Tag: Biochemistry

Prions, rogue proteins that cause incurable brain infections such as Mad Cow disease and its human equivalent, variant Creutzfeldt-Jakob Disease, are difficult to inactivate, resisting extreme heat, chemical disinfectants, and irradiation. Until now, scientists did not know whether prions entering sewers and septic tanks from slaughterhouses, meatpacking facilities, or private game dressing, could survive and pass through conventional sewage treatment plants. Joel Pedersen and colleagues used laboratory experiments with simulated wastewater treatment to show that prions can be recovered from wastewater sludge after 20 days, remaining in the “biosolids,” a byproduct of sewage treatment sometimes used to fertilize farm fields. Although emphasizing that prions have never been reported in wastewater treatment plant water or biosolids, the researchers note that existing tests are not sufficiently sensitive to detect the extremely low levels of prions possible in those materials. Their report appears in a paper scheduled for the July 1 issue of the American Chemical Society’s Environmental Science & Technology.

Science Daily
July 8, 2008

Original web page at Science Daily

Viruses are true experts at importing genetic material into the cells of an infected organism. This trait is now being exploited for gene therapy, in which genes are brought into the cells of a patient to treat genetic diseases or genetic defects. Korean researchers have now made an artificial virus. As described in the journal Angewandte Chemie, they have been able to use it to transport both genes and drugs into the interior of cancer cells. Natural viruses are extremely effective at transporting genes into cells for gene therapy; their disadvantage is that they can initiate an immune response or cause cancer. Artificial viruses do not have these side effects, but are not especially effective because their size and shape are very difficult to control—but crucial to their effectiveness. A research team headed by Myongsoo Lee has now developed a new strategy that allows the artificial viruses to maintain a defined form and size.

The researchers started with a ribbonlike protein structure (β-sheet) as their template. The protein ribbons organized themselves into a defined threadlike double layer that sets the shape and size. Coupled to the outside are “protein arms” that bind short RNA helices and embed them. If this RNA is made complementary to a specific gene sequence, it can very specifically block the reading of this gene. Known as small interfering RNAs (siRNA), these sequences represent a promising approach to gene therapy. Glucose building blocks on the surfaces of the artificial viruses should improve binding of the artificial virus to the glucose transporters on the surfaces of the target cells. These transporters are present in nearly all mammalian cells. Tumor cells have an especially large number of these transporters. Trials with a line of human cancer cells demonstrated that the artificial viruses very effectively transport an siRNA and block the target gene.

Science Daily
June 10, 2008

Original web page at Science Daily

Using a common virus as a tool for investigating abnormal cell proliferation, a team led by scientists at Cold Spring Harbor Laboratory (CSHL) has succeeded in clarifying an intricate series of biochemical steps that shed light on a way that cancer can begin. The team’s findings are the latest in a long and distinguished line of research at CSHL involving adenovirus, a type of virus that causes the common cold in people, but whose genome contains known oncogenes — genes whose expression can promote cancer under certain conditions. “Adenovirus carries a number of cooperating genes that modulate cell growth in ways we’re interested in,” said William Tansey, Ph.D., who, along with CSHL professors Scott Lowe, Ph.D., and Gregory Hannon, Ph.D., is one of the team’s co-leaders and corresponding author of a paper in Proceedings of the National Academy of Sciences. Other team members include molecular biologists from Stony Brook University in New York.

The team focused on an adenoviral oncogene called E1A, and a protein that it codes for with the same name. “Both have received a great deal of attention over the years,” said Dr. Tansey, “and to understand why, it helps to understand why viruses — in this case, adenovirus, a DNA tumor virus — is useful to us. We use them as you would use a flashlight, to illuminate important processes inside the cell that help us understand what goes awry in oncogenesis.” Viruses can’t reproduce on their own. A DNA virus like adenovirus is little more than a tiny, double-stranded segment of DNA enclosed within a protein shell. It must find a way to enter the nucleus of a living cell and hijack the cell’s reproductive machinery in order to reproduce itself. “It’s not adenovirus itself, but the things it does when it enters a cell, that really interest us,” Dr. Tansey explained. “By looking, in particular, at the activity of the proteins adenovirus codes for — proteins like E1A — we are tapping into a kind of natural growth-control mechanism.”

“The utility of DNA tumor viruses for cancer research is based on the premise that they’ve evolved to target the minimum number of cellular pathways necessary for virus propagation,” said Dr. Lowe. When things go awry, understanding how a tumor virus like adenovirus promotes cancer can reveal, in turn, “the most vulnerable pathways and nodes that are linked to tumorigenesis,” Dr. Hannon added. Because a tumor virus needs to commandeer the reproductive machinery of a living cell to survive, it must force the host cell to enter the reproductive, or S-phase, of its cycle. Past research has demonstrated that a protein called E2F is central in the process by which S-phase is activated. When the cell is not reproducing, E2F is known to be inhibited by its binding to another protein, called Rb, or retinoblastoma protein. “It’s this regulated association of E2F and Rb that is one of the primary mechanisms through which cells normally progress into S-phase,” Dr. Tansey said. The E1A protein, after binding Rb, is capable of physically pulling it off the E2F molecule. This unleashes the cell to replicate its DNA. And this, in turn, can promote transformations associated with cancer.

Recently, it’s been shown that E1A’s cancer-promoting activity is more extensive, also involving a gene-regulating protein called p400. Until the CSHL/Stony Brook team published its current paper, no one knew how E1A’s binding with p400 affected the process. The team knew from prior studies that when the E1A and p400 proteins were bound to one another, cellular growth control was disrupted. The question was why this potentially oncogenic effect occurred. What mechanisms were set in motion by the binding of these two proteins? They hypothesized that the answer could be found in the activity of yet another protein, called Myc, which Dr. Tansey has spent much of his career studying. Myc is an oncoprotein: one that is important in a great many regulatory processes in the cell, and which, when overexpressed, can cause dysregulation that leads to cancer. Prior work had shown that when E1A was present in a cell, the potentially dangerous Myc protein was stable — it did not degrade naturally. In new experiments, Tansey and colleagues found that E1A’s stabilization of the Myc protein was accomplished not, as was suspected by some, by directly inhibiting its degradation in a cellular component called the proteosome, which destroys proteins. Rather, E1A stabilized Myc by promoting its binding with p400.

To recap the complex sequence of events: E1A, when present in a cell, binds to p400. That protein, in turn, forms a complex with Myc which accounts for Myc’s stability in cells in which E1A is present. Close study showed that “the piece of the E1A protein that was important for stabilizing Myc was the same piece that bound to p400,” Dr. Tansey said. And just as E1A can pull the Rb protein away from E2F, initiating a cascade of pathologies potentially leading to oncogenesis, so does the ability of E1A to bind p400 — and via that connection to engage Myc — stabilize that oncoprotein and open the door to tumorigenesis. “We know now that the interaction of E1A and p400 is very important in terms of regulating cell growth in normal and cancer cells,” Dr. Tansey said. “So we’re taking a cue from the history of work on adenoviruses and we’re leaving E1A behind to concentrate on Myc and p400. For us, now, the next step is to learn more about the p400-Myc connection.”

Science Daily
May 13, 2008

Original web page at Science Daily

Researchers from the University of Pittsburgh and the University of Chicago were able to control heart muscle function in a new way after discovering the previously unknown role of two enzymes in heart muscle contraction, as detailed in the April 11 cover story of the Journal of Biological Chemistry. Although in the early stages, the research provides fresh knowledge of how heart muscle functions and also holds early potential as a treatment for various heart diseases—including congestive heart failure—that is possibly less taxing on the heart than current regimens. Experiments on slivers of heart muscle revealed that heart muscle contractions can be regulated by the enzymes histone acetyltransferases (HATs) and histone deacetylases (HDACs), explained Pitt professor Sanjeev Shroff, the Gerald McGinnis Chair of Bioengineering in the Swanson School of Engineering. Shroff and Pitt research associate Stephen Smith collaborated with Mahesh Gupta, an associate professor of surgery at the University of Chicago, and his research associate Sadhana Samant. The team found that HATs and HDACs influence acetylation of certain heart muscle proteins, a process wherein a radical cluster of atoms called an acetyl group attach to a protein and change its function. HATs facilitate acetylation, and HDACs remove the acetyl group. The team discovered that acetylation renders the muscle fiber more sensitive to calcium, which causes the muscle to contract.

“This is a completely new process in the area of heart muscle contraction,” Shroff said. “Acetylation is widely known to regulate such events inside the cell nucleus as gene regulation, but it’s never before been associated with heart muscle contraction.” Furthermore, Shroff and his colleagues could intervene in this microscopic process to control heart muscle contraction. By inhibiting HDACs, they increased the calcium sensitivity of the muscle fibers and strengthened contraction. As a possible treatment for such conditions as congestive heart failure, this technique could present an alternative to current therapies that counteract heart muscle weakness by boosting cellular calcium content, Shroff said. The heightened calcium improves muscle contraction but also results in more energy consumption in hearts that often are energy-starved to begin with. In contrast, inhibiting HDAC alters a natural process to make heart muscle more sensitive to the prevailing level of calcium, he said.

“We did not create this process—we are just manipulating what is already there,” Shroff explained. “The physiology to block HDAC is already there, and we just took advantage of that. This perturbation does not require greater mobilization of calcium, so we won’t end up with increased cardiac energy consumption. That’s been the Achilles heel of treatment so far.” The team’s next step involves examining HAT- and HDAC-driven regulation of cardiac contraction in the whole animal rather than just muscle samples. Then it can better determine the overall significance of the newly discovered process to the intact heart function and its therapeutic potential. “We want to see how much protein acetylation matters when operating alongside all the other processes in the heart and the body,” Shroff said. “If this process is shown to be significant under these conditions, it will be an exciting finding.”

Science Daily
April 29, 2008

Original web page at Science Daily

Degterev, Yuan and colleagues have discovered that RIP1 kinase is a specific cellular target of necrostatins, unique small molecules previously shown to reduce damage from necrosis. Research published in Nature Chemical Biology reveals that three specific inhibitors of a cell death pathway, termed necroptosis, all target and inhibit RIP1 kinase, a protein that can direct cells into necrosis. Induced by trauma such as a heart attack or stroke, this form of necrotic death can result in tissue damage contributing to death or long-term disability. The findings present a novel avenue for drug development. “Our research found that RIP1 kinase can be inhibited by three small molecules: necrostatin-1, -3 and -5,” reports first author Alexei Degterev, PhD, assistant professor at Tufts University School of Medicine and member of the biochemistry program faculty of the Sackler School of Graduate Biomedical Sciences. “Overall, these data establish RIP1 kinase as a new target for therapeutic drug development for human diseases involving necrotic tissue injury, and they establish necrostatins as first-in-class potent and selective inhibitors of RIP1 kinase,” the authors write, including last author, Junying Yuan, PhD, professor at Harvard Medical School.

Necrosis is relevant to many diseases, particularly those that involve an acute event, such as heart attack or stroke. “Cells are programmed to die when they reach the end of their lifecycle and that regulated process is called apoptosis. Cells can also be killed through pathways not controlled by apoptosis. Until recently, this type of cell death, termed necrosis, was believed to be unregulated, a type of accidental cell death caused when cells are placed under extreme stress such as during a heart attack, stroke or organ failure,” says Degterev. Previous research, as well as research by others, determined that necroptosis, one type of necrosis, is regulated (like apoptosis) and has a specific biochemical pathway. “Through our previous work, we have developed potent and specific small molecules capable of preventing necroptosis in isolated cells,” explains Degterev. This work, done in Yuan’s lab at Harvard, was subsequently awarded patents. “The next step, then, was to look for the target of the inhibition by necrostatins to understand how they inhibit necroptosis. We were particularly interested in RIP1 kinase because it was previously reported by other groups to be important for necroptosis and necrostatin-1 looked similar to known kinase inhibitors.”

The researchers employed several molecular techniques to determine the role these necrostatins played in inhibiting necroptosis. To test whether necrostatin-1 is a RIP1 kinase inhibitor in vitro, researchers added necrostatin-1 in incremental doses to purified RIPI kinase, and observed a dose-dependent decrease in its activity (phosphorylation). To validate their discovery, the researchers made small and specific structural changes to necrostatin-1, to see if loss of the RIP1 kinase inhibition resulted in the inability of the necrostatin-1 analogs to prevent necrosis. Similar experiments were conducted for necrostatin-3 and necrostatin-5 with similar results, which was surprising as the structure of these two molecules are very different from necrostatin-1. Further, based on the results of their analysis, researchers put forward the model describing mechanisms of RIP1 inhibition by necrostatin-1. “Next, research needs to determine the cellular pathway initiated by RIP1 kinase activity, develop better tools to further investigate its role in human disease, and establish how necrostatins are able to prevent RIP1 kinase from signaling the cell to kill itself,” says Degterev. “This may one day result in effective therapies, currently not available, for many life-threatening diseases.”

“These findings on RIP1 kinase inhibitors suggest entirely new possibilities to investigating the role of necroptosis in disease and indicate that these inhibitors may provide ways to prevent extensive tissue damage,” says Naomi Rosenberg, PhD, dean at the Sackler School of Graduate Biomedical Sciences and vice dean for research at Tufts University School of Medicine. “Discoveries like this reveal how basic science research provides the foundation to our understanding of disease and can point toward possible novel therapeutic strategies to ease the burden of those diseases.”

Science Daily
April 29, 2008

Original web page at Science Daily

One day soon a biosensing nanodevice developed by Arizona State University researcher Wayne Frasch may eliminate long lines at airport security checkpoints and revolutionize health screenings for diseases like anthrax, cancer and antibiotic resistant Staphylococcus aureus (MRSA). Even more incredible than the device itself, is that it is based on the world’s tiniest rotary motor: a biological engine measured on the order of molecules. Frasch works with the enzyme F1-adenosine triphosphatase, better known as F1- ATPase. This enzyme, only 10 to 12 nanometers in diameter, has an axle that spins and produces torque. This tiny wonder is part of a complex of proteins key to creating energy in all living things, including photosynthesis in plants. F1-ATPase breaks down adenosine triphosphate (ATP) to adenosine diphospahte (ADP), releasing energy. Previous studies of its structure and characteristics have been the source of two Nobel Prizes awarded in 1979 and 1997.

It was through his own detailed study of the rotational mechanism of the F1-ATPase, which operates like a three-cylinder Mazda rotary motor, that Frasch conceived of a way to take this tiny biological powerhouse and couple it with science applications outside of the human body. An article authored by Frasch and his colleagues in the ASU School of Life Sciences details the technology that would allow this. Their publication “Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors” was recently published in the journal Lab on a Chip, and featured in the online journal Chemical Biology. What Frasch and his colleagues show is that the enzyme can be armed with an optical probe (gold nanorod) and manipulated to emit a signal when it detects a single molecule of target DNA. This is achieved by anchoring a quiescent F1-ATPase motor to a surface. A single strand of a reference biotinylated DNA molecule is then attached to its axle. The marker protein, biotin, on the DNA is known to bind specifically and tightly to the glycoprotein avidin, so an avidin-coated gold nanorod is then added. The avidin-nanorod attaches to the biotinylated DNA strand and forms a stable complex.

When a test solution containing a target piece of DNA is added, this DNA binds to the single complementary reference strand attached to the F1-ATPase. The DNA complex, suspended between the nanorod and the axle, forms a stiff bridge. Once ATP is added to the test solution, the F1-ATPase axle spins, and with it, the attached (now double-stranded) DNA and nanorod. The whirling nano-sized device emits a pulsing red signal that can then be detected with a microscope. According to Frasch, the rotation discriminates fully assembled nanodevices from nonspecifically bound nanorods, resulting in a sensitivity limit of one zeptomole (600 molecules). Simply put, if it’s not moving and flashing, it simply isn’t relevant. Moreover, Frasch says, “Studies with the F1-ATPase in my laboratory show that since it can detect single DNA molecules, it far exceeds the detection limits of conventional PCR [polymerase chain reaction] technology.” Such a detection instrument based on the F1-ATPase enzyme would also be “faster and more portable,” he adds. Frasch will transfer his work from the bench to biotech, through establishment of a local company that utilizes the nano-sized F1-ATPase to produce a DNA detection instrument.

A prototype of the DNA detector is already in development. It is roughly the size of a small tissue box. Sampling would be as simple as taking a swab from an infected wound or a piece of baggage, dissolving it in a solution and placing a drop on a slide bearing reference F1-ATPases and their nanorods. Once in the instrument, red blinking signals emitted by rotating nanorods would let a computer know there’s trouble, literally, in a flash. SFAz funding has also enabled Frasch to extend the method to do protein detection at the single molecule level. This is novel because, unlike DNA, proteins can not be amplified artificially to improve the chances of detection. “Rapid and sensitive biosensing of nucleic acids and proteins is vital for the identification of pathogenic agents of biomedical and bioterrorist importance,” notes Frasch, who is also with the Center for Bioenergy and Photosynthesis in the College of Liberal Arts and Sciences. “It also provides a new avenue through which to analyze genotypes and forensic evidence.”

Science Daily
April 15, 2008

Original web page at Science Daily

The thousands of proteins found in nature are simply strings of amino acids, assembled by genes, and scientists have long believed that they automatically fold themselves into uniquely fixed, 3-dimensional shapes to fire the engine of life. In the era of genetic research, identifying those shapes and their functions has become a worldwide focus of biomedical science. Now, researchers at the Medical College of Wisconsin in Milwaukee have found that a protein, lymphotactin, which plays a vital role in the body’s immune response, can rapidly shift its shape –up to ten times a second– between two totally unrelated structures, each with a unique role in defending the body. Their discovery, published in the Proceedings of the National Academy of Science, March 17, alters a fundamental concept of biochemistry established in the 1960s. It may also inspire the search for other proteins with the ability to change form, and help address diseases of misfolded proteins such as Alzheimer’s, Parkinson’s, ALS, mad cow disease and many cancers.

“While our discovery raises more questions on the protein folding enigma, we hope it generates intensified research to learn the complex processes of these devastating diseases,” says team leader Brian Volkman, Ph.D., associate professor of biochemistry. Dr. Volkman’s team is using highly sensitive nuclear magnetic resonance (NMR) spectroscopy to solve three-dimensional protein structures. NMR provides information on the number and type of chemical entities in a molecule, and can measure distances between pairs of atoms within the molecule to produce a computer-generated 3-D model of its structure. They discovered that human lymphotactin, a regulatory protein released by the immune system to attract and activate white blood cells, exists naturally in two distinct structures, and that the newly-identified form has no similarity to any other known protein. They also learned that each form has a unique role, one attaching to the interior wall of the blood vessel, and the other reaching out to grab white blood cells. This means that converting from one lymphotactin structure to the other is likely essential for its activation, according to Dr. Volkman.

Science Daily
April 1, 2008

Original web page at Science Daily

Researchers at the University of California, Santa Cruz, have determined the three-dimensional structure of an RNA enzyme, or “ribozyme,” that carries out a fundamental reaction required to make new RNA molecules. Their results provide insight into what may have been the first self-replicating molecule to arise billions of years ago on the evolutionary path toward the emergence of life. In all forms of life known today, the synthesis of DNA and RNA molecules is carried out by enzymes made of proteins. The instructions for making those proteins are contained in genes made of DNA or RNA (nucleic acids). The circularity of this process poses a challenge for theories about the origins of life. “Which came first, nucleic acids or proteins? This question once seemed an intractable paradox, but with the discovery of ribozymes, it is now possible to imagine a prebiotic ‘RNA World’ in which self-replicating ribozymes accomplished both tasks,” said William Scott, associate professor of chemistry and biochemistry at UC Santa Cruz. Scott and postdoctoral researcher Michael Robertson determined the structure of a ribozyme that joins two RNA subunits together in the same reaction that is carried out in biological systems by the protein known as RNA polymerase. Their findings are published in the March 16 issue of the journal Science.

“An RNA-dependent RNA polymerase ribozyme is the foundation of the entire RNA World hypothesis,” Robertson said. “With that, you would have an RNA capable of making copies of itself; mutations or errors in some copies would result in variations that would be acted on by Darwinian natural selection, and the molecules would evolve into bigger and better ribozymes. That’s what makes this structure so interesting.”

Robertson and Scott determined the structure of a ribozyme that is not an entirely self-replicating RNA molecule, but it does carry out the fundamental reaction required of such a molecule–a “ligase” reaction creating a bond between two RNA subunits. Robertson obtained the ligase ribozyme through a kind of test-tube evolution when he was a graduate student at the University of Texas, Austin, working in the lab of biochemist Andrew Ellington. Starting with a mixture of randomly synthesized RNA molecules and selecting for the desired properties, researchers are able to evolve RNA enzymes from scratch. In the Ellington lab, Robertson evolved the ligase ribozyme (called the L1 ligase) and determined which parts were critical for its function and which parts could be removed to create a “minimal construct.”

At UC Santa Cruz, he began trying to grow crystals of the ribozyme so that he could use x-ray crystallography to determine its structure. Crystallizing RNA molecules is extremely difficult, and Robertson tried dozens of different versions of the ribozyme under different conditions before he succeeded. Using x-ray crystallography–which involves shining a beam of x-rays through the crystals and analyzing the resulting diffraction patterns–Robertson and Scott were then able to determine the three-dimensional structure of the ribozyme. The ribozyme has three stems that radiate from a central hub. The active site where ligation occurs is located on one stem, and the structure shows that the molecule folds in such a way that parts of another stem are positioned over the ligation site, forming a pocket where the reaction takes place. A magnesium ion bound to one stem and positioned in the pocket plays an important role in the reaction, Robertson said. The structure indicates that this artificially selected ribozyme uses reaction mechanisms that are much like those used by naturally occuring enzymes, Robertson said. “The L1 ligase appears to use strategies of transition-state stabilization and acid-base catalysis similar to those that exist for natural ribozymes and protein enzymes,” he said.

Science Daily
April 3, 2007

Original web page at Science Daily

An artificial nose could be a real benefit at times: this kind of biosensor could sniff out poisons, explosives or drugs, for instance. Researchers at the Max Planck Institute for Polymer Research and the Max Planck Institute of Biochemistry recently revealed a technique for integrating membrane proteins into artificial structures. Membrane proteins have several important functions in the cell, one of which is to act as receptors, passing on signals from molecules in the air, for example, to the cell interior. They are thus ideal biosensors, but until now were difficult to access in the lab. However, Max Planck scientists have now managed to incorporate in-vitro synthesized membrane proteins directly into artificial lipid membranes (Angewandte Chemie, International Edition, January 15, 2007). The senses of living organisms function using various mechanisms, among other things utilizing membrane proteins as receptors. Researchers at the Max Planck Institute for Polymer Research and the Max Planck Institute of Biochemistry have now succeeded in creating biosensors by incorporating such proteins into artificial structures. The membrane proteins are synthesized in-vitro directly from the genetic information introduced to the cell extract.

Previous attempts to create biosensors from membrane proteins failed due to an idiosyncrasy of these proteins: they are not water soluble. In the past, researchers tried to remove the proteins from their biological membranes by solubilising the latter using detergents. However, this destroys the natural folding structure of the protein membranes, which is precisely what makes the proteins so special. “We quickly realized how difficult it is to isolate such membrane proteins. Neither we, nor other research groups, were able to work with them using conventional methods,” explains Dr. Eva-Kathrin Sinner of the Max Planck Institute for Polymer Research in Mainz. Yet the Max Planck researchers found a way around this: they succeeded in incorporating the proteins in an artificial matrix, just as they would be integrated in a natural cell membrane. They achieved this by introducing the developing membrane proteins to artificial lipid membrane systems that mimic natural cell membranes from a statu nascendi, and the membrane proteins actually did simply insert themselves into the artificial membranes. The odorant receptors selected by the researchers were a type of G-protein coupled receptor taken from brown rats.

The scientists were also able to prove that the odorant receptors maintained their biological functions by demonstrating the binding of odorants to the receptors. “We now have something akin to an instruction manual on how membrane proteins that were previously difficult to access can be produced and analyzed in their active structure,” says Sinner. The new procedure developed by Eva-Kathrin Sinner and her research group makes it possible, for the first time, to examine the natural functions of such membrane proteins in situ. This is of considerable importance to pharmaceuticals research, as it means that new active agent screenings can now be carried out using receptors that were inaccessible until now.

Science Daily
March 6, 2006

Original web page at Science Daily

A complex molecule and snake venom may provide researchers with a more reliable method of diagnosing human diseases and developing new drugs. Purdue University researchers bound a complex nanomolecule, called a dendrimer, with a glowing identification tag that was delivered to specific proteins in living venom cells from a rattlesnake. The scientists want to find a better way to ascertain the presence, concentration and function of proteins involved in disease processes. They also hope the new method will facilitate better, more efficient diagnosis in living cells and patients.

Most diagnostic methods must be done on minute dead blood or tissue cell samples in a laboratory dish, said Andy Tao, a Purdue biochemist and senior author of the study. Because molecular interactions and protein functions are disturbed when samples are collected, researchers can’t obtain an accurate picture of biochemical mechanisms related to illnesses such as cancer and heart disease. Tao and his research team used dendrimers because they can pass through cell walls efficiently with little disturbance to the cells and then label specific proteins with isotopic tags while cells are still alive. This allows the scientists to determine the activities of proteins that play roles in specific diseases. Proteins carry genetic messages throughout the cell causing biochemical changes that can determine whether a cell behaves normally or abnormally. Proteins also are important in directing immune responses.

The Purdue scientists report on their new strategy to discover proteins and protein levels, called soluble polymer-based isotopic labeling (SoPIL), in the current issue of the journal Chemical Communications. The study also is featured in the journal’s news publication Chemical Biology. “The problem with the current method of using proteomics – protein profiling – is that we use very small sample amounts so sensitive that we can’t effectively use existing technologies to study them,” Tao said. “In addition, to study a specific protein and its function, we want to preserve its natural environment and see where two molecules meet and what the interaction is when they bind. “Taking small samples of blood, cells or tissue to study extracted proteins in laboratory dishes damages the sample and the natural environment is destroyed.” The dendrimers would carry one of the stable isotopic or fluorescent labels to identify the presence or absence of a protein that can be further developed for use as a disease indicator, or biomarker. Snake venom cells were used because they have a very high concentration of proteins similar to some found in human blood, Tao said. The proteins apparently are part of the biochemical process that affects blood clotting or hemorrhage. Understanding how the proteins behave could help determine predisposition to heart disease and cancer and also be useful in diagnosis and drug development.

Science Daily
March 6, 2007

Original web page at Science Daily

Vitamin A is no ordinary dietary supplement. Without it, the body’s immune system deteriorates, fetuses develop birth defects, and adults go blind. Now, researchers have identified the molecular lock that enables vitamin A to enter cells. The findings solve a longstanding mystery about vitamin A metabolism and could help scientists develop new ways to fight vitamin-A deficiency in the developing world. Vitamin A, also known as retinol, is made in the liver. From there, it’s carried through the blood and delivered to tissues by a molecule called retinol-binding protein (RBP). Just how vitamin A gets into cells, however, has remained a mystery. Several teams of biochemists have tried, over 3 decades, to isolate a receptor–or lock–for RBP’s key. Because they always came up empty, some researchers argued that a receptor did not exist. Biochemist Hui Sun of the University of California, Los Angeles, was convinced it was out there.

Most molecular keys fit tightly into their locks; so biochemists can often use the key to fish the receptor’s lock out of a solution. But based on previous evidence, Sun suspected that RBP maintained only loose contact with its receptor. To improve the chances of pulling them out together, Sun’s team added a chemical called a crosslinker to 400 ground-up cow eyeballs. Crosslinkers help proteins stick together, and cow eyeballs are chock full of Vitamin A (and therefore, theoretically, the RBP receptor). The strategy worked. When the team pulled RBP from the mix, it was attached to another protein, which resembled a receptor. To make sure they had the right receptor, called STRA6, the researchers injected its gene into cultured cells. Injected cells took up 15 times as much Vitamin A as cells without the added receptor gene, the team reports online today in Science. What’s more, when the researchers used genetic tricks to reduce the amount of RBP receptor–or to make mutant receptors–the cells took up less vitamin A. And when they measured receptor levels throughout the body, the receptor was most concentrated in tissues such as the retina, brain, and spleen that gobble up vitamin A.

ScienceNow
February 6, 2007

Original web page at ScienceNow

Carnegie Mellon University scientists have married bright fluorescent dye molecules with DNA nanostructure templates to make nanosized fluorescent labels that hold considerable promise for studying fundamental chemical and biochemical reactions in single molecules or cells. The work, published online Jan. 26 in “The Journal of the American Chemical Society,” improves the sensitivity for fluorescence-based imaging and medical diagnostics. “Our DNA nanotags offer unprecedented densities of fluorescent dyes and, thus, the potential for extremely bright fluorescent labels,” said graduate student Andrea Benvin, who developed the nanotags in the laboratory of Bruce Armitage, associate professor of chemistry in the Mellon College of Science (MCS) at Carnegie Mellon. “We’ve put it all into a very small package, which will allow us to detect molecules with great sensitivity without interfering with the biological processes we are trying to understand.”

The high brightness of the nanotags should be of great help in detecting rare cancer cells within tissue biopsies, for example, which is important in determining whether treatments have been successful or if recurrence is likely, according to Armitage. In addition, DNA nanotags offer the opportunity to perform multicolor experiments. This feature is extremely useful for imaging applications, Armitage says, because the multiple colors can be seen simultaneously, requiring only one experiment using one laser and one fluorescence-imaging machine. “For example, two different populations of cells, one healthy and the other cancerous, could be distinguished based on labeling them with different color fluorescent nanotags,” Armitage said.

Benvin, Armitage and colleagues at Carnegie Mellon’s Molecular Biosensor and Imaging Center modeled their DNA nanotags on the structure of phycobiliproteins. Found in certain types of algae, such as the red and blue algae in fresh and marine waters, these proteins contain multiple, fluorescent pigments that work together to absorb light energy that is then transferred to chlorophyll, where it is used for photosynthesis. The Carnegie Mellon team has mimicked this efficient light-harvesting process in the design of their DNA nanotags to create incredibly bright, fluorescent labels. “The primary advantages of our system are the simplicity of its design combined with the ease with which the fluorescence brightness and color can be tuned,” Armitage said.

To achieve greater brightness, the Carnegie Mellon team assembled well-defined nanostructured DNA templates that bind multiple fluorescent dye molecules between base pairs in the DNA helix. This arrangement keeps dyes far enough away from each other to avoid canceling out each other’s fluorescence. The DNA templates can also be modified to bind to other molecules or to the surface of a cell of interest. The innovative design creates nanotags with large light-harvesting capabilities and very high light-emission (fluorescence) intensities. Because the DNA can accommodate one dye for every two base pairs, a DNA nanostructure with 30 base pairs can bind up to 15 fluorescent dye molecules. The resulting dye-DNA complexes are approximately 15 times brighter than an individual dye molecule. And they can be made even brighter by simply increasing the number of base pairs in the DNA nanostructure.

Multicolor experiments are possible because the DNA nanotags contain “light-harvesting” dyes within the DNA helix that are excited by one wavelength of light and then transfer that excitation energy to “light-emitting” dyes on the nanotag’s surface. The light-emitting dyes can fluoresce at a different color from the light-harvesting dye. For example, one type of DNA nanotag can act as an antenna that efficiently harvests blue light and transfers that light energy to another dye within the nanostructure. The second dye then emits orange, red or even infrared light. Changing the light-harvesting dyes allows even more variation in the fluorescence color, Armitage said. The nanotags are easily assembled by mixing commercially available DNA strands and fluorescent dyes. And while the work described by the Carnegie Mellon team relied on a relatively simple two-dimensional DNA nanostructure, Armitage notes that the rapidly growing field of DNA nanotechnology is generating increasingly intricate three-dimensional nanostructures that should lead to further improvements in brightness. “We really feel that this is the tip of the iceberg and that nanotags 100 times brighter than existing labels can be developed in any color,” he said.

Science Daily
February 6, 2007

Original web page at Science Daily

By joining a sugar to a short-chain fatty acid compound, Johns Hopkins researchers have developed a two-pronged molecular weapon that kills cancer cells in lab tests. The researchers cautioned that their double-punch molecule, described in the December issue of the journal Chemistry & Biology, has not yet been tested on animals or humans. Nevertheless, they believe it represents a promising new strategy for fighting the deadly disease. “For a long time, cancer researchers did not pay much attention to the use of sugars in fighting cancer,” said Gopalan Sampathkumar, a postdoctoral fellow in the university’s Department of Biomedical Engineering and lead author of the journal article. “But we found that when the right sugar is matched with the right chemical partner, it can deliver a powerful double-whammy against cancer cells.”

Sampathkumar and his colleagues built upon 20-year-old findings that a short-chain fatty acid called butyrate can slow the spread of cancer cells. In the 1980s, researchers discovered that butyrate, which is formed naturally at high levels in the digestive system by symbiotic bacteria that feed on fiber, can restore healthy cell functioning. Efforts to use butyrate as a general drug for tumors elsewhere in the body, however, have been hindered by the high doses of the compound needed to effectively eradicate cancer. To get around this problem, scientists have tried to make butyrate more potent by modifying it or joining it to other compounds. Usually, the results have been disappointing because the molecular partner added to butyrate to improve delivery to the cancer cells often produced unsafe side effects.

In some of the less successful experiments, designed to avoid toxic side effects, researchers used innocuous sugar molecules such as glucose to carry butyrate into the cells. The Johns Hopkins team tried a different tack. “We didn’t think they chose the right partner molecule,” said Kevin J. Yarema, an assistant professor of biomedical engineering who supervised the project. “Our insight was to select the sugar partner to serve not just as a passive carrier but as additional ammunition in the fight against cancer.”

The researchers focused on a sugar called N-acetyl-D-mannosamine, or ManNAc, for short. The team created a hybrid molecule by linking ManNAc with butyrate. The hybrid easily penetrates a cell’s surface, then is split apart by enzymes inside the cell. Once inside the cell, ManNAc is processed into another sugar known as sialic acid that plays key roles in cancer biology, while butyrate orchestrates the expression of genes responsible for halting the uncontrolled growth of cancer cells. Although the study of the exact molecular mechanism is in its early stages, the researchers believe the separate chemical components work together to bolster the cancer-fighting power of butyrate. The double attack triggers cellular suicide, also called apoptosis, in the cancer cells.

To find out whether this butyrate-ManNAc hybrid alone would produce the positive results, the researchers tested three other sugar-butyrate combinations and a butyrate salt compound with no sugar attached. The four other formulas and the butyrate-ManNAc hybrid were each added to lab dishes containing cancer cells. After three to five days, cancer growth had slowed in all of the dishes. After 15 days, however, cancer growth had resumed in dishes treated with four of the compounds. But in samples treated with the butyrate-ManNAc hybrid, all of the cancer cells had died. The researchers also wanted to find out whether administering the two parts of the hybrid independently would achieve the same result. But in these experiments, the cancer cells did not self-destruct. The researchers suspect this is because the hybrid molecules more easily penetrate the surface of the cell than the individual chemicals. Once the components are inside, the researchers believe the partners help enzymes to resume the normal assembly of sugar molecules and correct aberrant gene expression patterns, two processes that go awry when cancer occurs.

Now that they’ve identified the butyrate-ManNAc molecule as a potential cancer fighter, the Johns Hopkins team members are expanding their research, looking for new drug-delivery methods and preparing for animal testing. The researchers believe the hybrid molecule will have minimal effect on healthy cells. Through the Johns Hopkins Technology Transfer Office, they have filed an application for a U.S. patent covering this class of compounds.

Science Daily
January 23, 2007

Original web page at Science Daily

Attempts to identify potential drugs that interfere with the action of one particular enzyme linked to heart disease and similar health problems led scientists at Johns Hopkins to create a new tool and new experimental approach that allow them to see multiple, real-time chemical reactions in living cells. Their report on the work is published July 21 in the journal ACS Chemical Biology. The team built a protein biosensor that indicates if an enzyme located nearby is turned on or off. The sensor is made from a protein that glows, originally isolated from jellyfish. When PKA is turned off, the biosensor glows blue. When PKA is turned on and is physically close to a biosensor, PKA itself changes the shape of the biosensor, causing it to glow green instead.

Most current drug development operations test chemicals on enzymes isolated from their normal environs and then take further steps to see if the chemical can get into the cell to do its work, and figure out how poisonous the chemical is to a cell. “Living cells are critical to our work because they show us how and what is actually happening in a normal context and time span when a chemical is added,” says Jin Zhang, Ph.D., an assistant professor of pharmacology and molecular sciences in Hopkins’ Institute for Basic Biomedical Sciences. Testing chemicals on enzymes in living cells provides the opportunity to find potential drugs that work in new ways. For example, using living cells allows researchers to “see” where in the cell chemicals do their work. Scientists could then design new drugs to go to specific places within cells to work more efficiently. Also, streamlining the one-at-a-time approach offers the chance to study – and rule out or in – many potentially useful chemicals at once.

What Zhang’s team developed is a biosensor and simple testing procedure that tells if a particular enzyme – called PKA – that acts like a “switch” is “on” or “off” in a living cell. The group has been focused on trying to understand and interfere with this enzyme switch, because if the enzyme is turned on at the wrong time or at the wrong place within cells, it can lead to cells misbehaving, which ultimately can lead to heart disease. In the course of their work, the team built a protein biosensor that indicates if an enzyme located nearby is turned on or off. The sensor is made from a protein that glows, originally isolated from jellyfish. When PKA is turned off, the biosensor glows blue. When PKA is turned on and is physically close to a biosensor, PKA itself changes the shape of the biosensor, causing it to glow green instead.

Manipulating the sensor allows the researchers to direct it to specific locales within cells. That allows the researchers to see where in the cell the active enzyme is located. So this PKA sensor not only indicates whether the enzyme is on or off, but also locates where PKA is being turned on or off within the cell. “Proteins aren’t spread out evenly in cells,” says Zhang, “but tend to cluster together in order to do specific jobs, and we now can see how different clusters are regulated differently.” When the researchers put their new sensor into living mammalian cells growing in the lab, they were able to test the effects of 160 different chemicals at once and see if any of these chemicals could turn on or off the PKA enzyme by looking for green or blue glowing cells. Of the 160 chemicals tested, three caused cells to turn on the switch and two others caused cells to turn off the switch.

The 160 chemicals tested are from the Johns Hopkins Clinical Compound Library, a collection of about 3,300 chemicals. Most of them are drugs already approved by the U.S. Food and Drug Administration, while others are drugs approved by regulatory agencies in other countries or are other clinically relevant chemicals. “If we can find a new activity for a known drug, this may lead to a new use or a new way of thinking about that drug,” says Zhang, who hopes to test the rest of the chemicals in the collection soon for their ability to interfere with the enzyme tested in this study. Finding a drug that can tame this enzyme could lead to new treatments for heart disease, diabetes, memory disorders and certain cancers, for example. Zhang says the “high throughput” potential of the sensor may have wide-reaching applications that could be adapted to testing various chemicals to test chemicals for their ability to interfere with other enzymes related to PKA – which as a family are known as kinases – that are widely implicated in diseases and an emerging class of drug targets.

Science Daily
August 1, 2006

Original web page at Science Daily

UCLA physicists report a significant step toward a new approach to protein engineering in the June 8 online edition, and in the July print issue, of the Journal of the American Chemical Society. “We are learning to control proteins in a new way,” said Giovanni Zocchi, UCLA associate professor of physics and co-author of the study. Zocchi said the new approach could lead ultimately to “smart medicines that can be controlled” and could have reduced side effects. Mimicking one essential cellular control mechanism, Zocchi’s laboratory has completed an important preliminary step.

Zocchi and UCLA physics graduate student Brian Choi report one representative example where the chemical mechanism by which the cell controls the function of its proteins can be effectively replaced, in vitro, by mechanical control. Specifically, they show how an enzyme complex called Protein Kinase A (PKA) — which plays a fundamental role in the cell’s signaling and metabolic pathways, and is controlled in the cell by a ubiquitous messenger molecule called cyclic AMP — can instead be controlled mechanically by a nanodevice that the researchers attached to the enzyme complex. The nanodevice is essentially a molecular spring made of DNA. “Molecular biologists have been trained for 50 years to think that because the sequence of amino acids determines a protein’s structure and the structure determines its function, if you want to change the structure, the way to do so is to change the sequence of amino acids. While that approach is correct, it is not the only way. We are introducing the notion that you can keep the sequence but change the structure with mechanical forces.

“This research has many ramifications, and may lead to a better fundamental understanding, as well as new directions for biotechnology and perhaps new approaches to medical treatments.” PKA, a complex of four protein molecules, contains two regulatory subunits and two catalytic subunits. Zocchi and Choi mechanically activated PKA by placing a controlled mechanical stress on two specific points in the regulatory subunit, which causes that subunit to fall off from the catalytic subunit, activating the enzyme. In order to obtain the desired effect, the mechanical tension is applied at specific locations in the regulatory subunit, Choi said. Knowing those locations requires a detailed understanding of the structure of the enzyme.

Proteins, the molecular machines that perform all tasks in the living cell, are switched on and off in living cells by a mechanism called allosteric control; proteins are regulated by other molecules that bind to their surface, inducing a change of conformation, or distortion in the shape, of the protein, making the protein either active or inactive, Zocchi explained. Cyclic AMP (cAMP) binds to PKA’s regulatory subunit and induces a change of conformation that leads to the catalytic subunit’s detaching from the regulatory subunit; this separation of the two subunits is how the enzyme complex is turned on in the cell, Zocchi said.

“We can activate the enzyme mechanically, while leaving intact the natural activation mechanism by cAMP,” said Zocchi, a member of the California NanoSystems Institute. “We believe this approach to protein control can be applied to virtually any protein or protein complex.” Zocchi’s group first demonstrated mechanical control of protein conformation last year, when the physicists attached a controllable molecular spring, made of a short piece of DNA, to a protein and used it to inhibit its function. In the new research, the group succeeded in activating the enzyme PKA through the same principle, by using the molecular spring to induce the change in conformation that, in the cell, is induced by the natural activator of PKA (the signaling molecule cAMP).

Zocchi’s group can mimic with mechanical tension the natural allosteric mechanism by which PKA is regulated by cAMP. PKA is significantly more complex than the protein that Zocchi’s group used last year. “I want to see whether we can make molecules which kill a cell based on the genetic signature of the cell,” Zocchi said. “Cancer cells would be an obvious application. This will however require many further steps. So far, we have only worked in vitro. The exciting part is, from the outside, cancer cells can look like normal cells, but inside they carry a genetic mark. “In the future, perhaps we can control more complicated molecular machines such as ribosomes. Many antibiotics work by blocking the ribosome of bacteria.”

Science Daily
July 3, 2006

Original web page at Science Daily