Rockefeller University, January 5, 2010 – Performance enhancers are the currency of a competitive society. But there’s one that we have always had: For millions of years, segments of our DNA have improved the performance of our genome, revving up protein production at those times we need it most. New research from Rockefeller University and the University of Michigan Medical School now show that these genome enhancers regulate how our bodies make germ-fighting antibodies, molecules that keep savvy viruses and bacteria at bay.
The research, which appears this month in the Journal of Experimental Medicine, represents a major technological advance that will allow scientists to understand the role of enhancers in the immune system — work that has stymied researchers for decades. “Many people left the field because working with antibody enhancers was so difficult,” says F. Nina Papavasiliou, head of the Laboratory of Lymphocyte Biology at Rockefeller. “It seemed like there was no way around the problem.”
Enhancers are short swaths of DNA that regulate genes from a distance, often megabases away. Generally, this distance from the genes they regulate makes enhancers hard to study. But immunoglobulin enhancers have been particularly problematic because of an additional twist: they are close to chromosome ends, which makes altering their local sequence especially difficult.
Instead of tampering with enhancers in place, Wesley Dunnick, a professor at the University of Michigan Medical School, devised a way to move the entire locus — the enhancers along with adjacent antibody genes that contain information about foreign invaders — onto an artificial bacterial chromosome. Placement on the artificial chromosome allows for the modification of the excised locus with relative ease: using tools pioneered at Rockefeller by Peter Model and Nathaniel Heintz, Dunnick could delete or mutate enhancer sequences at will. He then re-inserted these modified chromosomes into the mouse. Like a bubble floating in the genome, these artificial chromosomes would land randomly onto the mouse genome and get incorporated into it, now allowing him to study the effect of these modified enhancers on the generation of antibodies.
In response to the unlimited number of foreign antigens (bits of microbes, chemicals and other substances) that can invade our bodies, the immune system must be able to tailor-make an unlimited number of antibodies. However, the amount of DNA in a cell is limited, so antibody-producing B cells must mutate and re-arrange their antibody genes to step up to the challenge (using processes called somatic hypermutation and class switch recombination, respectively).
In collaboration with the Papavasiliou lab, Dunnick discovered that mice carrying the artificial chromosomes with the antibody genes behave in ways that are indistinguishable from unmanipulated mice: they recombine and mutate their antibody genes to generate highly specific attacks on foreign invader. But for that, they absolutely need their enhancers: without them, the cell’s machinery can transcribe and translate the antibody genes, but can’t rearrange or mutate them, suggesting that the enhancers function as a loading dock for a common initiator molecule, which is then hauled to the antibody genes.
The experiments show that the enhancers of antibody genes are vital in springing the immune system to action, and suggest that mutations in the enhancers may make an individual more susceptible to infections, even infections for which he should have been vaccinated. “The main goal of vaccination is to produce, in a short amount of time, antibodies that are diversified to be most effective against a particular virus or bacterium,” says Dunnick. “We were fortunate to identify the control elements that are critical for this antibody diversification.”
Story Source: Adapted from materials provided by Rockefeller University.
UCLA’s Erin Greiner, Xiaofeng Gu and X. William Yang examine Huntington’s disease in two mouse models. (Credit: UCLA)
NIH/National Institute of Neurological Disorders and Stroke, Jan. 5, 2010 – In Huntington’s disease, a mutated protein in the body becomes toxic to brain cells. Recent studies have demonstrated that a small region adjacent to the mutated segment plays a major role in the toxicity. Two new studies supported by the National Institutes of Health show that very slight changes to this region can eliminate signs of Huntington’s disease in mice.
Researchers do not fully understand why the protein (called mutant huntingtin) is toxic, but one clue is that it accumulates in ordered clumps of fibrils, perhaps clogging up the cells’ internal machinery.
“These studies shed light on the structure and biochemistry of the mutant huntingtin protein and on potentially modifiable factors that affect its toxicity,” said Margaret Sutherland, Ph.D., a program director at NIH’s National Institute of Neurological Disorders and Stroke (NINDS). “They reveal sites within the huntingtin protein and within broader disease pathways that could serve as targets for drug therapy.”
Both studies were published online this week. One study, published in the Journal of Cell Biology, was led by Leslie Thompson, Ph.D., and Joan Steffan, Ph.D., of the University of California, Irvine. The other study, in Neuron, was led by X. William Yang, M.D., Ph.D., of the University of California, Los Angeles in collaboration with Ron Wetzel, Ph.D., of the University of Pittsburgh School of Medicine.
Huntington’s disease is inherited, and usually strikes in middle age, producing uncontrollable movements of the legs and arms, a loss of muscle coordination, and changes in personality and intellect. It is inexorably progressive and leads to death of affected persons usually within 20 years after symptoms first appear. Individuals with the disease carry mutations that affect the huntingtin protein. The mutations involve a triple repeat DNA sequence, a type of genetic miscue similarly found in Friedreich’s ataxia, Kennedy’s disease, fragile X syndrome, and other neurodegenerative disorders.
The normal huntingtin protein consists of about 3,150 amino acids (which are the building blocks for all proteins). In individuals with Huntington’s disease, the mutated protein contains an abnormally long string of a single amino acid repeat; lengthier chains are associated with worse symptoms and earlier onset of the disease. In recent years, however, researchers have begun looking at the effects of other, nearby amino acids in this large protein — and in particular, biochemical changes to those amino acids.
In their study, Drs. Steffan and Thompson investigated how a process called phosphorylation affects huntingtin. Phosphorylation is the attachment of chemical tags, known as phosphates, onto the amino acids in a protein. The process occurs naturally and is a way of marking proteins for destruction by cellular waste handling systems. The researchers liken it to putting a sign on a pile of junk that tells the garbage collectors to take it away. Their study shows that phosphorylation of just two amino acids, located at one end of huntingtin, targets the protein for destruction and protects against the toxic effects of the mutant protein.
“Clearance of mutant huntingtin is likely regulated at many levels, but our data establish that these two amino acids are critical,” Dr. Steffan said.
Could boosting phosphorylation of those two amino acids reduce the buildup of huntingtin and improve symptoms of the disease? In parallel with the UC Irvine research, Dr. Yang and his team at UCLA were asking that question using an animal model of Huntington’s disease. Previously, Dr. Yang had created mice that carry the mutant huntingtin gene. These mice develop symptoms reminiscent of Huntington’s disease in humans, including poor coordination, mental changes such as increased anxiety, loss of brain tissue, and accumulation of clumps of huntingtin in brain cells.
Through further genetic engineering, Dr. Yang altered the same two critical amino acids at the end of the mutant huntingtin protein to either mimic phosphorylation (phosphomimetic) or resist it (phosphoresistant). Mice with the phosphoresistant version of the protein developed symptoms of Huntington’s, but mice with the phosphomimetic version remained free of symptoms and huntingtin clumps up to one year.
Meanwhile, test tube experiments by Dr. Wetzel’s group in Pittsburgh showed that phosphomimetic modification of a huntingtin fragment reduced its tendency to form clumps. Together, data from the mouse and test tube experiments provide strong support for the idea that phosphorylation acts as a molecular switch to alter clumping of the mutant protein, the researchers said.
The nearly complete lack of any signs of disease in the phosphomimetic Huntington mice may point toward new strategies to treat the disorder someday. Dr. Yang said, “Drugs that enhance or mimic the effects of phosphorylation may help to detoxify the mutant huntingtin protein.”
If such drugs could be developed, Drs. Steffan and Thompson theorize, they would likely be most effective at early stages of the disease, but less so at later stages, when the clearance machinery appears to run down. Dr. Yang said he plans to examine older mice carrying the phosphomimetic version of mutant huntingtin to determine how long they are protected from the disease.
The researchers received major funding from NINDS, with additional support from the National Institute on Aging, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of General Medical Sciences. Several nonprofit foundations also contributed to the research, including the Hereditary Disease Foundation, the Fox Family Foundation and CHDI Inc.
Co-authors of the Journal of Cell Biology study included J. Lawrence Marsh, Ph.D. and Lan Huang, Ph.D., at UC Irvine; Ana Maria Cuervo, M.D., Ph.D., at Albert Einstein College of Medicine, New York City; Donald C. Lo, Ph.D. at Duke University, Durham, N.C.; Paul H. Patterson, Ph.D., at California Institute of Technology, Pasadena; and Steven Finkbeiner, M.D., Ph.D., at the University of California, San Francisco.
Co-authors of the Neuron study included Xiaofeng Gu, M.D., Ph.D., and Erin Greiner at UCLA; Rakesh Mishra and Ravindra Kodali, Ph.D., at the University of Pittsburgh; Alex Osmand, Ph.D., at the University of Tennessee, Knoxville; and Dr. Finkbeiner at UCSF.
Story Source: Adapted from materials provided by NIH/National Institute of Neurological Disorders and Stroke
BiologyNews.net, January 5, 2010 — Researchers at MIT and Alnylam Pharmaceuticals report this week that they have successfully used RNA interference to turn off multiple genes in the livers of mice, an advance that could lead to new treatments for diseases of the liver and other organs.
Since the 1998 discovery of RNA interference – the naturally occurring phenomenon in which the flow of genetic information from a cell’s nucleus to the protein-building machinery of the cell is disrupted – scientists have been pursuing the tantalizing ability to shut off any gene in the body. Specifically, they have been trying to silence malfunctioning genes that cause diseases such as cancer.
The new delivery method, described in the Proceedings of the National Academy of Sciences, is orders of magnitude more effective than previous methods, says Daniel Anderson, senior author of the paper and a biomedical engineer at the David H. Koch Institute for Integrative Cancer Research at MIT.
“This greatly improved efficacy allows us to dramatically decrease the dose levels, and also opens the door to formulations that can simultaneously inhibit multiple genes or pathways,” says Anderson.
The key to success with RNA interference is finding a safe and effective way to deliver the short strands of RNA that can bind with and destroy messenger RNA, which carries instructions from the nucleus.
Anderson and his colleagues believe the best way to do that is to wrap short interfering RNA (siRNA) in a layer of fat-like molecules called lipidoids, which can cross cells’ fatty outer membrane. Using one such lipidoid, the researchers were able to successfully deliver five snippets of RNA at once, and Anderson believes the lipidoids have the potential to deliver as many as 20.
How they did it: The team at MIT, along with Alnylam researchers, have developed methods to rapidly produce, assemble and screen a variety of different lipidoids, allowing them to pick out the most effective ones.
In a previous study, the researchers created more than 1,000 lipidoids. For their latest study, they picked out one of the most effective and used a novel chemical reaction to create a new library of 126 similar molecules. The team focused on one that appeared the most promising, dubbed C12-200.
Using C12-200, the researchers achieved effective gene silencing with a dose of less than 0.01 milligrams of siRNA per kilogram of solution, and 0.01 milligrams per kilogram in non-human primates. If the same dosing were translated to humans, a potential therapy would only require an injection of less than 1 milliliter to specifically inhibit a gene, compared with previous formulations that would have required hundreds of milliliters, says Anderson.
Other authors from MIT include Kevin T. Love, Kerry P. Mahon, Christopher G. Levins, Kathryn A. Whitehead and Institute Professor Robert Langer.
Next steps: The MIT/Alnylam team hopes to start clinical trials within the next couple of years, after figuring out optimal doses and scaling up the manufacturing capability so they can produce large amounts of the siRNA-lipidoid complex.
Howard Hughes Medical Institute, January 5, 2010 — Terrible and swift as anthrax appears to its victims, the deadly toxin takes its time breaking into their cells. The entry of anthrax toxin into its cellular target is part of a carefully-planned, two-pronged attack, scientists have found. Howard Hughes Medical Institute international research scholar Gisou van der Goot has identified for the first time the cell signaling event that sets the deadly strike in motion.
In a paper published the week of Dec. 28, 2009, in the journal, Proceedings of the National Academy of Sciences, van der Goot and her colleagues at the Global Health Institute of the École Polytechnique Fédérale de Lausanne in Switzerland reveal how the anthrax toxin carefully times its attack, slowly assembling its component parts on the surface of a target cell before suddenly hijacking the cell’s own signals. The van der Goot lab reports that the assembled toxin, moored to a receptor outside the cell, directly activates an enzyme family inside the cell, called src-like kinases. The receptors-with the anthrax toxin attached-are pulled by the kinases into the cytoplasm for digestion. But once inside, the toxin instead slices up the cell.
Scientists had known that anthrax toxin hovers outside of cells, but no one knew how it got inside the cells. Van der Goot and her colleagues identified anthrax’s two step strategy. In the first stage, the bacterium, Bacillus anthracis, pumps out two molecules: edema factor (EF) and lethal factor (LF). But EF and LF don’t become dangerous to individual cells until stage two, when the anthrax bacteria creates a protective antigen (PA). PA is the key that allows EF and LF inside the cell through two common cell surface receptors, TEM8 and CMG2. Together, those three components-PA, EF, and LF-make up the anthrax toxin.
Once docked onto one of the receptors, PA begins assembling a seven-molecule structure -called a heptamer-that ensures EF and LF are correctly positioned to get inside the cell. It also signals the src-like kinases to pull the heptamer inside, buried inside a cellular bubble called an endosome. Once EF and LF are safely in the cell, they burst into the cytoplasm and cut important cellular proteins.
This effort to assemble the heptamer explains why anthrax toxin seems to hesitate on the cell’s surface before invading, van der Goot says. If PA enters the cell too early-before the assembly is complete-PA opens up the cell but EF and LF are left outside and the toxin won’t work, she says.
Van der Goot is especially intrigued by the anthrax toxin’s use of the host’s cell receptor CMG2. Mutations in CMG2 have also been implicated in systemic hyalinosis, a rare genetic disorder that manifests itself in newborns as severe joint problems, recurrent intestinal illnesses, and pulmonary infections. The CMG2 receptor may be forced into service as an anthrax toxin receptor, van der Goot says, “but it’s not its job to be the anthrax receptor. It’s there to do something else.” This study is the first to reveal a connection between hijacked CMG2 receptor and src-like kinases-more commonly involved with responding to epithelial growth factors-and may help scientists figure out the cause of systemic hyalinosis.
This research is just the beginning of understanding how the anthrax toxin works. “It’s likely much more complex,” van der Goot says. Anthrax has a long and resilient evolutionary history, she says, and it is likely to employ more than one signaling pathway to enter host cells. Her lab is pursuing other pathways that might explain how the ingested toxin moves inside the cell. They are also investigating how the cytoskeleton is co-opted into moving the deadly freight into the cell.
Van der Goot says it is unlikely that drugs that block src-like kinases will be a treatment for anthrax. Src-like kinase blockers exist but they might be unsafe for humans, she explains. They are used primarily by labs studying tumors where mutant src genes have long been identified as cancer-associated genes. “Still this is the first evidence that src-like kinases are important in anthrax,” she says.
About the Research Scholar…………………..
F. Gisou van der Goot, Ph.D.
Dr. van der Goot received her Ph.D. in 1990 from the University of Paris for her study of water channels. She went did postdoctoral work at the European Molecular Biology Laboratory in Heidelberg. In 1993, she joined the University of Geneva, where, in 2001, she was named associate professor; the same year, she received an EMBO Young Investigator Award. In 2006, she accepted her current position as professor at the Global Health Institute of the École Polytechnique Fédérale de Lausanne.
RESEARCH ABSTRACT SUMMARY:
Gisou van der Goot wants to understand the mechanisms by which anthrax toxin manages to delay the onset of normal immune responses. She uses a variety of cell biological, morphological and biochemical techniques, including an RNAi screen, to analyze the molecular mechanisms that govern the delivery and presentation of the toxin and its enzymes in the cell.
Examiner.com, January 5, 2010, Dick Pelletier — The Target Health Global Blog has long believed what computer scientists are now talking about. Computer scientists compare the Internet to Earth growing a global brain. As users, we represent the neurons; our emails, IMs, and blogs act as synaptic actions; and electromagnetic waves through the sky become neural pathways. Like germinating seeds, this wonder-tech continues to evolve and as many predict, will not stop until it achieves human-like consciousness.
Forward thinkers believe that sentience can emerge from information systems like the Internet. Belgium artificial intelligence expert, Francis Heylighen believes that the worldwide web empowers itself from billions of human interactions it observes daily, and one day, feelings and self-awareness will rise from this worldwide digital creation.
Feelings are a lower level of consciousness in a system’s environment. In that sense, the global brain is just beginning to understand events that affect its goals. Higher consciousness and self-awareness levels would enable the Internet to reflect on its own functioning. Although today’s algorithms make the web more intelligent, it still cannot monitor itself; but quantum computing and artificial intelligence advances expected in the 2020s, will greatly enhance our global brain.
Could tomorrow’s global brain connect directly to human minds? At present, information exchanged between humans and computers only occur with mouse, keyboard or voice. However, futurists believe that one day technology will enable us to interface wirelessly with machines (and each other) in a “thought-talking” mode. Imagine commanding machines with our thoughts and communicating mind-to-mind with each other.
Nearly 25% of humanity is now connected to this worldwide wonder, according to the forthcoming 2009 State of the Future report, which will be available mid-August at www.stateofthefuture.org. Of the 4 billion mobile phones in use around the world, most are “increasingly becoming personal electronic companions, combining the functions of a Web-connected computer, telephone, camera, music player, TV, and library.”
But today’s Internet needs help, experts say, so President Obama has allocated $7.2 billion in stimulus money to enhance America’s broadband capabilities. A faster Internet will allow a richer surfing experience, more lifelike teleconferencing, and outsourcing of more services to the Web, this according to a recent report from the Information Technology & Innovation Foundation.
Web applications are sprouting up everywhere. Carnegie Mellon professors teach classes digitally to satellite campuses around the world, MIT instructors upload lectures to YouTube, and The Teaching Company is researching ways for making telelectures profitable for universities.
Videoconferencing allows doctors to monitor patient health around the clock. The Renaissance Computing Institute in North Carolina has developed an Outpatient Health Monitoring System (OHMS) for asthma patients. OHMS uses multiple wireless sensors to monitor both patient’s condition and environmental factors.
Looking ahead, by 2020 or before, with video resolution four times sharper than today’s HDTV and haptic technologies providing realistic touch sensations, our wondernet will deliver holographic images of lifelike virtual interactions indiscernible from reality. We could organize meetings with friends or relatives from anywhere in the world with no travel involved. People will hug, kiss and reminisce as if everyone was actually together.
Finally, by mid-2030s, when artificial intelligence is expected to surpass human intelligence, positive futurists believe that this futuristic communication system will become fully conscious and self-aware as it guides humanity through this mind-boggling “magical future” time. Comments welcome.
In 1831 Erhard Friedrich Leuchs (1800-1837) described the diastatic action of salivary ptyalin (amylase) on starch. The modern history of enzymes began in 1833 when French chemists described the isolation of an amylase complex from germinating barley and named it diastase. In 1862 Danielewski separated pancreatic amylase from trypsin
An amylase is an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into di- and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. As diastase, amylase was the first enzyme to be discovered and isolated (by Anselme Payen in 1833). Specific amylase proteins are designated by different Greek letters. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. It will start to denature at around 60C.
Enzyme Basics 101
Science and History
Enzymes are proteins that are catalysts. This means they speed up chemical reactions in living organisms, but they aren’t consumed in those reactions. Here are some important things you should know about enzymes:
- Enzymes are effective in minute amounts (often too small to be detected by ordinary chemical tests) because they are not used up in the reaction that they catalyze.
- Enzymes are specific to the reactions they catalyze, that is, each enzyme only catalyzes one specific chemical reaction.
- Enzymes do not affect the direction of the reaction but make the reaction reach equilibrium sooner.
The living cell is a “container” for a multitude of biochemical activities that are lumped under the word metabolism. Both synthesis and decomposition of molecules in a living system would normally proceed too slowly to be useful to metabolic survival. Hence, the presence and activity of enzymes are vital to the life-supporting activities of cellular metabolism. Enzymes make up a substantial portion of the total protein of the cell. A typical cell contains about 3000 different kinds of enzyme molecules and many copies of each kind. Some of these biochemical activities are the basis of cell reproduction, conversion of compounds for cellular energy, and the synthesis of specific compounds that can be used within the cell or are secreted for extracellular reactions. Within a cell, chemical reactions take place within a narrow temperature and pH range. This is possible because enzymes generally lower the activation energy of a reaction through a variety of mechanisms. Many advances in controlling bacterial diseases come from an understanding of how certain enzymes within the bacterial cell operate. Interfering with these enzymes interferes with vital metabolic functions within the bacterium. Conversely, changing the genetics of a bacterium alters the production of various secreted substances, some of which turn out to be useful antibiotics. Certain vitamins, some of which are produced by bacteria (as in our intestine), are proteins that work in conjunction with other enzymes. The vitamins are then called coenzymes.
Some of the earliest studies on inorganic catalysts were done by the Swedish chemist Jöns Jakob Berzelius in 1835. Even though the catalytic activity of enzymes as found in yeast (the word enzyme is ancient Greek for “in yeast”) had been used for centuries, it was not until 1926 that the first enzyme was obtained in pure form (crystalline). This was done by James B. Sumner of Cornell University. He later (1946) received the Nobel Prize for his work with the enzyme urease, extracted from the jack bean. Urease is an enzyme that catalyzes the conversion of urea to ammonia and carbon dioxide. Certain bacteria that convert urea to ammonia as part of the nitrogen cycle contain this enzyme.
Herders who made canteens from the stomachs of goats and sheep found that when they filled them with milk instead of water the milk soon clumped into cheese. The agent for this transformation is rennin, an enzyme produced in ruminant stomachs and unique to mammals. It is used for the decomposition of milk protein. These same stomachs contain both bacteria and protozoans that produce additional enzymes for the chemical decomposition of the cellulose found in the cell wall of plants such as grass that ruminants depend upon for a source of energy. The cellulose is first converted into its constituent sugars through enzyme activity; the sugars are then metabolized for energy.
Yogurt, an ancient food with modern popularity, is prepared through the action of enzymes produced by several bacteria, especially Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus acidophilus.