Tag: Biochemistry

Tiny chemical cages created by researchers at Rutgers, The State University of New Jersey, show potential for delivering drugs to organs or tissues where they’re needed without causing harm elsewhere. These cage-like molecules, called nanocontainers or nanoscale capsules because they measure a mere 3.2 nanometers (billionths of a meter) wide, also could make pesticides less hazardous to handle, filter toxic substances out of wastewater and regulate the pace of reactions in chemical production. “While the concept of chemical cages is not new, we’ve created new components and advanced the assembly process to increase the chance that they’ll become practical,” said Ralf Warmuth, associate professor of chemistry and chemical biology at Rutgers and lead researcher. “We’ve shown a way to securely link molecules together in a cage using an efficient, one-step process.”

In an article slated for an upcoming issue of the chemistry journal Angewandte Chemie International Edition, Warmuth and colleagues describe how they’ve used common organic chemicals and straightforward techniques to create nanocontainers. These octahedral (eight-sided) capsules, with their cavity volume of almost two cubic nanometers, could enclose one or more molecules of a medicine, pesticide or intermediate in a chemical manufacturing process that, if left uncaged, might prematurely decay or interact with other substances in passing.

Previous techniques for assembling molecular cages involved tradeoffs. With one approach, the synthesis technique was straightforward, but the pieces of the molecular cages were not bound as tightly to each other. Another approach resulted in tighter bonds, but the process required several carefully orchestrated steps. The Rutgers advance is a one-step process that creates tight chemical bonds, surpassing earlier approaches in simplicity and efficiency.

The Rutgers process involves combining six larger bowl-shaped molecules with 12 smaller linear molecules, or bridges, that link the bowls together, insides facing each other. Atoms at four sites along each bowl’s rim bond to atoms on the ends of the bridges. The atomic structure and properties of these molecules ensure that they naturally assemble themselves into capsules and do so with high yield when combined in proper proportions. Early research suggests that the connections between the bridges and bowls can be broken and reattached under controlled conditions to introduce “payload” molecules – such as a drug or pesticide – into the cage and extract them when needed. The Rutgers team consisted of Warmuth, chemistry graduate students Xuejun Liu and Yong Liu, and undergraduate student Gina Li.

Science Daily
February 14, 2006

Original web page at Science Daily

University of Pittsburgh researcher Alexander Star and colleagues at California-based company Nanomix, Inc., have developed devices made of carbon nanotubes that can find mutations in genes causing hereditary diseases, they reported in the Jan. 16 issue of the journal Proceedings of the National Academy of Science. This method is less expensive and takes less time than conventional techniques. Carbon nanotubes are rolled-up sheets of graphite only a few nanometers wide-about the width of a molecule of DNA. The researchers used these nanotubes’ electrical properties to find a particular mutation in the gene that causes hereditary hemochromatosis, a disease in which too much iron accumulates in body tissues.

“The size compatibility between the detector and the detected species-DNA molecules in this case makes this approach very attractive for further development of label-free electronic methods,” said Star, who is an assistant professor of chemistry at Pitt. Star and his colleagues at Nanomix also tested fluorescently labeled DNA molecules in order to confirm that DNA had attached to the nanotube surfaces and was subsequently hybridized, or matched to its complementary DNA. “We have found that electrical measurement of carbon nanotube devices produce sensor results that are comparable to state-of-the-art optical techniques,” Star said.

He added, “The applications of our method for detection of other, more serious genetic diseases can be seen.” Label-free electronic detection of DNA has several advantages over state-of-the-art optical techniques, including cost, time, and simplicity. “Our technology can bring to market hand-held, field-ready devices for genetic screening, as opposed to laboratory methods using labor-intense labeling and sophisticated optical equipment,” Star said.

Science Daily
February 14, 2006

Original web page at Science Daily

Scientists from Imperial College London and the University of Surrey have developed a new technique for crystallising proteins, a discovery which could help speed up the development of new medicines and treatments. Crystallisation is the process which converts materials, such as proteins, into three dimensional crystals, thus enabling their atomic structure to be studied. The three dimensional structure of the crystals indicates the proteins function, and from this, researchers hope to be able to develop more effective treatments. However, production of high quality crystals has long posed a major bottleneck for X-ray crystallography. This problem has become increasingly acute with the advent of structural genomics and proteomics which aim to determine the structures of thousands of proteins. Protein crystallography plays a major role in this understanding because proteins, being the major machinery of living things, are often targets for drugs.

To direct the proteins to become crystals, researchers use a substance called a nucleant, which does this by encouraging protein molecules to form a crystal lattice. The research published online in Proceedings of the National Academy of Sciences, shows how the team, consisting of bio-medical scientists, material scientists and physicists, collaborated to develop a theory concerning the design of porous materials for protein crystallisation and put it into practice. The theory is based on the rational that the porous structure of a material, traps the protein molecules, and encourages them to crystallise. They tested the theory using BioGlass, a substance developed by Imperial’s material scientists, as a scaffold to trap and encourage the growth of protein crystals. BioGlass is a porous material, with a variety of different size pores able to trap different size proteins. They found BioGlass induced the crystallisation of the largest number of proteins ever crystallised using a single nucleant.

Professor Naomi Chayen, from Imperial College London, who led the research, said: “The first step in obtaining a good crystal is to get it to nucleate in an ordered way. The ‘holy grail’ is to find a ‘universal nucleant’ which would induce crystallisation of any protein. Although there has been considerable research in search of a universal nucleant, this is the first time we have designed one which works on a large number of materials.” The researchers plan to commercialise this discovery using Imperial Innovations, the College’s technology transfer company.

Science Daily
January 31, 2006

Original web page at Science Daily

Duke University biochemists aided by Duke computer scientists and computational chemists have identified the likely way two key enzymes dock in an intricate three-dimensional puzzle-fit to regulate cell division. Solving the docking puzzle could lead to anticancer drugs to block the runaway cell division behind some cancers, said the researchers. Significantly, their insights arose not just from meticulous biochemical studies, but also from using sophisticated simulation techniques to perform “chemistry in the computer.” In a paper published Nov. 24, 2005 online in the journal Biochemistry, members of the interdisciplinary collaboration described how they discovered the probable orientation required for a Cdc25B phosphatase enzyme to “dock” with and activate a cyclin-dependent kinase protein complex that also functions as an enzyme, known as Cdk2-pTpY–CycA. The work was funded by the National Institutes of Health.

Detailed study of such docking is important because uncontrolled overreaction of the Cdc25 family of enzymes has been associated with the development of various cancers. Anti-cancer drugs that jam the enzyme, preventing its docking with the kinase, could halt cell over proliferation to treat such cancers. However, developing such drugs has been hampered by lack of detailed understanding of how the Cdc25s fit with their associated kinases. “To me this is the culmination of my six years here at Duke,” said Johannes Rudolph, the Duke assistant professor of chemistry and biochemistry who led the research. “It’s very exciting. I think it’s a really hard problem.”

A successful docking between the two enzymes not only requires the “active sites” — where chemical reactions occur –on the phosphatase and the kinase to link precisely, Rudolph said. The two molecules’ component parts, or “residues,” must also orient in a tongue-and-groove fit at a few other special places, which the researchers dubbed ‘hot spots,” on the irregular molecular surfaces. Only when active sites and hot spots fit correctly can this brief docking accomplish its role in the cell division cycle, said Rudolph. That biochemical role is for the enzyme to remove the phosphates from two phosphate-bearing amino acids on the protein. Those removals alter electrical charges in a way that allows the protein to pick up other phosphate-containing chemical groups to pass along as part of a molecular bucket brigade.

Rudolph initially knew the kinase’s and phosphatase’s general topographies as well as the locations of their active sites. “But it was literally a guessing game trying to find which residues might be important in this interaction,” he said. “Somehow these two large complicated molecules had to also interact specifically somewhere other than the site where the chemistry occurs.” Biochemists traditionally answer such questions by laboriously making “mutant” versions of a protein in which a single residue is altered and lab-testing whether the resulting subtle change in the protein’s shape or chemistry changes the way the molecules interact with each other, he said. If there is no change, they then move on to the next residue. “So my students started to make these mutants randomly and test their activities, one at a time,” Rudolph said. “Each of these experiments is pretty hard, and pretty tedious.”

After this trial-and-error search remained fruitless, Rudolph, his graduate students Jungsan Sohn, Kolbrun Kristjansdottir and Alexias Safi and his post-doctoral investigator Gregory Burhman began collaborating with a team led by computer science and mathematics professor Herbert Edelsbrunner. Edelsbrunner, who has developed techniques and computational programs for modeling and analyzing complex molecular shapes, used a large cluster of computers and custom software to analyze about one thousand trillion different conceivable shape match-ups between the molecules. That initial mega-analysis reduced the potential molecular combinations to about 1,000 possibilities, which Rudolph called both “encouraging” and “discouraging.”

Edelsbrunner’s group, which included programmer Paul Brown, then began narrowing that search further. They did so by using a different software program that could identify the highest and lowest places on the molecules’ surfaces, and where “highest” on one might fit into the “deepest” on the other. “That’s not easy, because there is no point of reference on those complicated shapes,” Rudolph said. The researchers finally winnowed the possibilities to what Rudolph called “one reasonable guess” by enlisting another Duke group led by chemistry professor Waitao Yang.

Wang’s team, including his graduate student Jerry Parks, uses another bank of computers to calculate how components of molecules behave in small spaces — in this case “how they wiggle,” Rudolph said. By allowing both molecules to move — as they would in the real world — the researchers could evaluate whether match-ups that looked right when motionless were actually off the mark. “Tiny little shifts can change these things,” Rudolph said.

The interdisciplinary group’s Biochemistry paper, whose first author was Rudolph’s graduate student Sohn, confirmed the calculations with extensive biochemical evaluations of the two hot spot residues the study identified, one residue on the phosphatase and the other on the kinase. Both hot spots are located some distance from the molecules’ active sites, Rudolph noted. Overexpression of the Cdc25 group of enzymes has been associated with the development of numerous cancers. But “drug discovery targeting these phosphatases has been hampered by lack of structural information about how Cdc25s interact with their native protein substrates,” the authors wrote in their Biochemistry paper.

With the study’s results in hand, scientists can now search for potential inhibiting drug molecules shaped so they can overlap — and thus interfere — with the active sites as well as outlying hot spots the research identified, Rudolph said. He credited the study’s success to the power of interdisciplinary scientific collaborations, noting that he and Edelsbrunner initially met “by coincidence” in Duke’s Levine Science Research Center building, where they both have separate labs in separate wings.

Science Daily
January 3, 2006

Original web page at Science Daily

Ohio State University researchers have invented a process for uncoiling long strands of DNA and forming them into precise patterns. DNA strands fluoresce in these microscope images from Ohio State University. Researchers here have invented a process for uncoiling DNA strands and forming them into precise patterns — a prelude to biologically based electronics and medical devices. The squares in the lower right image measure approximately 10 micrometers (millionths of a meter) across. Ultimately, these DNA strands could act as wires in biologically based electronics and medical devices, said L. James Lee, professor of chemical and biomolecular engineering at Ohio State University.

In the early online edition of the Proceedings of the National Academy of Sciences, Lee and postdoctoral researcher Jingjiao Guan describe how they used a tiny rubber comb to pull DNA strands from drops of water and stamp them onto glass chips. Other labs have formed very simple structures with DNA, and those are now used in devices for gene testing and medical diagnostics. But Lee and Guan are the first to coax strands of DNA into structures that are at once so orderly and so complex that they resemble stitches on a quilt. “These are very narrow, very long wires that can be designed into patterns for molecular electronics or biosensors,” Lee said. “And in our case, we want to try to build tools for gene delivery, DNA recombination, and maybe even gene repair, down the road.”

The longest strands are one millimeter (thousandths of a meter) long, and only one nanometer (billionths of a meter) thick. On a larger scale, positioning such a long, skinny tendril of DNA is like wielding a human hair that is ten meters (30 feet) long. Yet Lee and Guan are able to arrange their DNA strands with nanometer precision, using relatively simple equipment. In this patent-pending technology, the researchers press the comb into a drop of water containing coils of DNA molecules. Some of the DNA strands fall between the comb’s teeth, so that the strands uncoil and stretch out along the surface of the comb as it is pulled from the water.

They then place the comb on a glass chip surface. Depending on how they place the comb, they leave behind strands of different lengths and shapes. “Basically, we’re doing nanotechnology using only a piece of rubber and a tiny droplet of DNA solution,” Guan said. Computer chips that bridge the gap between the electronic and the biological could make detection of certain chemicals easier, and speed disease diagnosis. But first, researchers must develop technologies to mass produce DNA circuits as they produce chip circuits today. The technique that Lee and Guan used is similar to a relatively inexpensive chip-making technology called soft lithography, where rubber molds press materials into shape.

In this study, they arranged the DNA into rows of “stitches,” pinstripes and criss-cross shapes. The pinstripes presented the researchers with a mystery: for some reason, thorn-like structures emerged along the strands at regular intervals. “We think the ‘thorns’ may be used as interconnects between nanowires, or they could connect the nanowires with other electronic components,” Guan said. “We are not trying to eliminate them, because we do not think they are defects. We also believe their formation is controllable, because they are almost completely absent in some experiments but abundant in others. Although we currently do not know exactly how the thorns form, maybe new and useful nanostructures may be created if we can better understand and control this process.”

The university will license the technology for further development. Lee and Guan are working on their first application – building the wires into sensors for detecting disease biomarkers. In the meantime, they are collaborating with researchers in the Department of Electrical and Computer Engineering at Ohio State to measure the electrical properties of the DNA wires. They are also using this technique to produce DNA-based nanoparticles for gene delivery.

Science Daily
January 3, 2006

Original web page at Science Daily

A novel “Flu Chip” developed at the University of Colorado at Boulder that can determine the genetic signatures of specific influenza strains from patient samples within hours may help world health officials combat coming epidemics and pandemics. Tests last month on the new technology by the Centers for Disease Control and Prevention in Atlanta showed the CU-Boulder Flu Chip can determine the genetic make-up of types and subtypes of the flu virus in about 11 hours, said CU-Boulder Professor Kathy Rowlen of the chemistry and biochemistry department. Current methods for characterizing flu subtypes infecting patients take about four days. The Flu Chip is expected to be in wide use in laboratories within a year, said Rowlen, who has led the two-year CU-Boulder research effort.
Rowlen, who is working on the Flu Chip development with CU-Boulder chemistry Professor Robert Kuchta and a team of postdoctoral researchers and students, said they are conferring with CU’s Technology Transfer Office and plan to make the Flu Chip genetic sequences freely available to interested researchers.

There currently are less than 200 facilities worldwide that provide detailed strain analysis of influenza, said Rowlen. Strain identification is critical for tracking emerging strains and in determining which flu strains are most likely to infect people the following year in order to develop annual, preventative vaccines, she said. “This new technology should help provide better global influenza surveillance by making it easier for more laboratories to swiftly identify severe flu strains, which in turn may aid health officials to stem potential flu epidemics and even pandemics,” Rowlen said. The chip, which can be configured to test for all known flu virus strains as well as new variant strains, was evaluated for three primary subtypes of flu in the October CDC test — the avian flu strain H5N1, and two of the most common human flu types worldwide in recent winters, H1N1 and H3N2. The chip was more than 90 percent accurate and will be tested again “side by side” with standard flu-virus culturing methods for accuracy and speed at the CDC’s Atlanta headquarters next month. “This was the first time a version of the Flu Chip was tested outside of our lab, and it exceeded our expectations,” she said.

The Flu Chip fits on a microscope slide and contains an array of microscopic spots, Rowlen said. Genetic bits of information that are complimentary to known, individual influenza strains are “spotted” robotically in an array, where each row of three spots contains a specific sequence of “capture” DNA. Each spot is approximately one-hundredth of an inch in diameter. The microarray is then immersed in a wash of influenza gene fragments obtained from the fluid of an infected individual. RNA fragments from the infected fluid bind to specific DNA segments on the microarray like a key in a lock, indicating both a match and that the virus signature is present, she said. The captured RNA is then labeled with another complimentary sequence that also contains a fluorescent dye, and such “hits” light up like a pinball machine when the chip is inserted into a laser scanner.

The Flu Chip also should be able to recognize mutations that might occur in avian flu H5N1, which has been spreading rapidly from bird to bird in Asia, Russia and parts of Europe, said Kuchta. While the avian virus does not now spread effectively from person to person, world health officials are fearful the strain will mutate and become transmittable between humans, possibly triggering a worldwide pandemic. “If an unusual flu subtype surfaces that has characteristics of both avian and human flu types, we could detect it rapidly using this technology,” Kuchta said.

Standard laboratory culturing techniques by the CDC and WHO currently take four days to five days to determine flu strains afflicting patients, said Kuchta. While commercial tests like rapid antigen testing can detect influenza in less than an hour, none provide genetic information about various flu subtypes, he said. Rowlen said that within a few years, the technology could be downsized to fit into a hand-held portable device the size of a cell phone or PDA and taken into remote areas around the world to test for lethal strains of flu. “We can make it small and simple enough to take into rural areas in places like the Congo, Cambodia or Indonesia that may lack lab facilities,” she said. “One of our goals has been to address the needs of developing nations by providing an inexpensive, field-portable test kit for respiratory illnesses to the World Health Organization for global screening of respiratory illness. “Kuchta said the team hopes to cut down on the 11-hour virus identification process. “We are now looking at ways to amplify the fluorescent signal after we capture the RNA on the microarray, which could shorten the identification time to just a couple of hours,” he said. Rowlen said the Flu Chip could also play a significant role in alerting government officials to an “engineered” influenza virus arising from terrorism.

Hurricane Katrina displayed the vulnerability of the United States to natural catastrophes, she said. “A flu pandemic is inevitable since the virus continually mutates and is naturally spread by migratory birds. Whether this year or 10 years from now, it is important to be prepared for such an event.” Most experts agree that preparations for a flu pandemic include early identification, vaccine development, the wide availability of pharmaceuticals and planning for possible local quarantine events. During the “Spanish Flu” pandemic of 1918-1919, between 20 million and 40 million people died from influenza in less than a year and an estimated one-fifth of the world’s population became infected. The flu chip also could be used to swiftly test for the avian flu virus at large, remote bird farms in Asia, Europe and Russia, said Kuchta. The chip also could be easily reconfigured to use for the global surveillance of any RNA virus, including SARS, measles, HIV and hepatitis C, the researchers said.

Science Daily Health & Medicine
December 6, 2005

Original web page at Sciece Daily Health & Medicine

Researchers studying how proteins called helicases travel along strands of DNA have found that when the proteins hit an obstacle they snap back to where they began, repeating the process over and over, possibly playing a preventative role in keeping the genome intact. Taekjip Ha, a professor of physics at the University of Illinois at Urbana-Champaign and a Howard Hughes Medical Institute investigator, likens the biological scenario to Boston Red Sox baseball; the team rolls along only to hit a late-season obstacle called the New York Yankees. Then, like the always-anticipated annual cry from Chicago Cubs fan, it’s back to square one next year. However, instead of causing more misery, as is the case for a baseball fan, this motor protein’s starting over may serve a beneficial purpose, clearing other, undesired proteins from the DNA, Ha said. The research was done in vitro, using purified proteins and studied with a technique that visualizes individual molecules on DNA. Whether the scenario plays out in real cells in not known and under exploration.

The discovery appears in the Oct. 27 issue of the journal Nature, along with a separate “News & Views” article by Eckhard Jankowsky, a biochemist at the Center for RNA Molecular Biology in Case Western University’s School of Medicine, who wrote about the potential importance of the findings. Ha’s postdoctoral fellow Sua Myong led the study, looking at the Rep helicase from an E-coli bacterium. Rep is known to be involved in restarting DNA replication stalled by DNA damage. As a single protein, a monomer, Rep can travel one way along a single strand of DNA but by itself cannot unzip it. Rep’s progress was visualized using the single molecule fluorescence resonance energy transfer (FRET) technique that Ha had developed.

By tagging the protein and DNA with green and red dyes, Myong measured FRET changes as Rep traveled along single DNA strands, which are short segments extending out from double strands. Each time the protein reached either the junction of the full double-stranded DNA or hit an artificially created protein obstacle, Rep instantly returned to near the beginning of the single strand on which it had initially bound. Upon closer examination using FRET, researchers discovered that Rep’s configuration gradually closed as it reached the obstacle in its path.
Then, conformational changes of Rep allow it to grab and transfer to the end of the single-stranded DNA, leading to the next cycle. “Although the very flexible single strand of DNA likely bombards the protein constantly, the protein doesn’t seem to pay attention to this overture until it hits a physical blockade,” Ha said.

Researchers had theorized that obstacles would force motor proteins to disengage from DNA. “The finding was totally unexpected and may indicate a new function for the protein,” Ha said. Jankowsky wrote that scientists “should not immediately search for the helix that the enzyme unzips, but instead remember how Rep snaps back.” In cells, single strands of DNA often occur when something is wrong, Ha said. The recycling action, he said, may represent a desirable function of the protein by keeping it engaged on a single strand, allowing time for repairs that allow normal DNA replication.

The human body has more than 200 types of helicases involved in replication, transcription, repair and other genetic processes, Ha said. Defective helicases have been linked to increased cancer risks and premature aging.

Science Daily
November 22, 2005

Original web page at Science Daily

Scientists continue to be puzzled by how proteins fold into their three-dimensional structures. Small single-domain proteins may hold the key to solving this puzzle. These proteins often fold into their three-dimensional structures by crossing only a single barrier. The barrier consists of an ensemble of extremely short-lived transition state structures which cannot be observed directly. However, mutations that slightly shift the folding barrier may provide indirect access to transition states. Researchers from the Max Planck Institute of Colloids and Interfaces and the University of California, San Francisco have suggested a novel method to construct transition state structures from mutational data (PNAS, July, 2005).

Proteins are chain molecules assembled from amino acids. The precise sequence of the twenty different types of amino acids in a protein chain is what determines which structure a protein folds into. The three-dimensional structures in turn specify the functions of proteins, which range from the transport of oxygen in our blood, to the conversion of energy in our muscles, and the strengthening of our hair. During evolution, the protein sequences encoded in our DNA have been optimised for these functions.

The reliable folding of proteins is a prerequisite for them to function robustly. Mis-folding can lead to protein aggregates that cause severe diseases, such as Alzheimer’s, Parkinson’s, or the variant Creutzfeldt-Jakob disease. To understand protein folding, research has long focused on metastable folding intermediates, which were thought to guide the unfolded protein chain into its folded structure. It came as a surprise about a decade ago that certain small proteins fold without any detectable intermediates. This astonishingly direct folding from the unfolded state into the folded state has been termed ‘two-state folding’. In the past few years, scientists have shown that the majority of small single-domain proteins are ‘two-state folders’, which are now a new paradigm in protein folding.

The characteristic event of two-state folding is the crossing of a barrier between the unfolded and folded state. This folding barrier is thought to consist of a large number of extremely short-lived transition state structures. Each of these structures is partially folded and will either complete the folding process, or will unfold again, with equal probability. Transition state structures are thus similar to a ball on a saddle point, which has the same probability, 0.5, of rolling to either side of the saddle. Since transition state structures are highly instable, they cannot be observed directly. To explore two-state folding, experimentalists instead create mutants of a protein. The mutants typically differ from the original protein — the wild type — in just a single amino acid. The majority of these mutants still fold into the same structure, however the mutations may slightly change the transition state barrier and, thus the folding time; that is, the time an unfolding protein chain on average needs to cross the folding barrier.

The central question is: can we reconstruct the transition state from the observed changes in the folding times? Such a reconstruction clearly requires experimental data on a large number of mutants. In the traditional interpretation, the structural information is extracted for each mutation, independent of the other mutations. If a mutation does not change the folding time, then the mutated amino acid traditionally is interpreted to be still unstructured in the transition state. In contrast, if a mutation changes the folding time, the mutated amino acid is interpreted to be partially or fully structured in the transition state, depending on the magnitude of the change.

This traditional interpretation is however often not consistent. For example, twenty single-residue mutations in the a-helix of the protein Chymotrypsin Inhibitor 2 (CI2) have very different effects on the folding time. Naïvely interpreted, these differences seem to indicate that some of the helical residues are unstructured in the transition state, while other residues, often direct neighbours, are highly structured. This naïve interpretation contradicts the fact that the folding of helices is co-operative, and can only occur if several consecutive helical turns are structured, stabilizing each other.

In a recent article in PNAS, a research team from the Max Planck Institute of Colloids and Interfaces and the University of California, San Francisco has suggested a novel interpretation of the mutational data. Instead of considering each mutation on its own, the new interpretation collectively considers all mutations within a cooperative substructure, such as a helix. In case of the a-helix of the protein CI2, this leads to a structurally consistent picture, in which the helix is fully formed in the transition state, but has not yet formed significant interactions with the ß-sheet. In the future, the Max Planck researchers hope to construct complete transition states from mutational data. An important step is to identify the cooperative subunits of proteins, which requires molecular modelling. In a similar way to how a mountain pass shows us how to cross the landscape, the transition states eventually may help us to understand how proteins navigate from the unfolded into the folded structure.

Science Daily
November 8, 2005

Original web page at Science Daily

Chemists who have trouble predicting how some large, complex biological molecules will react with others may soon have a solution from the world of computational quantum physics, say Purdue University researchers. Physicist Jorge H. Rodriguez and his Purdue research team are using powerful computers to probe the effects that spin, a quantum property of elementary particles, has on certain biochemical reactions. With further refinement, Rodriguez’s efforts could permit scientists to simulate and predict the results of reactions between complex substances in a virtual environment without the need to combine them physically in the lab. This technique could reduce drug development time and costs for pharmaceutical companies and expand our knowledge of life’s fundamental processes.

Using powerful supercomputers to analyze the interplay of the dozens of electrons that whirl in clouds about these molecules, a team of physicists led by Purdue’s Jorge H. Rodriguez has found that the quantum property of electrons called “spin” needs to be considered to obtain a complete and fundamental picture of how many biochemical reactions take place. In particular, a class of metal-based proteins that includes hemoglobin and chlorophyll, and their reactions in plants and animals, can be better understood with the technique.

Not only will this discovery sharpen our basic knowledge of biology, Rodriguez said, but it also could help scientists with a number of practical problems – such as selecting the best potential new drug compounds from a vast group of candidates, a process that can cost pharmaceutical companies years of work and millions of dollars. “Whereas we have had to be satisfied with observing the chemistry in living things and describing it afterward without complete understanding, we are developing computational tools that can predict what will happen between molecules before they meet in the test tube,” said Rodriguez, who is an assistant professor of physics in Purdue’s College of Science. “Not only does this research open up a new field of science that reveals how metalloproteins and their constituent particles interact, but the quantum theory behind it also should allow us to model and predict these behaviors accurately with computer simulation alone. It is an example of how much can be accomplished with interdisciplinary science.”

Rodriguez is pioneering a new field he calls “quantum biochemistry” – a field that involves both biochemistry and particle physics, which are often cited among the more formidable subjects science students tackle. Ordinarily, the two disciplines share little common ground. Although biochemistry deals with interactions among the complex molecules that our bodies use for the fundamental processes of life, these microscopically small molecules are nonetheless gargantuan entities in comparison with the tinier subatomic particles such as protons and electrons that physicists study.

“Despite these differences, there is one point of overlap between chemistry and physics that has interested me, and that is in the elementary particles that whirl about these molecules – the electrons,” Rodriguez said. “Physicists have long known that, according to the laws of quantum mechanics, there are some chemical reactions in our bodies that are ‘forbidden’ – such as hemoglobin’s binding oxygen in our lungs when we breathe. But they do happen nonetheless. So, because these reactions involve electron spin, we decided to take a closer look at them.” Charge is a familiar property of an electron, but it is not the only one. Electrons also have another quantum property called spin, and though they are all negatively charged, they can spin in one of two opposing directions – up or down.

“Nature loves balance, and you see evidence of it in both charge and spin,” Rodriguez said. “For example, electrons of opposite spin like to pair up with each other as they fly around the nucleus. This allows their spins to balance one another, just as positive and negative charges do between protons and electrons. Even when you have hundreds of electrons forming an immense cloud around a complex molecule, you still see balance in both charge and spin; we call this balance ‘conservation,’ and it’s something we count on in both chemistry and physics to help us understand these tiny objects. “But sometimes the electrons in metalloproteins seem to be playing a trick on us. As we see with hemoglobin, nature appears to be conserving electronic charge while sacrificing this conservation in spin.”

Hemoglobin’s active center contains iron, one of the so-called transition metals. These metals are noted for the way several of their electrons can fly around the nucleus unpaired. When a red blood cell encounters oxygen in our lungs, its hemoglobin is able to grasp some of the oxygen with some of these unpaired electrons, carrying it to the rest of our body. But in the process, the cumulative spin of the system changes in a way that is not conserved, which to a physicist looks as strange as a ball hitting the water without making a splash. “This chemistry is vital for life, but physicists wonder how it can happen,” Rodriguez said. “The charge between the electrons in the bonded oxygen and hemoglobin is balanced in the end, which makes sense to chemists. But the electronic spin of the entire system is not conserved, making a physicist frown at what appears to be a formally forbidden process. Of course, we needed to learn more about nature at the microscopic level.”

As many of these supposedly forbidden reactions involve biomolecules centered upon transition metals, which can flip back and forth between different spin states under certain conditions, Rodriguez theorized that it was this variability in spin state that was influencing the rate of these reactions. To explore whether this effect, which Rodriguez calls spin-dependent reactivity, was indeed the decisive factor, the team is modeling the reaction rates with a supercomputer, the only tool capable of keeping track of the motion of so many particles at once. “Supercomputers have allowed us to check our models against our understanding of spin’s effect on a reaction, and our models have been closely checked by experiment,” Rodriguez said. “The results suggest that our understanding of electron behavior is sufficient to create virtual models of molecules that we can then ‘react’ with one another in simulations that accurately predict what will happen when they meet in the physical world.”

Rodriguez said the approach, though still in its nascent stages, could provide insight into far more biologically important molecules when it is further developed. “We are at the point where we have developed computational tools to analyze the spin-dependent processes of biomolecules and have applied them to a few important test cases,” he said. “But our methods are based on approaches that are valid for any molecular system. Therefore, hundreds more metalloproteins that are of great scientific and practical interest may be studied in the future with the methods we have developed.”

For example, Rodriguez is planning to study the manganese involved in photosynthesis to understand how water is broken down to produce molecular oxygen. But for now, he is happy that the four years of work his team has put into the project have produced such encouraging results. “We are creating a new field that attempts to understand biochemical processes at the most fundamental level – that of quantum mechanics,” he said. “It could be the most important step toward making biochemistry a predictive science rather than a descriptive one.”

Science Daily
October 11, 2005

Original web page at Science Daily

Two Brown Medical School biologists have figured out the fate of healthy protein when it comes in contact with the infectious prion form in yeast: The protein converts to the prion form, rendering it infectious. In an instant, good protein goes bad. This quick-change “mating” maneuver sheds important light on the mysterious molecular machinery behind prions, infectious proteins that cause fatal brain ailments such as mad cow disease and scrapie in animals and, in rare cases, Creutzfeldt-Jacob disease and kuru in humans.

Because similar protein self-replication occurs in neurodegenerative diseases, the findings, published in the latest issue of Nature, may also help explain the progression of Alzheimer’s, Parkinson’s and Huntington’s diseases. Graduate student Prasanna Satpute-Krishnan and Assistant Professor Tricia Serio, both in Brown’s Department of Molecular Biology, Cell Biology and Biochemistry, conducted the research using Sup35, a yeast protein similar to the human prion protein PrP.

The researchers tagged a non-prion form of Sup35 with green fluorescent protein in one group of cells and “mated” these cells with another group that contained the prion form. When the two forms came in contact in the same cell, the green-glowing, healthy protein changed pattern – a visual sign that it converted to the prion form. These results were confirmed in a series of experiments using different biochemical and genetic techniques.

Because proteins can’t replicate like DNA and RNA – the genetic material in bacteria, viruses and other infectious agents – the research helps explain the puzzling process of how prions multiply and spread infection. Satpute-Krishnan said the speed of protein conversion was surprising. “The prions were taking all the existing protein and refolding it immediately,” she said. “It’s a very, very rapid change.” After the conversion, the yeast cells remained healthy but had new characteristics. This survival supports the theory that prions have endured through evolution because shape-shifting is advantageous, allowing cells to avoid stress by rapidly adjusting to a new environment. “Our studies provide some insight into how the appearance of a misfolded protein – a rare event – can lead to devastating neurological diseases,” said Serio. “Just a small amount of prion-state protein can rapidly convert healthy protein into a pathogenic form.”

Source: Brown University

Bio.com
September 27, 2005

Original web page at Bio.com

Space radiation preferentially destroys specific forms of amino acids, the most realistic laboratory simulation to date has found. The work suggests the molecular building blocks that form the “left-handed” proteins used by life on Earth took shape in space, bolstering the case that they could have seeded life on other planets. Amino acids are molecules that come in mirror-image right- and left-handed forms. But all the naturally occurring proteins in organisms on Earth use the left-handed forms – a puzzle dubbed the “chirality problem”. “A key question is when this chirality came into play,” says Uwe Meierhenrich, a chemist at the University of Nice-Sophia Antipolis in France. One theory is that proteins made of both types of amino acids existed on the early Earth but “somehow only the proteins of left-handed amino acids survived”, says Meierhenrich. Meierhenrich and colleagues have a different theory. “We say the molecular building blocks of life were already created in interstellar conditions,” he told New Scientist.

The team believes a special type of “handed” space radiation destroyed more right-handed amino acids on the icy dust from which the solar system formed. This dust, along with the comets it condensed into, then crashed into Earth and other planets, providing them with an overabundance of left-handed amino acids that went on to form proteins. The radiation is called circularly polarised light because its electric field travels through space like a turning screw, and comes in right- and left-handed forms. It is thought to be produced when dust grains become aligned in the presence of magnetic fields threading through regions of space much larger than our solar system. Circularly polarised light is estimated to make up as much as 17% of the radiation at any given point in space.

In 2000, an experiment showed that when circularly polarised ultraviolet light of a particular handedness was shone on an equal mix of right- and left-handed amino acids, it produced an excess of 2.5% by preferentially disintegrating one type. But that experiment was done using amino acids in a liquid solution, which behave differently than those in the solid conditions of icy dust in space. To avoid absorption by water molecules, it was also necessary to use light at a wavelength of 210 nanometres – significantly longer than the peak of 120 nm radiation actually measured in space.

Now, Meierhenrich’s team has performed a similar experiment. The group shone circularly polarised light at a wavelength of 180 nm on a solid film of both right- and left-handed forms of the amino acid leucine. It found that left-handed light produced an excess of 2.6% left-handed amino acids. “Going towards greater realism by exploring another wavelength of light and solid samples is definitely a good thing and a logical step forward,” says chemist Max Bernstein of NASA’s Ames Research Center in California, US, who is not part of the team. He says the research adds to previous measurements of an excess of left-handed amino acids in two meteorites. “If it is thanks to meteorites that our amino acids are left handed, then the same bias should exist at least across our solar system”, he told New Scientist.

But other solar systems may harbour right-handed amino acids if they are subjected to the other type of circularly polarised light, says Meierhenrich. “The chiral amino acids might have been delivered to other planets, to other solar systems,” he adds. “The probability that life arose somewhere else is increased with this experimental result.” Meierhenrich will continue to reduce the wavelength of the experimental radiation by using a synchrotron facility, due to begin operating in 2006. But the real test of his theory may come in 2014, when the European Space Agency’s Rosetta spacecraft lands a probe on Comet 67P/Churyumov-Gerasimenko. He designed an instrument for the lander that will measure the handedness of any amino acids it finds. “If we identify left-handed amino acids on the cometary surface, this would underline the hypothesis that the building blocks of proteins were created in interstellar space and were delivered via comets or micrometeorites to early Earth,” he says.

Journal reference: Angewandte Chemie International Edition (vol 44, p 2)

New Scientist
September 13, 2005

Original web page at New Scientist

Brown University biologists have solved the structure of a critical piece of synapse-associated protein 97 (SAP97) found in abundance in the heart and head, where it is believed to play a role in everything from cardiac contractions to memory creation. Results are published in The Journal of Biological Chemistry. Dale Mierke, associate professor of medical science at Brown, said that knowing how a piece of SAP97 is built is an important step. Now that part of the protein’s structure is solved, scientists can create a molecule to disable it. That, in turn, will allow them to fully understand SAP97’s role in the body. And that will point drug makers to targets for developing new ways to treat cardiac or neurological diseases. “To arrive at a solution, you need to understand the problem,” Mierke said. “Solving protein structures opens doors for effective treatments.”

SAP97 is found mainly in the central nervous system and is known as a “scaffolding” protein. In this role, it serves as a sort of tether, grabbing proteins inside the cell critical to nerve signaling and keeping them close to N-methyl-D-asparate (NMDA) receptors at the cell surface. NMDA receptors help usher in a neurotransmitter called glutamate that is essential for learning and memory and also plays a role in drug addiction. A similar scaffolding mechanism is at work in the heart, where it affects basic functions, including the heartbeat.

SAP97 is a complex protein made up of five “domains” similar to a train comprised of an engine and four boxcars. In their experiments, Mierke, graduate student Lei Wang and postdoctoral research fellow Andrea Piserchio – all colleagues in the Department of Molecular Pharmacology, Physiology and Biotechnology – focused on the engine. This domain, known as PDZ1, is where the protein links to NMDA receptors. The team used high-resolution nuclear magnetic resonance spectroscopy to solve the structure of PDZ1, as well as a small portion of the receptor to which it binds. Mierke said the group is now developing a molecule that can inhibit PDZ1 as well as PDZ2, the first boxcar on the multi-domain protein.

Science Daily
September 13, 2005

Original web page at Science Daily

Researchers studying zebrafish that die from anemia have discovered a new pathway for the synthesis of heme, the deep red, iron-containing molecule that is a component of hemoglobin and myoglobin. The research suggests that defects in this pathway may be an overlooked cause of anemia in humans. The research team led by Leonard I. Zon, a Howard Hughes Medical Institute investigator at Children’s Hospital Boston and Harvard Medical School, published its findings in the August 18, 2005, issue of the journal Nature. Zon and his colleagues in Boston collaborated on the studies with researchers from the University of Rochester Medical Center and the University of Utah School of Medicine. The researchers began their studies hoping to learn why a zebrafish mutant known as shiraz (sir) failed to produce hemoglobin. The sir mutant zebrafish, which were first isolated by Zon and colleagues in the Tübingen Screen Consortium in Germany, intrigued the researchers because they die from anemia caused by lack of hemoglobin.

Over the years, Zon and his colleagues have discovered many zebrafish mutants that fail to make hemoglobin because of defects in iron metabolism. As they have teased out the causes of these defects, they have learned that the biochemical pathway involved in hemoglobin synthesis in zebrafish has been largely conserved over the 300 million years of evolution between fish and humans. According to Zon, the easily manipulable fish constitutes an excellent model organism for studying the regulation of heme formation. In the current study, the researchers traced the hemoglobin defect to the gene for an enzyme known as glutaredoxin 5 (grx5). But the researchers found early on that the enzyme was not directly connected to hemoglobin production. “Nobody had worked on this gene in vertebrates before, but we found in the scientific literature that this gene in yeast was required for the production of iron-sulfur clusters in the mitochondria,” said Zon. Iron-sulfur clusters are incorporated into certain proteins to enable their enzymatic functions. In further experiments, the researchers confirmed that versions of grx5 in zebrafish, yeast, mice and humans are functionally equivalent. “It seemed like the whole process was evolutionarily conserved,” said Zon. “But the difference is that yeast do not make hemoglobin. So we needed to figure out a mechanism that would explain why these fish that have problems making iron-sulfur clusters could not make hemoglobin.”

Other researchers’ studies had indicated that the presence of iron-sulfur clusters in the cell is important for controlling an enzyme called iron regulatory protein 1 (IRP1). In turn, IRP1 regulates another enzyme called ALAS2 that plays a key role in heme synthesis. Indeed, experiments by Zon and his colleagues demonstrated that the loss of grx5 in the mutant zebrafish inappropriately activates IRP1, which blocks the synthesis of ALAS2, and thus heme production. For example, when they restored ALAS2 by injecting into the sir mutants a truncated form of ALAS2 that lacked the portion of the molecule sensitive to IRP1, they complete restored the mutant zebrafish hemoglobin production.

“People have always thought that hemoglobin synthesis required only enough iron in the cell for heme production to proceed and then just the addition of the globin protein to form hemoglobin,” said Zon. “Now, we’ve added a fourth component, iron-sulfur clusters, which are required for heme production. This is a very interesting and unpredicted finding from what we had known before, and our experiments have really defined a new pathway for hemoglobin production,” he said. Zon said that the findings could apply to developing new treatments for a rare form of anemia, known as sideroblastic anemia, in which elevated IRP1 activity causes a deficiency of ALAS2. In most cases, an increase in IRP1 is likely caused by a mutation in a transporter for iron-sulfur clusters that traps them in mitochondria, where they cannot interact with IRP1 to control it.

In a search for possible treatments for the anemia, Zon and his colleagues are exploring the genetic machinery of hemoglobin production in zebrafish for targets of drugs that could restore normal levels of iron-sulfur clusters. “The pathway that we have found is very sensitive, so our findings might be extended to enable treatments for other forms of anemia,” said Zon.

Science Daily
Septermber 13, 2005

Original web page at Science Daily

The power and promise of microarrays are vast. Offering the ability to run tens of thousands of experiments in parallel on a small glass slide, gene chips have transformed functional genomics, whether through expression analysis or genotyping, from fantasy to reality with dizzying speed. And yet, longstanding doubts persist as to the worth of the torrent of data that microarrays produce. Experiments have proved difficult to reproduce, and the lists of genes found in similar studies often have only limited overlap. Myriad protocols and platforms, and the proliferation of homemade arrays, means that there is no one standardized set of procedures to follow when designing or replicating an experiment. Some analyses have found cross-platform reproducibility to be poor.

But, microarray technology isn’t as flawed as it was feared to be. At least, those are the findings of a trio of papers published in the May issue of Nature Methods, which together represent the most systematic effort yet to assess the reliability and reproducibility of microarray data. The authors made large-scale efforts to compare data across commercial and in-house platforms, to tease out the “platform effect” from the “lab effect,” and to discover how much biological signal really exists in the massive amounts of discordant data that microarrays often yield. “Three independent studies came up with complementary conclusions, saying microarrays are pretty close to being ready for prime time,” says John Quackenbush, who is a professor of biostatistics at Harvard’s Dana-Farber Cancer Institute, a member of the advisory board of the Microarray Gene Expression Data Society (MGED), and an author on two of the papers.

The recent studies confirmed three persistent criticisms: That the bewildering array of platforms and research protocols available can make results from different studies hard to compare; that, in the hands of less-experiened labs, homemade arrays are less dependable than commercial chips; and that different labs doing the same study can often get very different results. But the studies also had some surprisingly good news for the microarray community. First, they found that the various platforms, both commercial and homemade, could all deliver good results in experienced hands. They found also that standardized research protocols go a long way toward increasing reproducibility. And perhaps most importantly, they determined that the vast bulk of microarray data is driven by underlying biology, even in cases where platforms differ over a particular gene’s expression. Despite efforts in the three papers and other studies to crown a victor, no one microarray platform or set of experimental protocols has yet emerged as the best. Still, there are lessons to be learned.

If you’re just getting into the microarray research game, and you don’t have access to a core facility, consider using a commercial chip. The experimental protocols will be clearer and more standardized. Labs can achieve good results with in-house spotted arrays, but the recent studies indicate that success with homemade arrays depends largely on expertise and experience. And price is no longer a deal-breaker: Costs have come down in recent years to the point where paying more for a commercial chip might appear competitive with the cost of equipping a lab to produce spotted arrays.

“If you’re good at tinkering and making something work and you have a lot of time to do that, you might want to go with one of these homegrown platforms. You’ll save money and maybe get better results. But if you just want to stick something in some hole and press a button, you’re probably better off with an industrial product,” says Rafael Irizarry, an author on one of the Nature Methods papers and a biostatistician at the Johns Hopkins Bloomberg School of Public Health.

Don’t expect the magnitudes of individual gene expressions to be comparable across platforms; they’re usually not. You can make more valid comparisons by looking at relative, rather than absolute, gene expression. For a more biologically meaningful and statistically robust approach to data analysis, look at the level of the biological process (i.e., the Gene Ontology (GO) annotation) rather than the gene. “Our paper showed that the biological representations of the genes are more important than the genes themselves. Even if on a gene-by-gene basis we found inconsistency, when we looked at the processes themselves, those conserved processes are there,” says Weis.

Looking at the process level allows researchers to make sense of inherent variation, says Aviv Regev, a research fellow at Harvard’s Bauer Center for Genomics Research and coauthor of a recent Nature Genetics paper that proposed a process-level method of looking at microarray data. “When you analyze things at the level of gene sets, there is noise at the level of individual sets. This might not only be a result of methodological problems with your microarrays, they might be inherent biological variability,” she says.

One of the major barriers to the reproducibility of microarray data is a basic one: Given the plethora of platforms and protocols, it’s often hard to precisely reconstruct the experimental methods used in a study from the published paper. Several years ago, MGED developed its standard for data reporting, called Minimal Information about a Microarray Experiment (MIAME), to cut down on the widespread methodological confusion. The guidelines require researchers to go above and beyond what would ordinarily be required in a methods section, including detailed descriptions of the protocols used in RNA extraction, labeling, and hybridization; the data normalization algorithms used in preprocessing; and the design of the arrays themselves. MGED has also called for researchers to submit their raw data to public databases such as the Gene Expression Omnibus and ArrayExpress.

Although many specialized journals have yet to adopt MIAME, the standard is increasingly becoming the law of the land. Nature, Cell, and The Lancet adopted MIAME compliance requirements for their authors in 2002, and many other journals have followed suit. Several commercial array manufacturers and software developers have written protocols and software to help their users follow MIAME guidelines. (See, for instance, [http://www.mged.org/Workgroups/MIAME/miame_software.html] for a list) “Most of the requirements are difficult to comply with if you don’t have the tools,” says Regev.

But while standards for data reporting are becoming well established, the same is not true for data collection, says Chris Stoeckert, associate professor at the University of Pennsylvania’s Center for Bioinformatics and a member of MGED’s advisory board. As they seek to develop best practices for experimentation, MGED will be looking at studies like the ones recently published in Nature Methods to shed light on what remains a murky issue. “We all want to be able to look at a microarray experiment and assess whether it was done correctly or not. To a large degree we are unable to do that, because there are no standards yet as to how to assess it,” Stoeckert says. “We need standards on the informatics side – how do you evaluate the data that’s there – and also standards in terms of how to experimentally control for quality.”

The intense spotlight currently being cast on microarray research may shed light on other research as well. All researchers can learn a few lessons on the importance of the lab effect, says Irizarry. “It’s been known forever, not just in microarrays, that there is a lab effect. There’s a paper I cite where the speed of light is plotted from different labs across time, and the difference between labs is statistically significant,” he says. “I think microarrays are getting a bum rap because of one of their strengths, which is that they produce a lot of data. I think if other technology produced this much data, we would start seeing similar problems.” Irizarry sees the beginnings of hopeful trends toward standardization in the research community. For instance, many universities are moving towards using core facilities rather than processing microarrays in individual labs. “In academia, you are seeing a movement towards standardization,” he says. “Given what I’ve seen in terms of the lab effect, this is a move in the right direction.”

The Scientist
August 16, 2005

Original web page at The Scientist

The diagnostic methods for specific animal diseases are described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (the Terrestrial Manual) and the OIE Manual of Diagnostic Tests for Aquatic Animals (the Aquatic Manual). However, these manuals do not recommend specific diagnostic kits that can be used by Member Countries. Consequently the existence of several such kits has led to possible confusion in some Member Countries, which are not comfortable to choose approved diagnostic kits for trade or surveillance purposes.

In accordance with OIE missions to control animal diseases, to harmonise animal diseases control methods including the harmonisation of diagnostic methods, the OIE International Committee adopted during the 71st General Session in May 2003, Resolution No. XXIX, endorsing the principle of validation and certification of diagnostic assays (test methods) for infectious animal diseases by the OIE. The Resolution asked the Director General of the OIE to set up specific procedures to be followed for the validation and certification of diagnostic assays, based on the fundamental concept of ‘Fitness for Purpose’. The OIE has, with the technical support of the OIE Collaborating Centres of Vienna (Austria) and Fougères (France), finalised a formal procedure for the validation and certification of diagnostic kits. This procedure is open to both public and private laboratories producing diagnostic kits and which are desirous of having those kits approved and registered at the OIE.

Assessment of the diagnostic kit will be carried out by independent experts and the results submitted for consideration by the Biological Standards Commission before being endorsed by the OIE International Committee during the annual General Session. This procedure may last approximately for 135 days for each application. If the diagnostic test is validated and certified by the OIE, the producer would be authorised to use the OIE logo on any document or equipment associated with the test.

OIE
June 7, 2005

Original web page at OIE

Directed chemical synthesis can produce a vast range of molecular structures, but the intended product must be known at the outset. In contrast, evolution in nature can lead to efficient receptors and catalysts whose structures defy prediction. To access such unpredictable structures, researchers have prepared dynamic combinatorial libraries, in which reversibly-binding building blocks assemble around a receptor target. They selected for an acetylcholine receptor by adding the neurotransmitter to chloroform/dimethylsulfoxide solutions of dipeptide hydrazones [proline-phenylalanine or proline-(cyclohexyl)alanine], which reversibly combine through hydrazone linkages. At thermodynamic equilibrium, the dominant receptor structure was an elaborate [2]-catenane, consisting of two interlocked macrocyclic trimers. This complex receptor with a 100 nanomolar affinity for acetylcholine could be isolated on a preparative scale in 65% yield.

Science
March 29, 2005

Original web page at Science

Researchers have moved a step forward in understanding how calorie restriction is linked to lifespan extension in mammals. In this week’s issue of Nature, a group from the United States reports that SIRT1—the mammalian version of a protein linked to longevity in simpler organisms—controls glucose metabolism in mice in response to fasting.

Pere Puigserver of Johns Hopkins University and colleagues found that fasting signals induce the SIRT1 protein in the liver. This protein is one of the mammalian homologues of Sir2, known to extend lifespan in yeast and worms. SIRT1 then interacts with the coactivator PGC-1alpha, which, in turn, triggers glucose production, a key metabolic change associated with extended lifespan. “Our work provides a novel connection between PGC-1alpha, a protein involved in the food-deprivation response, and SIRT1, a protein linked to aging in lower organisms,” Puigserver told The Scientist.

SIRT1, which is an NAD+-dependent histone deacetylase, had already been associated with calorie restriction and longevity in mammals. Induced by food deprivation, it inhibits stress-induced apoptotic cell death in vitro and promotes fat mobilization in vitro and in vivo. However, it was unclear how SIRT1 might be involved in pathways such as gluconeogenesis and glycolysis, which are directly affected by calorie restriction in mammals.

In the Nature paper, the research team provides a connection between SIRT1 and these pathways. Moreover, they show that SIRT1 acts as a sensor of food deprivation. “During starvation, there is an increase in pyruvate, a nutrient signal that induces translation of SIRT1, and an increase in NAD+, which functions as a substrate and as an activator of SIRT1. The active SIRT1 interacts with PGC-1alpha, deacetylates it, and keeps it active, promoting glucose production in the liver,” explained Puigserver. With these results, the researchers showed that besides the hormonal control of PGC-1 through glucocorticoids and glucagon during fasting, there is a nutrient control as well, which targets SIRT1.

Marc Tatar of Brown University, who did not participate in the research, found the role of SIRT1 in nutrient sensing impressive. “There are hormonal inputs for sensing nutrients that are released systemically and circulate throughout the body,” Tatar told The Scientist. “But what we are beginning to see is that there are also systems in which every cell can sense the nutrient condition in their own neighborhood and adjust their metabolism to their local nutrient conditions.”

Tatar said this type of autonomous nutrient sensing could date back to times when organisms were only single celled and didn’t have hormone signals. “These are probably the roots, and the reason that you find [this sensing system] in yeast, nematodes and mammals, is because it is very ancestral. We are looking at it in flies,” said Tatar.

According to Leonard Guarente of the Massachusetts Institute of Technology, who was not involved in the study, the Nature paper provides a good example in which SIRT1 is influencing a key physiological aspect of calorie restriction in a mammal. “Although this is not the first example, it’s an important one,” he said. Guarente’s group recently reported how SIRT1 influences fat mobilization in mammals. “In fat cells, the target that SIRT1 is acting on is the nuclear hormone receptor PPAR-gamma, a critical regulator of fat; in this system, it’s PGC-1, which is a cofactor for PPAR-gamma. This suggests we are converging in a critical pathway here.”

“Calorie restriction really mitigates many diseases. Once we understand these pathways, we can think about developing drugs that can intervene pharmacologically and have implications to specific diseases,” explained Guarente. “The hypothesis linking low food to longevity and disease resistance through Sir2 is robust. The testing of the hypothesis is just beginning.”

BioMed Central
March 15, 2005

Original web page at BioMed Central