Healthy News for Healthy Nation

A multidisciplinary team – graduate student Abhijit Mishra, left; materials science and engineering professor Gerard Wong; and postdoctoral researcher Vernita Gordon – has solved the mystery of how stealthy HIV protein gets into cells. They are standing next to a small angle X-ray spectrometer. Their findings could improve the design of therapeutic agents that cross a variety of membrane types. (Credit: Photo by L. Brian Stauffer)

Scientists have known for more than a decade that a protein associated with the HIV virus is good at crossing cell membranes, but they didn’t know how it worked. A multidisciplinary team from the University of Illinois has solved the mystery, and their findings could improve the design of therapeutic agents that cross a variety of membrane types.

The TAT protein transduction domain of the HIV virus has some remarkable properties. First, it is a tiny part of the overall TAT protein, containing only 11 amino acids. Second, and more important, it has an uncanny knack for slipping across membranes, those lipid-rich bags that form the boundaries of cells and cellular components and are designed to keep things out.

“TAT is extremely good at getting through cell membranes,” said materials science and engineering professor Gerard Wong, who led the new study. “You can attach TAT to almost anything and it will drag it across the membrane. It can work for virtually all tissues, including the brain.”

The TAT protein’s versatility makes it desirable as a drug-delivery device. It is already being used for gene therapy. (TAT is not involved in transmitting the HIV virus; it only aids the passage of other materials across the membranes of infected cells.)

Because it has so many potential uses, scientists have long endeavored to understand the mechanism that allows the TAT protein to work. But their efforts have been stymied by some baffling observations.

Six of its 11 residues are arginine, a positively charged amino acid that gives the protein its activity.

Most membranes are composed of a double layer of neutral, water-repellent lipids on their interiors, with hydrophilic (water-loving) “head groups” on their internal and external surfaces.  The head groups generally carry a mildly negative charge, Wong said. Since opposites attract, it made sense to the researchers that the positively charged TAT protein would attract the negatively charged head groups on the surface of the membranes. This attraction could deform the membrane in a way that opened up a pathway through it.

If a short, positively charged protein was all that was needed for TAT to work, the researchers thought, then any positively charged amino acid should do the trick. But when they replaced the arginine in the protein with other positively charged amino acids, it lost its function. Clearly, a positive charge was not enough to make it work.

To get a better picture of the interaction of TAT with a variety of membranes, the researchers turned to confocal microscopy and synchrotron x-ray scattering (SAXS). Although sometimes used in biological studies, SAXS is more common to the fields of physics or materials science, where the pattern of X-ray scattering can reveal how atomic and nano scale materials are structured.

The researchers found that adding the TAT protein to a membrane completely altered its SAXS spectrum, a sign that the membrane conformation had changed. After analyzing the spectrum, the researchers found that TAT had made the membranes porous.

“The TAT sequence has completely reconstructed (the membrane) and made it into something that looks a little bit like a sponge with lots of holes in it,” Wong said.

Something about the TAT protein had induced a “saddle splay curvature” in the membrane. This shape resembles a saddle (like that of a Pringles potato chip), giving the openings, or pores, a bi-directional arc like that seen inside a doughnut hole.

The newly formed pores in the membrane were 6 nanometers wide, large enough to allow fairly sizeable proteins or other molecules to slip through. The pores would also make it easier for other biological processes to bring materials through the membrane.

Further analysis showed that the arginine was interacting with the head groups on the membrane lipids in a way that caused the membrane to buckle in two different directions, bringing on the saddle splay curvature that allowed the pores to form.

When another positively charged amino acid, lysine, was used instead of arginine, the protein bent the membrane in one direction only, forming a shape more like a closed cylinder that would not allow materials to pass through.

These findings will aid researchers hoping to enhance the properties of the TAT protein that make it a good vehicle for transporting therapeutic molecules into cells, Wong said.

A paper describing their findings appears this month in Angewandte Chemie.

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Dr. Curtis Nickel. (Photo courtesy of Queen’s University)

Kingston, ON – Queen’s University has been selected as the only Canadian site to test a new antidepressant drug approved by the FDA (U.S. Food and Drug Administration) for its potential to alleviate pain in two common bladder conditions that have no known cause and no effective therapy.
Funded by the U.S. National Institutes of Health (NIH), the research will be carried out at 10 medical centres in the United States and Canada. Dr. Curtis Nickel, professor of Urology at Queen’s and urologist at Kingston General Hospital, heads the Canadian study.

The researchers are recruiting adults newly diagnosed with either painful bladder syndrome (PBS) or interstitial cystitis (IC), to learn if the oral drug amitriptyline will reduce the pain and frequent urination associated with these conditions. An estimated 10 million people worldwide suffer from PBS and IC.

Although amitriptyline is primarily used for depression, the way it works makes it useful for treating the pain of fibromyalgia, multiple sclerosis, and other chronic pain syndromes, Dr. Nickel explains. “Prior small studies have suggested the drug may be a wise choice for this syndrome as well, because it blocks nerve signals that trigger pain and may also decrease muscle spasms in the bladder, helping to cut both pain and frequent urination.”

The researchers believe that 25 to 75 milligrams of amitriptyline a day may begin relieving IC pain within a week. In contrast, doses in the range of 150 to 300 milligrams are generally used to treat depression.

The 10 centres where testing will take place make up the Interstitial Cystitis Clinical Research Network, sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the NIH, which coordinates all government health care research in the United States.

In 2003, Dr. Nickel and his Kingston Genito-urinary Research Group were awarded an unprecedented four research grants from the NIH, totaling almost $8 million. These studies are now examining alternative, complementary and novel therapies for prostate and bladder disease, in both laboratory research and clinical trials involving more than 3,000 men and women throughout southern Ontario.

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Fibromyalgia is a chronic, widespread pain in muscles and soft tissues accompanied by fatigue. (Credit: iStockphoto/Ken Hurst)

Fibromyalgia, a chronic, widespread pain in muscles and soft tissues accompanied by fatigue, is a fairly common condition that does not manifest any structural damage in an organ. Twenty-five years ago, Muhammad B. Yunus, MD, and colleagues published the first controlled study of the clinical characteristics of fibromyalgia syndrome.
That seminal article, published in Seminars in Arthritis and Rheumatism, led directly to formal recognition of this disease by the medical community. In the June 2007 issue of Seminars in Arthritis and Rheumatism, Dr. Yunus once again makes an important contribution to the field of chronic pain and fatigue by meticulously synthesizing and interpreting the extensive body of scientific literature on fibromyalgia and his own insights into the concept of central sensitivity syndromes (CSS).

Fibromyalgia, affecting approximately 2% of the US population, is an example of a class of maladies called CSS. These diseases are based on neurochemical abnormalities and include irritable bowel syndrome, migraine and restless legs syndrome.

Incorporating a critical review of over 225 publications and the author’s broad experience in fibromyalgia and related diseases, Dr. Yunus describes 13 separate conditions that are related to central sensitization (CS), where the central nervous system (spinal cord and brain) becomes extremely sensitized on certain parts of the body, so that even mild pressure or touch would cause much pain. Such hypersensitivity may also be associated with other symptoms such as poor sleep and fatigue.

According to Dr. Yunus, “CSS are the most common diseases that are based on real neurochemical pathology and cause real pain and suffering. In some patients stress and depression may contribute to the symptoms but they are all based on objective changes in the central nervous system.”

Dr. Norman L. Gottlieb, Editor of Seminars in Arthritis and Rheumatism, believes that this article “advances our understanding of fibromyalgia, unifies and advances concepts, and suggests that this and several other common disorders have much in common in terms of their biopsychosocial development. This, hopefully, will expand both clinical and research interest in this group of diseases and lead to advances in therapy for many of them.”

In an accompanying editorial John B. Winfield, MD, comments, “Without question, Muhammad Yunus is the father of our modern view of fibromyalgia…. Yunus, who took a rather more biological approach to fibromyalgia in the past, now emphasizes a biopsychosocial perspective. In my view, this is tremendously important because it is the only way to synthesize the disparate contributions of such variables as genes and adverse childhood experiences, life stress and distress, posttraumatic stress disorder, mood disorders, self-efficacy for pain control, catastrophizing, coping style, and social support into the evolving picture of central nervous system dysfunction vis-a-vis chronic pain and fatigue ….Science and medicine now have a rational scaffolding for understanding and treating chronic pain syndromes previously considered to be ‘functional’ or ‘unexplained.’ …Neuroscience research will continue to reveal the mechanisms of CS, but only if informed through a biopsychosocial perspective and with the interdisciplinary collaboration of basic scientists, psychologists, sociologists, epidemiologists, and clinicians.”

Dr. Yunus concludes that CSS is an important new concept that embraces the biopsychosocial model of disease. He advocates further critical studies to fully test this concept which seems to have important significance for new directions for research and patient care involving physician and patient education. “Each patient, irrespective of diagnosis,” says Dr. Yunus, “should be treated as an individual, considering both the biological and psychosocial contributions to his or her symptoms and suffering.”

The article is “Fibromyalgia and Overlapping Disorders: The Unifying Concept of Central Sensitivity Syndromes” by Muhammad B. Yunus, MD, Professor of Medicine, Section of Rheumatology, The University of Illinois College of Medicine at Peoria, Peoria, Illinois. The accompanying editorial is “Fibromyalgia and Related Central Sensitivity Syndromes: Twenty-Five Years of Progress” by John B. Winfield, MD, University of North Carolina School of Medicine. Both appear in the June issue of Seminars in Arthritis and Rheumatism, Vol. 36:6, published by Elsevier.

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Fibromyalgia scan. Fibromyalgia patients had reduced mu-opioid receptor (MOR) availability within regions of the brain that normally process and dampen pain signals – specifically, the nucleus accumbens, the anterior cingulate and the amygdala. (Credit: Image courtesy of University of Michigan Health System)

People who have the common chronic pain condition fibromyalgia often report that they don’t respond to the types of medication that relieve other people’s pain.
New research from the University of Michigan Health System helps to explain why that might be: Patients with fibromyalgia were found to have reduced binding ability of a type of receptor in the brain that is the target of opioid painkiller drugs such as morphine.

The study included positron emission tomography (PET) scans of the brains of patients with fibromyalgia, and of an equal number of sex- and age-matched people without the often-debilitating condition. Results showed that the fibromyalgia patients had reduced mu-opioid receptor (MOR) availability within regions of the brain that normally process and dampen pain signals — specifically, the nucleus accumbens, the anterior cingulate and the amygdala.

“The reduced availability of the receptor was associated with greater pain among people with fibromyalgia,” says lead author Richard E. Harris, Ph.D., research investigator in the Division of Rheumatology at the U-M Medical School’s Department of Internal Medicine and a researcher at the U-M Chronic Pain and Fatigue Research Center.

“These findings could explain why opioids are anecdotally thought to be ineffective in people with fibromyalgia,” he notes. The findings appear in The Journal of Neuroscience. “The finding is significant because it has been difficult to determine the causes of pain in patients with fibromyalgia, to the point that acceptance of the condition by medical practitioners has been slow.”

Opioid pain killers work by binding to opioid receptors in the brain and spinal cord. In addition to morphine, they include codeine, propoxyphene-containing medications such as Darvocet, hydrocodone-containing medications such as Vicodin, and oxycodone-containing medications such as Oxycontin.

The researchers theorize based on their findings that, with the lower availability of the MORs in three regions of the brains of people with fibromyalgia, such painkillers may not be able to bind as well to the receptors as they can in the brains of people without the condition.

Put more simply: When the painkillers cannot bind to the receptors, they cannot alleviate the patient’s pain as effectively, Harris says. The reduced availability of the receptors could result from a reduced number of opioid receptors, enhanced release of endogenous opioids (opioids, such as endorphins, that are produced naturally by the body), or both, Harris says.

The research team also found a possible link with depression. The PET scans showed that the fibromyalgia patients with more depressive symptoms had reductions of MOR binding potential in the amygdala, a region of the brain thought to modulate mood and the emotional dimension of pain.

The study subjects were 17 women with fibromyalgia and 17 women without the condition.

The senior author of the paper was Jon-Kar Zubieta, M.D., Ph.D., the Phil F. Jenkins Research Professor of Depression in the U-M Department of Psychiatry and a member of U-M’s Molecular and Behavioral Neuroscience Institute, Depression Center and Department of Radiology. Other authors were Daniel J. Clauw, M.D.; David J. Scott, Ph.D.; Samuel A. McLean, M.D., MPH; and Richard H. Gracely, Ph.D.

The research was supported by grants from the Department of the Army; the National Center for Research Resources, a component of the National Institutes of Health; and the NIH. Harris was supported by an NIH–National Center for Complementary and Alternative Medicine Grant. McLean was supported by an NIH grant.

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