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In this section:
» Understanding Life Processes at the Molecular Level
» Basic Studies Illuminate Disease Mechanisms
» New Approaches to Therapeutics
» Promising Technologies
These science advances convey the breadth and significance of NIGMS-supported research. Although only the lead scientists are named, coworkers and collaborators contributed substantially to the achievements.
Understanding Life Processes at the Molecular Level
Discovering How Cell "Doors" Open and Close
Cells don't let just any substance slip through their protective external membranes. Most substances must first convince cells to open their doors to allow them in or out. Scientists have known for years that electrically charged particles, such as sodium and potassium ions, can pass across a cell's membrane through tunnel-like protein structures known as ion channels. Ion channels are critical to many biological processes, such as the beating of the heart, nerve impulses, digestion, and insulin release. But until recently, the precise mechanisms by which cells open and close these doors to the outside world have largely remained a mystery.
In a continuation of his Nobel Prize-winning ion channel research, Roderick MacKinnon has recently determined the three-dimensional structure of a "voltage-dependent" potassium channel. His findings have challenged the previously accepted view of how these channels sense voltage changes across the cell membrane. The segment of the channel that acts as the sensor turns out to extend into the membrane itself rather than being tucked in on the inner surface of the channel, as had been believed. The sensor is pulled from one side of the membrane to the other when the voltage changes. In turn—through a process not yet understood—the sensor pulls on other parts of the channel, causing it either to open or close. The sensor thus responds to voltage changes much like the button of an automatic door responds to pressure: It senses the stimulus and brings about a response.
Nigel Unwin, Ph.D., a cell biologist at The Scripps Research Institute in La Jolla, California, has been studying a different type of ion channel: one that responds to a chemical, rather than an electrical, stimulus. He has found that this channel, called the acetylcholine receptor, has door-like segments in its middle that, when closed, block the flow of ions. The doors are attached to the rest of the channel through hinge-like regions around which they can rotate. When the chemical messenger binds to the channel, the doors open, allowing ions to pass through.
A deeper understanding of the workings of ion channels may allow scientists to develop new drugs for conditions ranging from heart disease to diabetes.
Clues to Why Old Eggs Fail
Older women are more likely to give birth to children with Down syndrome, a form of mental retardation. The condition is caused by the presence of three copies, rather than the standard two copies, of chromosome 21 in human cells. This problem in chromosome distribution, called meiotic nondisjunction, occurs in the course of the cell division that gives rise to eggs (as well as to sperm). But no one knows how or why meiotic nondisjunction increases as eggs age. The question has been difficult to answer because scientists were unable to develop an animal model in which to study the condition.
Now, Sharon Bickel, Ph.D., a molecular biologist at Dartmouth College in Hanover, New Hampshire, has developed a method that uses fruit flies to gain insight into this puzzle of human biology. Fruit flies continuously produce eggs, but Bickel manipulated the diet of the flies in a way that suspended the maturation of their eggs, allowing them to "age." This mimicked the aging of human eggs.
Studying the "aged" fruit fly eggs, Bickel found that the incidence of meiotic nondisjunction jumped just as it does in older women. Her work also indicated that a back-up genetic system that normally helps to ensure proper chromosome separation and distribution deteriorates as fruit fly eggs age. No one yet knows if the same back-up system exists in humans, or if identical mistakes account for the increased risk of Down syndrome in the children of older mothers. But the fruit fly model system will allow Bickel and others to investigate these important questions.
RNA Changes Guide the Nervous System
The four chemical building blocks of DNA connect in various orders, or sequences, to form genes. Our genes carry the instructions for making proteins, which perform thousands of different tasks in our bodies. In reading a particular gene's instructions and making the protein it specifies, a cell creates an intermediate molecule called messenger RNA (mRNA). This molecule's sequence usually exactly reflects the gene's sequence. But in certain cases, cellular enzymes can "retype" a portion of an mRNA's sequence. This "edited" mRNA template causes the cell to produce a completely different protein than the original gene specified. The new protein often has a different function.
Geneticist Robert Reenan, Ph.D., of the University of Connecticut Health Center in Farmington has developed a new approach to search for edited genes. In doing so, he discovered that the process is not as rare as was once believed. Reenan compared the sequences of an edited gene in 18 species of experimental fruit flies. He noticed a preserved pattern, or signature, in all of the versions of the gene. Reenan suspects that the signature translates into a common RNA shape that is easily recognized by RNA editing machinery in the cell. Looking for other fly genes that may contain the editing signature, he evaluated the spellings of more than 900 genes in just two fruit fly species and discovered 16 additional genes that undergo editing. He found that all of the newly discovered editable genes spell out proteins used for super-quick electrical or chemical transmission by the fruit fly nervous system.
Reenan's discovery may help explain how the nervous system can adapt rapidly to changes in our surroundings by permitting cells to make quick edits to gene copies (in the form of mRNA) rather than to the original DNA. A more thorough interpretation of the language scripted in genes central to nervous system function may help researchers find new ways to diagnose and treat a variety of neurological diseases known to have a genetic component, such as Parkinson's disease, Alzheimer's disease, epilepsy, and many others.
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Basic Studies Illuminate Disease Mechanisms
Cell Death Research Uncloaks Deadly Virus Action
Death is vital to life, especially when your body needs to get rid of worn-out or unneeded cells. This essential housekeeping job is performed by an elaborate biochemical process called apoptosis, or programmed cell death. However, under certain conditions, apoptosis can go awry, leading to life-threatening illnesses.
Scientists studying fruit flies have discovered various proteins involved in apoptosis, including the aptly named Grim and Reaper proteins. To find out what proteins with similar functions exist in other species, pharmacologist Sally Kornbluth, Ph.D., of Duke University Medical Center in Durham, North Carolina, searched through databases cataloging the DNA sequences of proteins. What she found was a protein resembling Reaper in a family of infectious agents called Bunyaviruses.
It was an important finding, since Bunyaviruses are the major cause of two potentially fatal insect-transmitted diseases in humans: hemorrhagic fever and pediatric encephalitis. Like Reaper, the Bunyavirus protein dubbed appears to deal its deadly blow in part by promoting apoptosis. Studies in mice by Kornbluth's team show that causes massive death of brain cells—an observation that could help explain the severe brain inflammation in people with hemorrhagic fever and pediatric encephalitis.
Interestingly, the discovery of these potent proteins could also shed light on why viral encephalitis is so serious in infants. Based on her studies, Kornbluth suggests that Bunyaviruses may hijack the normal processes of cell death that are particularly active during the early wiring of young nervous systems. Further research is now under way in her lab to figure out the precise mechanism by which proteins act. By understanding these mechanisms, scientists hope they can one day develop better ways to treat viral encephalitis and other dangerous diseases in which these proteins may be involved.
Inherited Susceptibility to Meningococcal Disease
Meningococcal disease is a potentially fatal illness caused by the bacterium Neisseria meningitides. This bacterium is commonly found in the noses and throats of healthy people, but occasionally it invades the fluid surrounding the brain and spinal cord and causes meningitis. Worse still, the bacterium may seep into the bloodstream, causing meningococcal sepsis, an overwhelming, generalized blood infection. Neither scenario is good news: 2 percent of those who develop meningitis die from the infection, while the fatality rate for those who come down meningococcal sepsis is 12 percent. Roughly 2,400 cases of meningococcal disease occur in the United States annually, and most of its victims are infants or young adults1. Given that Neisseria meningitides is harmless to most of us, scientists have long suspected that a person's genes play a role in determining whether or not meningococcal disease will develop.
Bruce Beutler, M.D., an immunologist at The Scripps Research Institute, has been studying a family of genes that is involved in "innate" immunity, the body's initial, broad defense response to invading microbes. These genes, called the Toll-like receptors (TLRs), were discovered in fruit flies, and it was in this insect model system that were first found to be involved in providing immunity.
One TLR Beutler has taken a particular interest in, toll-like receptor 4 (TLR4), binds to a molecule on the exterior surface of Neisseria meningitides bacteria. TLR4 sounds an early alarm when the bacteria breach our barriers, summoning immune cells to destroy the invaders. A defect in TLR4 could potentially allow the bacteria to get a toe-hold and lead to an overwhelming infection.
By comparing the DNA sequences of 220 people who had meningococcal disease with the sequences of 283 healthy control subjects, and using special software he designed, Beutler identified a number of rare variations in TLR4 that correlated with susceptibility to the disease. His results suggest that these variant sequences predispose a person to meningococcal disease by impairing the ability to respond in the first minutes or hours of a bacterial invasion.
In addition to providing evidence for a genetic basis to meningitis susceptibility, Beutler findings pave the way for the development of diagnostic tests for those at risk. In the future, preventive treatment could be given to those with risk-increasing TLR4 mutations prior to surgery or travel to areas where meningococcal disease epidemics are in progress. In addition, the approach that Beutler has pioneered may be applied to the study of other diseases in which both genes and environment play a role.
Cell Stress May Contribute to Neurological Diseases
People respond to stress or heat by sweating; cells respond by activating heat shock proteins that help repair or clear damaged proteins. Research by cell biologist William Welch, Ph.D., of the University of California, San Francisco, has illuminated an important connection between heat shock proteins and nine devastating neurological diseases, including Huntington's disease.
Scientists know that all of the diseases are associated with mutant proteins that form clumps in nerve cells, leading to the cells' death. Welch used mouse cells to study one of these diseases, spinobulbar muscular atrophy (SBMA). In SBMA, clumping of the mutated proteins activates the cell's stress response system, generating heat shock proteins that would normally rid the cell of the malformed proteins. Welch found that the protein clumps grab the heat shock proteins before they can do their work, permanently activating the stress response system. This generates still more heat shock proteins, which are in turn pulled out of commission by the clumps.
In addition, Welch discovered that cells damaged in this way are extremely sensitive to various kinds of stress, including heat and different toxins. His experiments showed that only half of the cells with the clumped proteins survived a temperature increase that normal cells could tolerate. Moreover, the abnormal cells that did survive experienced further clumping. These results suggest that physiological or environmental stress may play a role in initiating and/or accelerating this group of neurological diseases. Welch is now using laboratory mice to test whether stressed animals do indeed develop more quickly than do non-stressed animals.
1 Rosenstein NE, Perkins BA, Stephens DS, et al., The Changing Epidemiology of Meningococcal Disease in the United States, 1992-1996. J Infect Dis 180:1894-1901, 1999.
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New Approaches to Therapeutics
Gene Silencing Studies Yield Potential New Class of Anticancer Drug
Everything from the genes you inherit to what you eat, the air you breathe, and how much you exercise influences the development of cancer. But the disease ultimately results from chemical changes to DNA that spur cells to grow and divide uncontrollably. One of these changes is called DNA methylation, which in normal cells "silences," or shuts off, genes that are not needed in particular cell types or under certain conditions.
In some cancer cells, however, abnormal DNA methylation shuts off tumor suppressor genes, which normally put the brakes on cell growth and division. Since turning these genes off opens the door to uncontrolled cell growth and cancer, scientists have long hoped that chemicals that inhibit DNA methylation would one day prove useful as anticancer drugs. But the toxicity and instability of these chemicals have dogged drug-development efforts.
Now, thanks to a study that began with a commonly studied laboratory fungus, a new DNA methylation inhibitor is under investigation as a potential anticancer drug. Molecular biologist Eric Selker, Ph.D., of the University of Oregon, Eugene, initially discovered that a molecule called zebularine reverses DNA methylation in the fungus. He went on to show that zebularine also reverses DNA methylation and reactivates a tumor suppressor gene in human bladder cancer cells grown in the laboratory. Selker then injected these cancer cells into mice and gave them zebularine orally. The molecule reduced tumor size in the mice, making zebularine the first methylation inhibitor to have such an effect in animals.
Zebularine is unique among the DNA methylation inhibitors that have been studied to date because it is chemically stable, can be administered orally, and appears to be minimally toxic. Its only side effect seems to be a slight weight loss in the mice given the chemical. These favorable properties suggest that zebularine may be a good candidate for drug development and that it could be a prototype for a new class of anticancer drugs. Selker's university has filed a patent on the potential uses of zebularine and is currently working to develop it into a marketable drug.
Copper Transporter Is Key to Cisplatin Drug Resistance
Nearly half a century ago, a stroke of luck led scientists to discover the cancer drug cisplatin. Researchers investigating the effect of electrical fields on bacterial growth discovered that a metal-containing chemical solution used to conduct current, not the electrical fields themselves, stopped the bacteria from dividing. Further tests proved that cisplatin , a platinum-containing substance produced when the metal electrodes reacted with the chemical solution, could also halt the growth of cancer cells. Today, cisplatin is an important component of the treatment for many advanced testicular and ovarian cancers. However, tumor cells can "learn" how to reject cisplatin and other chemotherapy drugs, allowing the cells to multiply and spread. Doctors cannot simply give more cisplatin to remedy this drug resistance problem because of the risk of serious ear and kidney toxicities caused by higher doses of the drug.
Basic researchers studying the role of metals in biology have made another surprise discovery about cisplatin. An interdisciplinary team of scientists found what is likely to be a main cause cisplatin resistance, and it has to do with how the body handles another metal: copper. Biochemist Dennis Thiele, Ph.D., of the University of Michigan Medical School in Ann Arbor and the late geneticist Ira Herskowitz, Ph.D., of the University of California, San Francisco, joined forces to discover that the Ctr1 protein, which transports metals into cells, takes in not only copper, but also cisplatin. The researchers found that yeast cells that lacked the metal transporter protein were highly resistant to cisplatin, suggesting that the cells had no way to absorb the drug. The scientists next created experimental cell lines using mice that lack the equivalent copper-intake protein. Mouse cells that did not contain any Ctr1 protein were eight times more resistant to cisplatin than their normal counterparts.
Since human Ctr1 is 92 percent identical to mouse Ctr1, it is highly likely that the metal transport protein works the same way in mice as it does in humans. The researchers reasoned that a defective or missing copy of the gene that codes for Ctr1 in some tumor cells may explain why certain people stop responding to cisplatin. The findings have important medical implications: If researchers can figure out a way to increase the amount or activity of Ctr1 in tumor cells, they may be able to extend the use of this effective chemotherapy drug. Future pharmacogenetic studies, in which scientists search for connections between genes and drug response, may help identify who will respond well or poorly to cisplatin treatment.
Botulinum Toxin Study May Lead to Inhaled Vaccine
Botulinum toxin (BT) is the single most poisonous substance known, with very small amounts causing paralysis and death2. Botulism, the illness caused by this bacterially produced toxin, typically results from eating contaminated food. Cases of botulism are rare, but concerns about the possible use of BT as a bioterrorism agent have brought a new urgency to research in this area. Of special interest is the effect of inhaling the toxin.
Biochemist Lance Simpson, Ph.D., of Jefferson Medical College in Philadelphia concluded that inhaling BT can cause poisoning when the toxin travels from the airways to the bloodstream, where it does widespread damage to the body. He also discovered that a piece of the BT protein called the heavy chain was an effective inhaled vaccine in experimental mice. Simpson vaccinated the mice with the BT heavy chain and then injected them with a dose of BT estimated to be the same as if the animals had inhaled large amounts of the toxin. The BT heavy chain protein did not appear to harm the mice, and it stimulated their immune systems to produce protective antibodies against the toxin.
Simpson's work has immediate clinical relevance in suggesting ways to manufacture a human vaccine against this potential bioterror weapon. Even better is that the vaccine might be able to be administered by inhalation, not injection. While an antitoxin to neutralize BT circulating in the bloodstream is available, quantities of this remedy are insufficient to rapidly treat large numbers of people. More importantly, an antitoxin cannot enter poisoned nerve cells, limiting the usefulness of such a strategy. A safe and effective inhalation vaccine could circumvent these problems.
2 Fact sheet on botulinum toxin produced by the Center for Biosecurity at the University of Pittsburgh Medical Center (2003)
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Promising Technologies
Making Microcircuits with Yeast Proteins
Sometimes serendipity, not necessity, can be the mother of invention. Case in point: the chance discovery of a new way to fabricate tiny electrical circuits from yeast proteins.
Susan Lindquist, Ph.D., a molecular biologist at the Whitehead Institute for Biomedical Research, is a leader in the study of prions. These misfolded forms of normal cellular proteins can cause serious illnesses, such as mad cow disease, in humans and animals. While studying prions in common brewer's yeast, she unexpectedly found one particular molecule called NM that can assemble itself into very thin fibers with surprising precision.
The prion fibers, which are quite similar to those found in the tangles within brains of people with Alzheimer's s disease, turned out to have some rather unusual—and potentially quite useful—properties. The fibers are exceptionally durable and have very specific sizes, nanometers wide and up to several hundred micrometers long. The physical properties of the fibers suggest that they might be used to create extremely small "biotemplated" devices such as electric circuits.
Teaming with materials scientists, Lindquist and her collaborators then decided to see if they could genetically modify NM proteins to bind tiny particles of gold. The resulting gold-coated fibers formed stable electrical wires with ideal characteristics: very high conductivity and low resistance. In addition, the team found that NM proteins can be fused with other proteins, creating hybrid molecules that potentially could form complex circuits with special chemical and biological functions, such as those of enzymes. Lindquist speculates that this technology may lead to a wide variety of nanodevices with applications in both the electronics and medical industries.
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Designing Protein Sensors
Unlike Sumo wrestlers—who can bind their opponents tightly using a number of moves and positions—proteins rely on the precise shape of their binding sites to attach to target molecules. Only through such specificity, for instance, can an antibody capture a virus. The ability of proteins to bind other molecules underlies innumerable life processes and medical treatments. For example, protein binding allows cell-to-cell communication, gives hormones their punch, and delivers chemotherapy to cancer cells.
Scientists study protein binding with the goal of finding ways to control it. An important step in this direction comes from computational biologist Homme Hellinga, Ph.D., of the Duke University Medical Center. His approach involves designing proteins that bind to new targets, which has potential applications across vast areas of medical science, toxic waste clean-up, and drug development.
Hellinga started with a known bacterial protein that binds to nutrients. He chose to modify this protein to bind molecules it would never encounter in nature, including the explosive TNT and the brain chemical serotonin. Hellinga used a cluster of 20 linked computers to explore all of the ways atoms could be arranged in the protein's binding site—an astounding 1076 possibilities (that's more than a quadrillion multiplied by itself five times). With a sophisticated computer algorithm, he pared down these virtually countless possibilities to 17 promising arrangements that could be tested directly. Hellinga then constructed these 17 altered proteins in the laboratory. To determine the ability of the synthetic proteins to attach to their new targets, he engineered the proteins to glow when binding took place. The experiments lit up the lab. When inserted back into living bacteria, the designer proteins continued to carry out their new functions, taking the research closer to real-world applications.
The TNT-grabbing protein could serve as a biosensor to detect land mines or undetonated underwater explosives. Similar redesigned proteins could sniff out pollutants or chemical warfare agents. In addition, the ability to bind serotonin suggests possible diagnostic uses, since conditions such as depression and anxiety cause fluctuations in serotonin levels in the brain. The newly designed proteins could prove a boon to the pharmaceutical industry as well, since they can differentiate between mirror-image forms of molecular compounds. One form may be biologically active, while the other is inactive or even harmful. The ability to distinguish one from the other could lead to safer drugs in less time and at lower cost.
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