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Nitric Oxide May Hold Key To First Treatment For Deadly Form Of Malaria, Duke Scientists Report

Duke University Medical Center

DURHAM, N.C. -- In a discovery that defines a new message system inside living cells, a team of researchers led by Duke University Medical Center reports finding that the chemical nitric oxide is a universal signaling molecule that can control basic life processes down to the level of the gene, the master plan of a cell.

The study findings, published in the Sept. 6 issue of the journal Cell, describes an entirely new cellular signaling mechanism -- the attachment of nitric oxide (NO) to a key portion of a protein called a thiol -- that can directly activate genes. The work was funded by the National Heart, Lung and Blood Institute, a division of the National Institutes of Health.

The researchers theorize that the process of flagging proteins with NO to activate them may prove to be as fundamental a biological process as flagging proteins with phosphate, a process now understood to be the primary on-off switch for thousands of proteins that control life processes.

Although the discovery was made in bacteria, the team believes that human cells also use NO molecules as a genetic signaling mechanism.

"It appears that life uses NO ubiquitously," said the study's leader, Dr. Jonathan Stamler, a cardiologist and pulmonologist. "Knowing that NO compounds have the ability to turn genes on is a phenomenal understanding that may lead to a new chapter of research and possible therapeutic uses."

The finding ties together the mountains of scientific studies in recent years linking NO with everything from blood vessel dilation to neurotransmission in the brain to intestinal contraction to penile erection. "Virtually everywhere scientists look in the body, they find NO has a role," Stamler said. "This study shows why. NO influences myriad body functions because NO is a universal signal."

The body keeps NO, which is toxic in excess, in balance by sensing how much NO is present and turning genes on and off to keep NO in equilibrium. If this equilibrium is upset, Stamler theorizes, it may lead to a number of diseases that have recently been associated with NO, including cancer, stroke, hardening of heart arteries, infectious disease, and arthritis.

The scientists say that an immediate application from the research may be a new way to disarm invasive bacteria that have become resistant to antibiotics -- a major problem in the treatment of infections, especially in hospital settings.

Other contributors to the work include Alfred Hausladen, a Duke biochemist, and Christopher Privalle and Teresa Keng, two former Duke researchers who are now scientists at Apex Bioscience Inc., Research Triangle Park, N.C. Joseph DeAngelo, director of research at Apex, also collaborated on the project.

This is the second recent discovery Stamler has made in NO research. Earlier this year, he found that an NO compound, combined with hemoglobin, is a major regulator of gas exchange, as well as blood pressure, in the circulatory system. That work was published March 21 in the British journal Nature.

The current discovery was made when the researchers were studying why some bacteria escape attack by immune system cells called macrophages, which are designed to disable and devour them. The question is of increasing importance in acute health care.

When bacteria invade the body, the immune system floods the bacterial cell with noxious oxygen and nitrogen-free radicals.

The researchers found that when NO enters a bacterium, the NO attaches to a particular place on proteins called a thiol, a protein subunit that contains sulfur. The NO-thiol coupling forms a new NO-compound called SNO (for S-nitrosothiol). SNOs are souped-up cousins of NO molecules, and they have a wider range of functions.

To defend themselves, bacteria deploy an army of small thiol mops that sop up the SNO barrage and then break SNO down into harmless compounds.This is a bacterium's first line of defense.

If too much SNO gets in, it disables the bacteria by damaging key proteins the bacteria need to survive. While the bacteria are busy trying to repair themselves, the macrophages can attack and destroy them.

But the scientists discovered that the bacteria have an additional defense system. SNO compounds also attach to a transcription factor, a protein that finds specific genes on the bacterial genome and activates them. The genes, in turn, are translated into proteins whose job it is to break down the excess SNO, rendering it harmless.

In this way, bacteria have evolved an ingenious second line of defense against an SNO attack, Stamler said in an interview. "The bacteria offer SNO a target, which turns out to be a genetic switch that leads to its own destruction." he said. "The moment SNO enters a bacterium, a race is on as to whether it will defend itself quickly enough to evade the immune attack, or whether the SNO will win by quickly disabling the cell, making it vulnerable to a macrophage attack. In some cases, then, the bacterium wins, forming resistance to an immune system attack."

The findings offer both practical and profound implications, Stamler said.

The immediate benefit of the research is to suggest a way that new antibiotic drugs might be developed, said Hausladen, the study's first author. These drugs could plug up the transcription factor, known as OxyR, preventing it from being switched on, or they could bind to and deactivate the proteins that are produced to break down SNO. "This would be a novel way to disarm bacteria that has never been exploited," Hausladen said.

The actions of NO in this experiment also resemble the process by which oxygen affects cell health and disease, the scientists say.

"Now we have demonstrated that an NO group, attached to a thiol within a cell, has regulatory function," Stamler said. "There has been growing evidence that NO does its work in the body by signaling genes, but no one has found proof before this."

Both oxygen and NO are vital to life processes, but too much of either can damage cells. To keep the amount of oxygen and NO in balance, cells have built-in systems to eliminate the excess. One way to do that is to have transcription factor sensors that get turned on when too much oxygen or NO is present.

In fact, bacterial cells attempt to control both excess oxygen and excess nitrogen with the same OxyR transcription factor.

In human cells, constant vigilance against excess oxygen and NO takes a toll over time. When the system is out of balance, perhaps when a transcription factor is mutated, disease can result, the scientists say.

"It is a parallel process to what is known as oxidative stress, in which an excess of oxygen in cells can lead to a host of diseases, as well as the cumulative damage we call aging," Stamler said. "NO has a similar deleterious function, which we call nitrosative stress. When a cell can't contain the flow of SNO, the nitrosative stress can well be theorized to contribute to cancer, arthritis, neurodegenerative diseases, stroke and hardening of the arteries -- all diseases associated with NO."

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