is “the key to success in drug discovery.” In Marfan syndrome, for example, investigators learned that a mutant gene leads to a malfunctioning aorta. Finding a cure flowed from understanding the underlying pathological processes. Scolnick proudly describes research on a gene involved with cholesterol buildup and an elevated risk for cardiovascular disease. This led to the development of statins, which has helped dramatically reduce the death rate in people with heart disease.

Scolnick offers a dramatic chronology of his pioneering work at Merck starting in 1981 to find an effective AIDS treatment, an effort leading to the protease inhibitor Crixivan. His timeline covers more than a decade of scientific collaboration to block the mechanism of HIV, and involves false starts, the death of a key scientist in the Lockerbie bombing, pressure from AIDS activists and corporate overseers, a “miracle” AIDS patient, breakthroughs in measuring viral protein, and more than one “twist of fate.”

In 2004, Scolnick turned in a new direction: toward mental illness, a field stalled for decades due to ignorance “about the underlying biochemistry and physiology of the disease.” Today, with the help of genomics and computative technologies, researchers are beginning to reveal the basic genetic architecture of schizophrenia and bipolar illness, says Scolnick. The “outline of their biochemistry” is starting to come clear for the first time, leading to the real possibility of novel therapeutics. While the challenges are formidable, he believes, consolidating MIT’s “first rate neuroscience, human genetics, chemistry (creates) a unique opportunity to do something in a field that desperately needs the kind of approach and change we were able to bring to the AIDS field.”

Vaccines Animation

2009-11-09T13:27:00.004+05:30

vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains a small amount of an agent that resembles a microorganism. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

Vaccines can be prophylactic (e.g. to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are also being investigated; see cancer vaccine).

Types
Vaccines are dead or inactivated organisms or purified products derived from them.

There are several types of vaccines currently in use.[4] These represent different strategies used to try to reduce risk of illness, while retaining the ability to induce a beneficial immune response.
Killed

Vaccines containing killed microorganisms - these are previously virulent micro-organisms which have been killed with chemicals or heat. Examples are vaccines against flu, cholera, bubonic plague, polio and hepatitis A.
Attenuated

Some vaccines contain live, attenuated virus microorganisms. These are live micro-organisms that have been cultivated under conditions that disable their virulent properties, or which use closely-related but less dangerous organisms to produce a broad immune response. They typically provoke more durable immunological responses and are the preferred type for healthy adults. Examples include yellow fever, measles, rubella, and mumps. The live Mycobacterium tuberculosis vaccine developed by Calmette and Guérin is not made of a contagious strain, but contains a virulently modified strain called "BCG" used to elicit immunogenicity to the vaccine.
Toxoid

Toxoids - these are inactivated toxic compounds in cases where these (rather than the micro-organism itself) cause illness. Examples of toxoid-based vaccines include tetanus and diphtheria. Not all toxoids are for micro-organisms; for example, Crotalus atrox toxoid is used to vaccinate dogs against rattlesnake bites.
Subunit

Protein subunit - rather than introducing an inactivated or attenuated micro-organism to an immune system (which would constitute a "whole-agent" vaccine), a fragment of it can create an immune response. Characteristic examples include the subunit vaccine against Hepatitis B virus that is composed of only the surface proteins of the virus (produced in yeast) and the virus-like particle (VLP) vaccine against human papillomavirus (HPV) that is composed of the viral major capsid protein.
Conjugate

Conjugate - certain bacteria have polysaccharide outer coats that are poorly immunogenic. By linking these outer coats to proteins (e.g. toxins), the immune system can be led to recognize the polysaccharide as if it were a protein antigen. This approach is used in the Haemophilus influenzae type B vaccine.
Experimental

A number of innovative vaccines are also in development and in use:

* Recombinant Vector - by combining the physiology of one micro-organism and the DNA of the other, immunity can be created against diseases that have complex infection processes
* DNA vaccination - in recent years a new type of vaccine called DNA vaccination, created from an infectious agent's DNA, has been developed. It works by insertion (and expression, triggering immune system recognition) of viral or bacterial DNA into human or animal cells. Some cells of the immune system that recognize the proteins expressed will mount an attack against these proteins and cells expressing them. Because these cells live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they will be attacked instantly by the immune system. One advantage of DNA vaccines is that they are very easy to produce and store. As of 2006, DNA vaccination is still experimental.
* T-cell receptor peptide vaccines are under development for several diseases using models of Valley Fever, stomatitis, and atopic dermatitis. These peptides have been shown to modulate cytokine production and improve cell mediated immunity.
* Targeting of identified bacterial proteins that are involved in complement inhibition would neutralize the key bacterial virulence mechanism[5].

While most vaccines are created using inactivated or attenuated compounds from micro-organisms, synthetic vaccines are composed mainly or wholly of synthetic peptides, carbohydrates or antigens.
Valence

Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism.[6] A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms.[7] In certain cases a monovalent vaccine may be preferable for rapidly developing a strong immune response.

Vaccine. (2009, November 8). In Wikipedia, The Free Encyclopedia. Retrieved 07:59, November 9, 2009, from http://en.wikipedia.org/w/index.php?title=Vaccine&oldid=324652602

Works structure of the body from cells

2009-11-08T15:03:00.000+05:30

Mismeasure of Man

2009-11-08T15:01:00.000+05:30

Ralph Horwitz, MD, professor of medicine at Stanford discusses how measurement can both strengthen and weaken clinical science and care. Often overlooked amid today's enthusiasm for quantifiable results, he says, are the real complexities of medicine.

Telomere Replication

2009-10-31T10:08:00.001+05:30

DNA Polymerase

2009-10-31T09:24:00.001+05:30

DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. The newly-polymerized molecule is complementary to the template strand and identical to the template's original partner strand. DNA polymerases use a magnesium ion for catalytic activity.

Function

DNA polymerase can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication.

Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3'->5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue.

Ref:

DNA polymerase. (2009, October 19). In Wikipedia, The Free Encyclopedia. Retrieved 03:55, October 31, 2009, from http://en.wikipedia.org/w/index.php?title=DNA_polymerase&oldid=320725948

PDB File

2009-10-31T09:06:00.000+05:30

Protein Modification (Golgi)

2009-10-31T08:51:00.000+05:30

NanoBees

2009-10-25T13:29:00.000+05:30

When bees sting, they pump poison into their victims. Now the toxin in bee venom has been harnessed to kill tumor cells by researchers at Washington University School of Medicine in St. Louis. The researchers attached the major component of bee venom to nano-sized spheres that they call nanobees.

In mice, nanobees delivered the bee toxin melittin to tumors while protecting other tissues from the toxin's destructive power. The mice's tumors stopped growing or shrank. The nanobees' effectiveness against cancer in the mice is reported in advance online publication Aug. 10 in the Journal of Clinical Investigation.

"The nanobees fly in, land on the surface of cells and deposit their cargo of melittin which rapidly merges with the target cells," says co-author Samuel Wickline, M.D., who heads the Siteman Center of Cancer Nanotechnology Excellence at Washington University. "We've shown that the bee toxin gets taken into the cells where it pokes holes in their internal structures."

Melittin is a small protein, or peptide, that is strongly attracted to cell membranes, where it can form pores that break up cells and kill them.

"Melittin has been of interest to researchers because in high enough concentration it can destroy any cell it comes into contact with, making it an effective antibacterial and antifungal agent and potentially an anticancer agent," says co-author Paul Schlesinger, M.D., Ph.D., associate professor of cell biology and physiology. "Cancer cells can adapt and develop resistance to many anticancer agents that alter gene function or target a cell's DNA, but it's hard for cells to find a way around the mechanism that melittin uses to kill."

The scientists tested nanobees in two kinds of mice with cancerous tumors. One mouse breed was implanted with human breast cancer cells and the other with melanoma tumors. After four to five injections of the melittin-carrying nanoparticles over several days, growth of the mice's breast cancer tumors slowed by nearly 25 percent, and the size of the mice's melanoma tumors decreased by 88 percent compared to untreated tumors.

The researchers indicate that the nanobees gathered in these solid tumors because tumors often have leaky blood vessels and tend to retain material. Scientists call this the enhanced permeability and retention effect of tumors, and it explains how certain drugs concentrate in tumor tissue much more than they do in normal tissues.

But the researchers also developed a more specific method for making sure nanobees go to tumors and not healthy tissue by loading the nanobees with additional components. When they added a targeting agent that was attracted to growing blood vessels around tumors, the nanobees were guided to precancerous skin lesions that were rapidly increasing their blood supply. Injections of targeted nanobees reduced the extent of proliferation of precancerous skin cells in the mice by 80 percent.

Overall, the results suggest that nanobees could not only lessen the growth and size of established cancerous tumors but also act at early stages to prevent cancer from developing.

"Nanobees are an effective way to package the useful, but potentially deadly, melittin, sequestering it so that it neither harms normal cells nor gets degraded before it reaches its target," Schlesinger says.

If a significant amount of melittin were injected directly into the bloodstream, widespread destruction of red blood cells would result. The researchers showed that nanoparticles protected the mice's red cells and other tissues from the toxic effects of melittin. Nanobees injected into the bloodstream did not harm the mice. They had normal blood counts, and tests for the presence of blood-borne enzymes indicative of organ damage were negative.

When secured to the nanobees, melittin is safe from protein-destroying enzymes that the body produces. Although unattached melittin was cleared from the mice's circulation within minutes, half of the melittin on nanobees was still circulating 200 minutes later. Schlesinger indicates that is long enough for the nanobees to circulate through the mice's bloodstream 200 times, giving them ample time to locate tumors.

"Melittin is a workhorse," says Wickline, also professor of medicine in the Cardiovascular Division and professor of physics, of biomedical engineering and of cell biology and physiology. "It's very stable on the nanoparticles, and it's easily and cheaply produced. We are now using a nontoxic part of the melittin molecule to hook other drugs, targeting agents or imaging compounds onto nanoparticles."

The core of the nanobees is composed of perfluorocarbon, an inert compound used in artificial blood. The research group developed perfluorocarbon nanoparticles several years ago and have been studying their use in various medical applications, including diagnosis and treatment of atherosclerosis and cancer. About six millionths of an inch in diameter, the nanoparticles are large enough to carry thousands of active compounds, yet small enough to pass readily through the bloodstream and to attach to cell membranes.

"We can add melittin to our nanoparticles after they are built," Wickline says. "If we've already developed nanoparticles as carriers and given them a targeting agent, we can then add a variety of components using native melittin or melittin-like proteins without needing to rebuild the carrier. Melittin fortunately goes onto the nanoparticles very quickly and completely and remains on the nanobee until cell contact is made."

The flexibility of nanobees and other nanoparticles made by the group suggests they could be readily adapted to fit medical situations as needed. The ability to attach imaging agents to nanoparticles means that the nanoparticles can give a visible indication of how much medication gets to tumors and how tumors respond.

"Potentially, these could be formulated for a particular patient," Schlesinger says. "We are learning more and more about tumor biology, and that knowledge could soon allow us to create nanoparticles targeted for specific tumors using the nanobee approach."

Reflections on the Current H1N1 Flu

2009-10-23T09:57:00.001+05:30

John M. Barry brings unsettling news from the frontlines of H1N1 research: this novel influenza virus is very hard to pin down. In spite of international scientific scrutiny, H1N1 continues to baffle and elude, worrying health officials defending against the pandemic, and challenging some ideas about influenza in general. Says Barry, “A lot of things we thought we knew, the virus demonstrates we knew wrong.”

Barry examines the current pandemic in both historic and scientific context. Most influenza viruses share certain features: They can jump to other species by way of mutation, or by mixing genetic components with another virus that happens to be infecting the same cell at the same time. Influenza pandemics go “as far back in history as we can look,” with 10 occurring in just the last 300 years. Four of the most recent pandemics appear to have rolled out in waves of varying lethality, infecting at peak times some 30% of the human population.

Before last year, the latest pandemic threat seemed to be H5N1, an avian flu jumping to humans. But, says Barry, “while we were all looking at H5N1, this H1N1 virus snuck up on us…and we have no idea yet how serious it will be.” The problem for researchers is that H1N1 simply won’t behave in predictable ways. When ordinary influenza viruses are transmissible between humans, novel molecular markers are present. The current H1N1 doesn’t bear these markers, yet is transmissible. There are conflicting reports on whether this flu is more infectious than the seasonal flu. There’s evidence that some people over 60 are resistant, perhaps because they carry antibodies to previous influenzas. And although H1N1 doesn’t exhibit conventional molecular tags for virulence, it is virulent. Unlike seasonal flu, when H1N1 kills, it targets younger people, and it does so through viral pneumonia, as opposed to complicating bacterial infections. “Depending on how you ask the question, it’s either extraordinarily mild, more mild than seasonal flu, or more than 100 times as virulent as seasonal influenza.”

While H1N1 seems stable for the moment, and to some, unthreatening, its path can’t yet be plotted. Some of the most infamous flu epidemics take two years to travel around the world, moving from sporadic activity to “blanketing the entire globe and causing enormous morbidity numbers.” If this flu takes off, history tells us, short of a “retreat on a Vermont mountain with shotguns,” there will be nowhere to hide, says Barry. “This virus is going to find me.”

Bacteriorhodopsin

2009-10-21T10:01:00.002+05:30

Bacteriorhodopsin is a protein used by archaea, most notably halobacteria. It acts as a proton pump, i.e. it captures light energy and uses it to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy.

Bacteriorhodopsin is an integral membrane protein usually found in two-dimensional crystalline patches known as "purple membrane", which can occupy up to nearly 50% of the surface area of the archaeal cell. The repeating element of the hexagonal lattice is composed of three identical protein chains, each rotated by 120 degrees relative to the others. Each chain has seven transmembrane alpha helices and contains one molecule of retinal buried deep within, the typical structure for retinylidene proteins.

It is the retinal molecule that changes its conformation when absorbing a photon, resulting in a conformational change of the surrounding protein and the proton pumping action. It is covalently linked to Lys216 in the chromophore by Schiff base action. After photoisomerization of the retinal molecule, Asp85 becomes a proton acceptor of the donor proton from the retinal molecule. This releases a proton from a "holding site" into the extracellular side (EC) of the membrane. Reprotonation of the retinal molecule by Asp96 restores its original isomerized form. This results in a second proton being released to the EC side. Asp85 releases its proton into the "holding site" where a new cycle may begin.

The bacteriorhodopsin molecule is purple and is most efficient at absorbing green light (wavelength 500-650 nm, with the absorption maximum at 568 nm).

The three-dimensional tertiary structure of bacteriorhodopsin resembles that of vertebrate rhodopsins, the pigments that sense light in the retina. Rhodopsins also contain retinal; however, the functions of rhodopsin and bacteriorhodopsin are different and there is only slight homology in their amino acid sequences. Both rhodopsin and bacteriorhodopsin belong to the 7TM receptor family of proteins, but rhodopsin is a G protein coupled receptor and bacteriorhodopsin is not. In the first use of electron crystallography to obtain an atomic-level protein structure, the structure of bacteriorhodopsin was resolved in 1990. It was then used as a template to build models of other G protein-coupled receptors before crystallographic structures were also available for these proteins.

Many molecules have homology to bacteriorhodopsin, including the light-driven chloride pump halorhodopsin (for whom the crystal structure is also known), and some directly light-activated channels like channelrhodopsin.

All other photosynthetic systems in bacteria, algae and plants use chlorophylls or bacteriochlorophylls rather than bacteriorhodopsin. These also produce a proton gradient, but in a quite different and more indirect way involving an electron transfer chain consisting of several other proteins. Furthermore, chlorophylls are aided in capturing light energy by other pigments known as "antennas"; these are not present in bacteriorhodopsin based systems. Lastly, chlorophyll-based photosynthesis is coupled to carbon fixation (the incorporation of carbon dioxide into larger organic molecules); this is not true for bacteriorhodopsin-based system. It is thus likely that photosynthesis independently evolved at least twice, once in bacteria and once in archaea.

"Bacteriorhodopsin." Wikipedia, The Free Encyclopedia. 23 Aug 2009, 19:24 UTC. 21 Oct 2009 <http://en.wikipedia.org/w/index.php?title=Bacteriorhodopsin&oldid=309646691>.

Elongation Factor EF-TU

2009-10-17T15:23:00.000+05:30

EF-Tu (elongation factor thermo unstable) mediates the entry of the aminoacyl tRNA into a free site of the ribosome. EF-Tu functions by binding an aminoacylated, or charged, tRNA molecule in the cytoplasm. This complex transiently enters the ribosome, with the tRNA anticodon domain associating with the mRNA codon in the ribosomal A site. If the codon-anticodon pairing is correct, EF-Tu hydrolyzes GTP into GDP and inorganic phosphate, and changes in conformation to dissociate from the tRNA molecule. The aminoacyl tRNA then fully enters the A site, where its amino acid is brought near the P-site polypeptide and the ribosome catalyzes the covalent transfer of the polypeptide onto the amino acid.

EF-Tu contributes to translational accuracy in three ways. It delays GTP hydrolysis if the tRNA in the ribosome’s A site does not match the mRNA codon, thus preferentially increasing the likelihood for the incorrect tRNA to leave the ribosome. It also adds a second delay (regardless of tRNA matching) after freeing itself from tRNA, before the aminoacyl tRNA fully enters the A site. This delay period is a second opportunity for incorrectly-paired tRNA (and their bound amino acids) to move out of the A site before the incorrect amino acid is irreversibly added to the polypeptide chain. A third mechanism is the less well understood function of EF-Tu to crudely check amino acid-tRNA associations, and reject complexes where the amino acid is not bound to the correct tRNA coding for it.

Exploring the Mitochondria

2009-10-17T14:37:00.001+05:30

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane).

Outer membrane

The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the ER membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria.

Intermembrane space

The intermembrane space is basically the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, as large proteins must have a specific signaling sequence to be transported across the outer membrane, the protein composition of this space is different than the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

Inner membrane

The inner mitochondrial membrane contains proteins with five types of functions:

1. Those that perform the redox reactions of oxidative phosphorylation
2. ATP synthase, which generates ATP in the matrix
3. Specific transport proteins that regulate metabolite passage into and out of the matrix
4. Protein import machinery.
5. Mitochondria fusion and fission protein

It contains more than 100 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in beef hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane does not contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain.

Cristae

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. In typical liver mitochondria, for example, the surface area, including cristae, is about five times that of the outer membrane. Mitochondria of cells that have greater demand for ATP, such as muscle cells, contain more cristae than typical liver mitochondria.These folds are studded with small round bodies known as F1 particles or oxysomes.

Matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins (see: protein biosynthesis). A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 total genes: 22 tRNA, 2 rRNA, and 13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus.

Histone Modification Animation

2009-10-03T09:47:00.003+05:30

Histones are subject to a wide variety of posttranslational modifications including but not limited to, lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation (Vasquero 2003). These modifications occur primarily within the histone amino-terminal tails protruding from the surface of the nucleosome as well as on the globular core region (Cosgrove 2004).

Histone modifications are proposed to affect chromosome function through at least two distinct mechanisms. The first mechanism suggests modifications may alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA. The second mechanism proposes that these modifications are binding sites for protein recognition modules, such as the bromodomains or chromodomains, that recognize acetylated lysines or methylated lysine, respectively.

The existence of these modifications and recognition modules led to a well established “histone code” hypothesis proposed by Strahl and Allis (2000). Overall, posttranslational modifications of histones create an epigenetic mechanism for the regulation of a variety of normal and disease-related processes.

Zinc Finger Domain Animation

2009-09-29T21:34:00.003+05:30

Zinc fingers are small protein domains that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins or small molecules. The name "zinc finger" was coined to describe the hypothesized structure of the repeated unit in Xenopus laevis transcription factor IIIA.

Classes

Zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. They can be classified by the type and order of these zinc coordinating residues (e.g. Cys2His2, Cys4, and Cys6). A more systematic method classifies them into different "fold groups" based on the overall shape of the protein backbone in the folded domain. The most common "fold groups" of zinc fingers are the Cys2His2-like (the "classic zinc finger"), treble clef, and zinc ribbon.

Cys2His2

The Cys2His2-like fold group is by far the best characterized class of zinc fingers and are extremely common in mammalian transcription factors. These domains adopt a simple ββα fold and have the amino acid Sequence motif: X2-Cys-X2,4-Cys-X12-His-X3,4,5-His This class of zinc fingers can have a variety of functions such as binding RNA and mediating protein-protein interactions, but is best known for its role in sequence specific DNA-binding proteins such as Zif268. In such proteins, individual zinc finger domains typically occur as tandem repeats with two, three or more fingers comprising the DNA-binding domain of the protein. These tandem arrays can bind in the major groove of DNA and are typically spaced at 3-bp intervals. The α-helix of each domain (often called the "recognition helix") can make sequence specific contacts to DNA bases; residues from a single recognition helix can contact 4 or more bases to yield an overlapping pattern of contacts with adjacent zinc fingers.

Gag knuckle

This fold group is defined by two short β-strands connected by a turn (zinc knuckle) followed by a short helix or loop and resembles the classical Cys2His2 motif with a large portion of the helix and β-hairpin truncated. The best characters members of this family are found in the retroviral nucleocapsid (NC) protein from HIV and other related retroviruses. The gag knuckle zinc finger in the HIV NC protein is the target of a class of drugs known as zinc finger inhibitors.

Treble clef

The treble clef motif consists of a β-hairpin at the N-terminus and an α-helix at the C-terminus that each contribute two ligands for zinc binding, although a loop and a second β-hairpin of varying length and conformation can be present between the N-terminal β-hairpin and the C-terminal α-helix. These fingers are present in a diverse group of proteins that frequently do not share sequence or functional similarity with each other. The best characterized proteins containing treble clef zinc fingers are the [nuclear hormone receptors].

Zn2/Cys6

The canonical members of this class contain a binuclear zinc cluster in which two zinc ions are bound by six cysteine residues. These zinc fingers can be found in several transcription factors including the yeast Gal4 protein.

"Zinc finger." Wikipedia, The Free Encyclopedia. 16 Sep 2009, 10:25 UTC. 16 Sep 2009 <http://en.wikipedia.org/w/index.php?title=Zinc_finger&oldid=314315524>.

Lysozyme Structure

2009-09-29T20:37:00.000+05:30

Lysozymes, also known as muramidase or N-acetylmuramide glycanhydrolase, are a family of enzymes (EC 3.2.1.17) which damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is abundant in a number of secretions, such as tears, saliva, human milk and mucus. It is also present in cytoplasmic granules of the polymorphonuclear neutrophils (PMN). Large amounts of lysozyme can be found in egg white. C-type lysozymes are closely related to alpha-lactalbumin in sequence and structure making them part of the same family.

Function

The enzyme functions by attacking peptidoglycans (found in the cell walls of bacteria, especially Gram-positive bacteria) and hydrolyzing the glycosidic bond that connects N-acetylmuramic acid with the fourth carbon atom of N-acetylglucosamine. It does this by binding to the peptidoglycan molecule in the binding site within the prominent cleft between its two domains. This causes the substrate molecule to adopt a strained conformation similar to that of the transition state[citation needed]. According to Phillips-Mechanism, the lysozyme binds to a hexasaccharide. The lysozyme then distorts the 4th sugar in hexasaccharide (the D ring) into a half-chair conformation. In this stressed state the glycosidic bond is easily broken.

The amino acid side chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whilst Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The glycosyl enzyme intermediate then reacts with a water molecule, to give the product of hydrolysis and leaving the enzyme unchanged.

Role in disease

Lysozyme is part of the innate immune system. Children fed infant formula lack lysozyme in their diet and have three times the rate of diarrheal disease. Since lysozyme is a natural form of protection from pathogens like Salmonella, E.coli and Pseudomonas, when it is deficient due to infant formula feeding, can lead to increased incidence of disease.

Whereas the skin is a protective barrier due to its dryness and acidity, the conjunctiva (membrane covering the eye) is instead protected by secreted enzymes, mainly lysozyme and defensin. However, when these protective barriers fail, conjunctivitis results.

Electron Transport in Mitochondria

2009-09-13T19:18:00.004+05:30

The cells of almost all eukaryotes contain intracellular organelles called mitochondria, which produce ATP. Energy sources such as glucose are initially metabolized in the cytoplasm. The products are imported into mitochondria. Mitochondria continue the process of catabolism using metabolic pathways including the Krebs cycle, fatty acid oxidation, and amino acid oxidation.

The end result of these pathways is the production of two kinds of energy-rich electron donors, NADH and succinate. Electrons from these donors are passed through an electron transport chain to oxygen, which is reduced to water. This is a multi-step redox process that occurs on the mitochondrial inner membrane. The enzymes that catalyze these reactions have the ability to simultaneously create a proton gradient across the membrane, producing a thermodynamically unlikely high-energy state with the potential to do work. Although electron transport occurs with great efficiency, a small percentage of electrons are prematurely leaked to oxygen, resulting in the formation of the toxic free-radical superoxide.

The similarity between intracellular mitochondria and free-living bacteria is striking. The known structural, functional, and DNA similarities between mitochondria and bacteria provide strong evidence that mitochondria evolved from intracellular bacterial symbionts

Four membrane-bound complexes have been identified in mitochondria. Each is an extremely complex transmembrane structure that is embedded in the inner membrane. Three of them are proton pumps. The structures are electrically connected by lipid-soluble electron carriers and water-soluble electron carriers.

Complex I

Complex I (NADH dehydrogenase, also called NADH:ubiquinone oxidoreductase; EC 1.6.5.3) removes two electrons from NADH and transfers them to a lipid-soluble carrier, ubiquinone (Q). The reduced product, ubiquinol (QH2) is free to diffuse within the membrane. At the same time, Complex I moves four protons (H+) across the membrane, producing a proton gradient. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of main sites of production of a harmful free radical called superoxide.

The pathway of electrons occurs as follows:

NADH is oxidized to NAD+, reducing Flavin mononucleotide to FMNH2 in one two-electron step. The next electron carrier is a Fe-S cluster, which can only accept one electron at a time to reduce the ferric ion into a ferrous ion. In a convenient manner, FMNH2 can be oxidized in only two one-electron steps, through a semiquinone intermediate. The electron thus travels from the FMNH2 to the Fe-S cluster, then from the Fe-S cluster to the oxidized Q to give the free-radical (semiquinone) form of Q. This happens again to reduce the semiquinone form to the ubiquinol form, QH2. During this process, four protons are translocated across the inner mitochondrial membrane, from the matrix to the intermembrane space. This creates a proton gradient that will be later used to generate ATP through oxidative phosphorylation.

Complex II

Complex II (succinate dehydrogenase; EC 1.3.5.1) is not a proton pump. It serves to funnel additional electrons into the quinone pool (Q) by removing electrons from succinate and transferring them (via FAD) to Q. Complex II consists of four protein subunits: SDHA,SDHB,SDHC, and SDHD. Other electron donors (e.g., fatty acids and glycerol 3-phosphate) also funnel electrons into Q (via FAD), again without producing a proton gradient.

Complex III

Complex III (cytochrome bc1 complex; EC 1.10.2.2) removes in a stepwise fashion two electrons from QH2 at the QO site and sequentially transfers them to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. The two other electrons are sequentially passed across the protein to the Qi site where quinone part of ubiquinone is reduced to quinol. A proton gradient is formed because it takes 2 quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site. (in total 6 protons: 2 protons reduce quinone to quinol and 4 protons are released from 2 ubiquinol). The bc1 complex does NOT 'pump' protons, it helps build the proton gradient by an asymmetric absorption/release of protons.

When electron transfer is hindered (by a high membrane potential, point mutations or respiratory inhibitors such as antimycin A), Complex III may leak electrons to oxygen resulting in the formation of superoxide, a highly-toxic species, which is thought to contribute to the pathology of a number of diseases, including aging.

Complex IV

Complex IV (cytochrome c oxidase; EC 1.9.3.1) removes four electrons from four molecules of cytochrome c and transfers them to molecular oxygen (O2), producing two molecules of water (H2O). At the same time, it moves four protons across the membrane, producing a proton gradient.

Coupling with oxidative phosphorylation

The chemiosmotic coupling hypothesis, as proposed by Nobel Prize in Chemistry winner Peter D. Mitchell, explains that the electron transport chain and oxidative phosphorylation are coupled by a proton gradient across the inner mitochondrial membrane. The efflux of protons creates both a pH gradient and an electrochemical gradient. This proton gradient is used by the FOF1 ATP synthase complex to make ATP via oxidative phosphorylation. ATP synthase is sometimes regarded as complex V of the electron transport chain. The FO component of ATP synthase acts as an ion channel for return of protons back to mitochondrial matrix. During their return, the free energy produced during the generation of the oxidized forms of the electron carriers (NAD+ and Q) is released. This energy is used to drive ATP synthesis, catalyzed by the F1 component of the complex.

Coupling with oxidative phosphorylation is a key step for ATP production. However, in certain cases, uncoupling may be biologically useful. The inner mitochondrial membrane of brown adipose tissue contains a large amount of thermogenin (an uncoupling protein), which acts as uncoupler by forming an alternative pathway for the flow of protons back to matrix. This results in consumption of energy in thermogenesis rather than ATP production. This may be useful in cases when heat production is required, for example in colds or during arise of hibernating animals. Synthetic uncouplers (e.g., 2,4-dinitrophenol) also exist, and, at high doses, are lethal.

Summary

The mitochondrial electron transport chain removes electrons from an electron donor (NADH or QH2) and passes them to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane. There are three proton pumps: I, III, and IV. The resulting transmembrane proton gradient is used to make ATP via ATP synthase.

The reactions catalyzed by Complex I and Complex III exist roughly at equilibrium. This means that these reactions are readily reversible, simply by increasing the concentration of the products relative to the concentration of the reactants (for example, by increasing the proton gradient). ATP synthase is also readily reversible. Thus ATP can be used to make a proton gradient, which in turn can be used to make NADH. This process of reverse electron transport is important in many prokaryotic electron transport chains.

Electron transport chain. (2009, September 7). In Wikipedia, The Free Encyclopedia. Retrieved 21:19, September 7, 2009, from http://en.wikipedia.org/w/index.php?title=Electron_transport_chain&oldid=312464432

Photosystem II Animation

2009-09-13T19:00:00.001+05:30

PSII is the membrane protein complex found in oxygenic photosynthetic organisms (higher plants, green algae and cyanobacteria), which harnesses light energy to split H2O into O2, protons and electrons. It drives one of the most oxidising reactions known to occur in nature and is responsible for the production of atmospheric oxygen, essential for aerobic life on this planet. In addition, by catalysing the first step of the photosynthetic electron transport chain, PSII is also involved in the production of a substantial proportion of the global biomass.

Photosystem II, the Evolution of Non-cyclic Photosynthesis

Photosynthesis first evolved as an anoxygenic process in bacteria that were similar to the current green sulphur bacteria, where the transmission of an electron from the photosystem is accompanied by the extraction of a proton from hydrogen sulphide (H2S), producing sulphur as a by-product. In order to rejuvenate the pigment for further use, the electron takes a circular path from the original excitation of the pigment, through the photosynthetic electron transport system, back to the pigment molecule. This type of photosynthesis involved just one photosystem (P700) and culminated in the synthesis of ATP, an unstable energy source.

The advent of oxygenic photosynthesis provided an organism not only with ATP, but also with a stable energy source in the form of organic compounds that can be stored for later use. The transition from anoxygenic to oxygenic photosynthesis involved an extension of the existing system, whereby new reactions were added on to existing ones. This was achieved through a remarkable increase in protein complexity with the development of a second photosystem, photosystem II (PSII). The incorporation of hydrogen atoms into carbon-containing compounds required a source of reducing power, which came from the oxidation of water. However, it takes significantly more energy to split a hydrogen atom from water than it does from H2S. PSII contains chlorophyll a, first developed amongst cyanobacteria 2.5 billion years ago, which absorbs a shorter wavelength of light (680nm) with a higher energy level, and which is referred to as P680.

Plants, algae and some bacteria use two photosystems, PSI with P700 and PSII with P680. Using light energy, PSII acts first to channel an electron through a series of acceptors that drive a proton pump to generate ATP, before passing the electron on to PSI. Once the electron reaches PSI it has used most of its energy in producing ATP, but a second photon of light captured by P700 provides the required energy to channel the electron to ferredoxin, generating reducing power in the form of NADPH. The ATP and NADPH produced by PSII and PSI, respectively, are used in the light-independent reactions for the formation of organic compounds. This process is non-cyclic, because the electron from PSII is lost and is only replenished through the oxidation of water. Hence, there is a constant flow of electrons and associated hydrogens from water for the formation of organic compounds. It is this stripping of hydrogens from water that produces the oxygen we breathe.

PPAR Inhibiors in Cancer Treatment

2009-09-06T18:39:00.001+05:30

Poly (ADP-Ribose) Polymerase (PARP) is a protein cells use to repair genetic injuries naturally. But cancer cells also use this protein to repair their own DNA damage. Inhibiting this action allows chemotherapy and radiation to do its job against cancers resulting from genetic mutation.

Alpha Helix

2009-09-05T12:30:00.003+05:30

Alpha helix (α-helix) is a right- or left-handed coiled conformation, resembling a spring, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier (i+4 \rightarrow i hydrogen bonding). This secondary structure is also sometimes called a classic Pauling-Corey-Branson alpha helix.

Structure

Geometry and hydrogen bonding

The amino acids in an α helix are arranged in a right-handed helical structure where each amino acid corresponds to a 100° turn in the helix (i.e., the helix has 3.6 residues per turn), and a translation of 1.5 Å (= 0.15 nm) along the helical axis. The pitch of the helix (the vertical distance between two points on the helix) is 5.4 Å (= 0.54 nm)which is the product of 1.5 and 3,6. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid four residues earlier; this repeated i+4 \rightarrow i hydrogen bonding defines an α-helix. Similar structures include the 310 helix (i+3 \rightarrow i hydrogen bonding) and the π-helix (i+5 \rightarrow i hydrogen bonding). These alternative helices are relatively rare, although the 310 helix is often found at the ends of α-helices, "closing" them off. Transient i+2 \rightarrow i helices (sometimes called δ-helices) have also been reported as intermediates in molecular dynamics simulations of α-helical folding.

Residues in α-helices typically adopt backbone (φ, ψ) dihedral angles around (-60°, -45°). More generally, they adopt dihedral angles such that the ψ dihedral angle of one residue and the φ dihedral angle of the next residue sum to roughly -105°. Consequently, α-helical dihedral angles generally fall on a diagonal stripe on the Ramachandran plot (of slope -1), ranging from (-90°, -15°) to (-35°, -70°). For comparison, the sum of the dihedral angles for a 310 helix is roughly -75°, whereas that for the π-helix is roughly -130°. The general formula for the rotation angle Ω per residue of any polypeptide helix with trans isomers is given by the equation.

The α-helix is tightly packed; there is almost no free space within the helix. The amino-acid side chains are on the outside of the helix, and point roughly "downwards" (i.e., towards the N-terminus), like the branches of an evergreen tree (Christmas tree effect). This directionality is sometimes used in preliminary, low-resolution electron-density maps to determine the direction of the protein backbone.

Stability

Helices observed in proteins can range from four to over forty residues long, but a typical helix contains about ten amino acids (about three turns). Short polypeptides generally do not exhibit much alpha helical structure in solution, since the entropic cost associated with the folding of the polypeptide chain is not compensated for by a sufficient amount of stabilizing interactions. The backbone hydrogen bonds of α-helices are generally considered slightly weaker than those found in β-sheets, and are readily attacked by the ambient water molecules. However, in more hydrophobic environments such as the plasma membrane, or in the presence of co-solvents such as trifluoroethanol (TFE), or isolated from solvent in the gas phase,[6] oligopeptides readily adopt stable α-helical structure.

F1 making ATP

2009-09-04T10:28:00.001+05:30

Photosystem I

2009-09-01T21:52:00.005+05:30

Photosystem I is a proteinaceous transmembrane structure composed of several proteins and embedded with pigment molecules. This structure is located inside chloroplasts and secured within the thylakoid membrane with exposure to the thylakoid lumen on one side and to the chloroplast stroma on the other side. PS I acts as an energy converter for various photosynthetic organisms.

Mechanics of Photosystem I

Light energy in the form of photons is converted into electrons to power the generation of ATP or the reduction of NADP+ to NADPH.[4] Photons are received by an antenna complex of pigment molecules. Antenna molecules become photoexcited and pass the energy as resonance energy (text). The resonance energy is transferred to the reaction center pigment chlorophyll a. The reaction center in turn transfers electrons to a primary electron acceptor and subsequent electron acceptors and carriers. Finally, the electrons reduce NADP+ or help generate ATP. Electrons may be recycled to increase the proton concentration in the thylakoid lumen in a process called cyclic electron flow. In cyclic electron flow electrons are passed from the PS I reaction center and then carried to a cytochromeb6f complex where they help transport protons into the thylakoid lumen thus creating ATP.Plastocyanin may accept electrons from the cytochrome b6f complex and pass them along to the reaction center in the antenna complex beginning the cycle again.

F1 component making ATP

2009-09-01T20:57:00.001+05:30

Cyclic Photophosphorylation

2009-09-01T20:47:00.002+05:30

In cyclic electron flow, the electron begins in a pigment complex called photosystem I, passes from the primary acceptor to plastoquinone, then to cytochrome b6f (a similar complex to that found in mitochondria), and then to plastocyanin before returning to chlorophyll. This transport chain produces a proton-motive force, pumping H+ ions across the membrane; this produces a concentration gradient which can be used to power ATP synthase during chemiosmosis. This pathway is known as cyclic photophosphorylation, and it does not produce O2, as well as ATP. Unlike non-cyclic photophosphorylation, NADP+ does not accept the electrons, but they are sent back to photosystem I. NADPH is NOT produced in cyclic photophosphorylation. In bacterial photosynthesis, a single photosystem is used, and therefore is involved in cyclic photophosphorylation.

Coenzyme Transporting Electrons to ETC

2009-08-31T21:26:00.001+05:30