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Online repository of biological information which aims to create a knowledge base for students by the provision of animations and lectures.
DNA Polymerase
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.
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
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NanoBees
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."
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Reflections on the Current H1N1 Flu
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.”
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Bacteriorhodopsin
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>.
"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>.
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Elongation Factor EF-TU
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.
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Exploring the Mitochondria
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.
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Histone Modification Animation
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.
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