Chemists Document Workings of Key Staph Enzyme -- And How to Block It

ScienceDaily (Jan. 26, 2011) — Researchers have determined the structure and mechanism of an enzyme that performs the crucial first step in the formation of cholesterol and a key virulence factor in staph bacteria.

Chemists led by Illinois professor Eric Oldfield, center, determined the structure of a key enzyme that could lead to more efficient drugs to treat staph infections, parasites and high cholesterol. The research team, from left, research scientist Yonghui Zhang, graduate student Fu-Yang Lin, research scientist Rong Cao and postdoctoral associate Ke Wang.

Chemists at the University of Illinois and collaborators in Taiwan studied a type of enzyme found in humans, plants, fungi, parasites, and many bacteria that begins the synthesis of triterpenes -- one of the most abundant and most ancient classes of molecules. Triterpenes are precursors to steroids such as cholesterol.

"These enzymes are important drug targets," said chemistry professor Eric Oldfield, who co-led the study. "Blocking their activity can lead to new cholesterol-lowering drugs, antibiotics that cure staph infections, and drugs that target the parasites that cause tropical maladies such as Chagas disease -- a leading cause of sudden death in Latin America."

For the study, the team picked a representative enzyme, dehydrosqualene synthase (CrtM), from the Staphylococcus aureus bacterium. Staph is one of the most common, yet notoriously hard to kill, bacterial infections. A key reason for staph's resilience is a golden-colored coating called staphyloxanthin that protects it from the body's immune system. CrtM catalyzes the first reaction in making staphyloxanthin, so inhibiting it would strip the bacteria of their protective coats and leave them vulnerable to attack by white blood cells.

The researchers already knew what CrtM looked like and its end product, but they didn't know how the enzyme did its job. Uncovering the mechanism of action would enable scientists to design better inhibitors, and even tailor them to other targets.

The team crystallized the enzyme and soaked it with intermediates and inhibitors. They then studied the complex structures by X-ray crystallography using the synchrotron at the Advanced Photon Source at Argonne National Laboratory.

They found that CrtM performs a two-step reaction, individually removing two diphosphate groups from the substrate. The substrate switches between two active sites within the enzyme as the reaction progresses. The most effective inhibitors bind to both sites, blocking the enzyme from any action.

"The leads that people have been developing for treating these diseases really haven't had any structural basis," said Oldfield, also a professor of biophysics. "But now that we can see how the protein works, we're in a much better position to design molecules that will be much more effective against staph infections and parasitic diseases, and potentially, in cholesterol-lowering."

The researchers' inhibitor technologies have been licensed to AuricX Pharmaceuticals, which recently received a grant from the Texas Emerging Technology Fund for preclinical testing in staph infections.

The team published its work in the Proceedings of the National Academy of Sciences. The work was sponsored by the National Institutes of Health and the National Science Council. Co-authors were U. of I. graduate students Fu-Yang Lin and Yi-Liang Liu, research scientists Rong Cao and Yonghui Zhang, and postdoctoral associate Ke Wang. Taiwan collaborators included Chia-I Liu, Wen-Yih Jeng, Tzu-Ping Ko and Andrew H. Wang.

New Map of Brain Connectivity Shows Changes During Development

ScienceDaily (Jan. 26, 2011) — Connected highways of nerve cells carry information to and from different areas of the brain and the rest of the nervous system. Scientists are trying to draw a complete atlas of these connections -- sometimes referred to as the "connectome" -- to gain a better understanding of how the brain functions in health and disease.

New research conducted at The Scripps Research Institute shows that this road atlas undergoes constant revisions as the brain of a young animal develops, with new routes forming and others dropping away in a matter of hours. "We have shown that the connectome is dynamic during development, but we expect it will also change according to an individual's experience and in response to disease," says Scripps Research Professor Hollis Cline, senior author of the study.

The study, published in the January 27, 2011 issue of Neuron, dispels some long-held notions of how connections between nerve cells are established, highlighting the interplay between formation and removal. The findings have implications for conditions in which these mechanisms may have gone awry, such as autism, schizophrenia, and perhaps Alzheimer's.

A Dynamic Map

Cline's group has been studying how experience -- the different sights and sounds and other environmental cues picked up by neurons -- change connections and activities in the brain through a process known as plasticity. "Based on our prior research we expected that the connectome would be dynamic," says Cline.

To start to document how the connectome changes and test current models of how the map is laid out, Cline and colleagues turned to the frog Xenopus laevis. They combined two new techniques to map in great detail all the connections that form during tadpole development in an area of the brain that receives and interprets signals from the eyes.

In the nervous system, information is handed from one nerve cell to another through two arms, called dendrites and axons, stretching out from opposite sides of each cell. The axon carries information away from a nerve cell, or neuron, and passes it to the dendrite of another; dendrites receive the information, which travels through the cell to the axon. The region where information is transferred from one neuron to another (and where axons and dendrites connect) is called the synapse.

The Cline group's approach relied on taking time-lapse images of growing axons and dendrites over several days. The researchers then zoomed in on synapses that formed using electron microscopy, a technique that magnifies objects up to 2 million times. This close-up view revealed some "surprising results about synapse formation and plasticity," says Cline.

Promiscuous Partners

Models of synapse formation typically show that as dendrites extend new branches, each branch forms an immature synapse with a target axon that is later either maintained or eliminated. But Cline's study shows instead the process is not as selective. Each growing dendrite samples not one but many possible partners before selecting one with which to maintain contact.

As new branches grow from dendrites, they form many immature synapses on axons. Then, as each new dendrite branch matures, most immature synapses are eliminated; the ones not eliminated mature into stable synapses. "We did not know that dendrites make so many connections that are then removed," says Cline. "It is always fun in science when you see that what was expected is not what actually happens."

Cline and colleagues also discovered that axon synapses don't form, as previously thought, on growing branches but rather at swellings, called boutons, located on stable axon branches.

Another surprise was that when growing dendrites go searching for potential partners, they reach out to axon boutons that had previously connected with other dendrites -- "as if they were attracted to a restaurant that already has a line at the door, rather than trying a brand new one," says Cline.

Over time, these boutons decrease the number of connected partners to form mature connections with single dendrites.

Shining the Spotlight on Elimination

Up until now, researchers had focused their work primarily on determining how new connections form and on finding ways to enhance such formation. But Cline's findings that so many immature connections are removed during development puts greater emphasis on the process of elimination, she says.

Several studies have shown that brain activity helps new connections to form. Thus, Cline asked whether activity is also required for synapse elimination to occur. To answer this question, Cline and colleagues shut off activity in the visual system by keeping some of the tadpoles in darkness. These animals had many more immature synapses than animals that could see, indicating brain activity is needed for selecting which synapses should be eliminated.

The team now plans to look in more detail at the molecular signals involved in the elimination process, hoping to identify any genes that might be responsible. "It is possible that some genetic diseases are caused by the inefficient elimination of synapses," explains Cline. For example, individuals with a disease known as fragile X, a leading cause of mental retardation, are thought to have too many synapses, suggesting elimination did not occur properly.

Potential 'Cure' for Type 1 Diabetes

ScienceDaily (Jan. 26, 2011) — Type 1 diabetes could be converted to an asymptomatic, non-insulin-dependent disorder by eliminating the actions of a specific hormone, new findings by UT Southwestern Medical Center researchers suggest.

These findings in mice show that insulin becomes completely superfluous and its absence does not cause diabetes or any other abnormality when the actions of glucagon are suppressed. Glucagon, a hormone produced by the pancreas, prevents low blood sugar levels in healthy individuals. It causes high blood sugar in people with type 1 diabetes.

"We've all been brought up to think insulin is the all-powerful hormone without which life is impossible, but that isn't the case," said Dr. Roger Unger, professor of internal medicine and senior author of the study appearing online and in the February issue of Diabetes. "If diabetes is defined as restoration of glucose homeostasis to normal, then this treatment can perhaps be considered very close to a 'cure.' "

Insulin treatment has been the gold standard for type 1 diabetes (insulin-dependent diabetes) in humans since its discovery in 1922. But even optimal regulation of type 1 diabetes with insulin alone cannot restore normal glucose tolerance. These new findings demonstrate that the elimination of glucagon action restores glucose tolerance to normal.

Normally, glucagon is released when the glucose, or sugar, level in the blood is low. In insulin deficiency, however, glucagon levels are inappropriately high and cause the liver to release excessive amounts of glucose into the bloodstream. This action is opposed by insulin, which directs the body's cells to remove sugar from the bloodstream.

Dr. Unger's laboratory research previously found that insulin's benefit resulted from its suppression of glucagon.

In type 1 diabetes, which affects about 1 million people in the U.S., the pancreatic islet cells that produce insulin are destroyed. As a countermeasure to this destruction, type 1 diabetics currently must take insulin multiple times a day to metabolize blood sugar, regulate blood-sugar levels and prevent diabetic coma. They also must adhere to strict dietary restrictions.

In this study, UT Southwestern scientists tested how mice genetically altered to lack working glucagon receptors responded to an oral glucose tolerance test. The test -- which can be used to diagnose diabetes, gestational diabetes and prediabetes -- measures the body's ability to metabolize, or clear, glucose from the bloodstream.

The researchers found that the mice with normal insulin production but without functioning glucagon receptors responded normally to the test. The mice also responded normally when their insulin-producing beta cells were destroyed. The mice had no insulin or glucagon action, but they did not develop diabetes.

"These findings suggest that if there is no glucagon, it doesn't matter if you don't have insulin," said Dr. Unger, who is also a physician at the Dallas VA Medical Center. "This does not mean insulin is unimportant. It is essential for normal growth and development from neonatal to adulthood. But in adulthood, at least with respect to glucose metabolism, the role of insulin is to control glucagon.

"And if you don't have glucagon, then you don't need insulin."

Dr. Young Lee, assistant professor of internal medicine at UT Southwestern and lead author of the study, said the next step is to determine the mechanism behind this result.

"Hopefully, these findings will someday help those with type 1 diabetes," Dr. Lee said. "If we can find a way to block the actions of glucagon in humans, then maybe we can minimize the need for insulin therapy."

Dr. Unger said anything that reduces the need for injected insulin is a positive.

"Matching the high insulin levels needed to reach glucagon cells with insulin injections is possible only with amounts that are excessive for other tissues," he said. "Peripherally injected insulin cannot accurately duplicate the normal process by which the body produces and distributes insulin. If these latest findings were to work in humans, injected insulin would no longer be necessary for people with type 1 diabetes."

Dr. May-Yun Wang, assistant professor of internal medicine at UT Southwestern, and researchers from the Albert Einstein College of Medicine also contributed to the work.

The study was supported in part by the VA North Texas Health Care System, the American Diabetes Association and the National Institutes of Health.

Discovery of a Biochemical Basis for Broccoli's Cancer-Fighting Ability

ScienceDaily (Jan. 26, 2011) — Scientists are reporting discovery of a potential biochemical basis for the apparent cancer-fighting ability of broccoli and its veggie cousins. They found for the first time that certain substances in the vegetables appear to target and block a defective gene associated with cancer.

Broccoli. Scientists are reporting discovery of a potential biochemical basis for the apparent cancer-fighting ability of broccoli and its veggie cousins. They found for the first time that certain substances in the vegetables appear to target and block a defective gene associated with cancer.

Their report, which could lead to new strategies for preventing and treating cancer, appears in ACS' Journal of Medicinal Chemistry.

Fung-Lung Chung and colleagues showed in previous experiments that substances called isothiocyanates (or ITCs) -- found in broccoli, cauliflower, watercress, and other cruciferous vegetables -- appear to stop the growth of cancer. But nobody knew exactly how these substances work, a key to developing improved strategies for fighting cancer in humans. The tumor suppressor gene p53 appears to play a key role in keeping cells healthy and preventing them from starting the abnormal growth that is a hallmark of cancer. When mutated, p53 does not offer that protection, and those mutations occur in half of all human cancers. ITCs might work by targeting this gene, the report suggests.

The scientists studied the effects of certain naturally-occurring ITCs on a variety of cancer cells, including lung, breast and colon cancer, with and without the defective tumor suppressor gene. They found that ITCs are capable of removing the defective p53 protein but apparently leave the normal one alone. Drugs based on natural or custom-engineered ITCs could improve the effectiveness of current cancer treatments or lead to new strategies for treating and preventing cancer.

The authors acknowledged funding from the Ruth L. Kirschstein National Research Service Award and a grant from the National Cancer Institute of the National Institutes of Health.

Food-Borne Bacteria Causes Potentially Fatal Heart Infection

ScienceDaily (Jan. 26, 2011) — Researchers at the University of Illinois at Chicago College of Medicine have found that particular strains of a food-borne bacteria are able to invade the heart, leading to serious and difficult-to-treat heart infections.

Listeria monocytogenes cardiac-invasive strain replicating and moving within infected heart cells. The bacteria (red rods) invade heart cells, multiply, and begin to move through the cell by rearranging cell structural proteins (green) that initially coat the bacteria (green and red rods) and then form long comet tails located directly behind moving bacteria.

The study is available online in theJournal of Medical Microbiology.

The bacteria Listeria monocytogenesis commonly found in soft cheeses and chilled ready-to-eat products. For healthy individuals, listeria infections are usually mild, but for susceptible individuals and the elderly, infection can result in serious illness, usually associated with the central nervous system, the placenta and the developing fetus.

About 10 percent of serious listeria infections involve a cardiac infection, according to Nancy Freitag, associate professor of microbiology and immunology and principle investigator on the study. These infections are difficult to treat, with more than one-third proving fatal, but have not been widely studied and are poorly understood.

Freitag and her colleagues obtained a strain of listeria that had been isolated from a patient with endocarditis, or infection of the heart.

"This looked to be an unusual strain, and the infection itself was unusual," she said. Usually with endocarditis there is bacterial growth on heart valves, but in this case the infection had invaded the cardiac muscle.

The researchers were interested in determining whether patient predisposition led to heart infection or whether something different about the strain caused it to target the heart.

They found that when they infected mice with either the cardiac isolate or a lab strain, they found 10 times as much bacteria in the hearts of mice infected with the cardiac strain. In the spleen and liver, organs that are commonly targeted by listeria, the levels of bacteria were equal in both groups of mice.

Further, the researchers found that while the lab-strain-infected group often had no heart infection at all, 90 percent of the mice infected with the cardiac strain had heart infections. The researchers obtained more strains of listeria, for a total of 10, and did the same experiment. They found that only one other strain also seemed to also target the heart.

"They infected the heart of more animals and were always infecting heart muscle and always in greater number," Freitag said. "Some strains seem to have this enhanced ability to target the heart for infection."

Freitag's team used molecular genetics and cardiac cell cultures to explore what was different about these two strains.

"These strains seem to have a better ability to invade cardiac cells," she said. The results suggest that these cardiac-associated strains display modified proteins on their surface that enable the bacteria to more easily enter cardiac cells, targeting the heart and leading to bacterial infection.

"Listeria is actually pretty common in foods," said Freitag. "And because it can grow at refrigerated temperatures, as foods are being produced with a longer and longer shelf life, listeria infection may become more common. In combination with an aging population that is more susceptible to serious infection, it's important that we learn all we can about these deadly infections."

The study was supported by a Public Health Service Grant; by Public Health Service post-doctoral training fellowships; and an American Heart Association Predoctoral Fellowship.