Scientists Can Now Block Heroin, Morphine Addiction

In a major breakthrough, an international team of scientists has proven that addiction to morphine and heroin can be blocked, while at the same time increasing pain relief.

Photp: Jens Langner at http://commons.wikimedia.org/wiki/File:PET-image.jpg

Laboratory studies have shown that the drug (+)-naloxone (pronounced: PLUS nal-OX-own) will selectively block the immune-addiction response.The team from the University of Adelaide and University of Colorado has discovered the key mechanism in the body's immune system that amplifies addiction to opioid drugs.

The results -- which could eventually lead to new co-formulated drugs that assist patients with severe pain, as well as helping heroin users to kick the habit -- will be published August 16 in the Journal of Neuroscience.

"Our studies have shown conclusively that we can block addiction via the immune system of the brain, without targeting the brain's wiring," says the lead author of the study, Dr Mark Hutchinson, ARC Research Fellow in the University of Adelaide's School of Medical Sciences.

"Both the central nervous system and the immune system play important roles in creating addiction, but our studies have shown we only need to block the immune response in the brain to prevent cravings for opioid drugs."

The team has focused its research efforts on the immune receptor known as Toll-Like receptor 4 (TLR4).

"Opioid drugs such as morphine and heroin bind to TLR4 in a similar way to the normal immune response to bacteria. The problem is that TLR4 then acts as an amplifier for addiction," Dr Hutchinson says.

"The drug (+)-naloxone automatically shuts down the addiction. It shuts down the need to take opioids, it cuts out behaviours associated with addiction, and the neurochemistry in the brain changes -- dopamine, which is the chemical important for providing that sense of 'reward' from the drug, is no longer produced."

Senior author Professor Linda Watkins, from the Center for Neuroscience at the University of Colorado Boulder, says: "This work fundamentally changes what we understand about opioids, reward and addiction. We've suspected for some years that TLR4 may be the key to blocking opioid addiction, but now we have the proof.

"The drug that we've used to block addiction, (+)-naloxone, is a non-opioid mirror image drug that was created by Dr Kenner Rice in the 1970s. We believe this will prove extremely useful as a co-formulated drug with morphine, so that patients who require relief for severe pain will not become addicted but still receive pain relief. This has the potential to lead to major advances in patient and palliative care," Professor Watkins says.

The researchers say clinical trials may be possible within the next 18 months.

This study has been funded by the National Institute on Drug Abuse (NIDA) in the United States and the Australian Research Council (ARC).

Source: University of Adelaide (2012, August 14). Scientists can now block heroin, morphine addiction. ScienceDaily. Retrieved April 24, 2013, from http://www.sciencedaily.com­/releases/2012/08/120814213246.htm

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Scientists pioneer new method for watching proteins fold

A protein's function depends on both the chains of molecules it is made of and the way those chains are folded. And while figuring out the former is relatively easy, the latter represents a huge challenge with serious implications because many diseases are the result of misfolded proteins. Now, a team of chemists at the University of Pennsylvania has devised a way to watch proteins fold in "real-time," which could lead to a better understanding of protein folding and misfolding in general. 

The research was conducted by Feng Gai, professor in the Department of Chemistry in the School of Arts and Sciences, along with graduate students Arnaldo Serrano, also of Chemistry, and Robert Culik of the Department of Biochemistry and Molecular Biophysics at Penn's Perelman School of Medicine. They collaborated with Michelle R. Bunagan of the College of New Jersey's Department of Chemistry. 

Their research was published in the international edition of the journal Angewandte Chemie, where it was featured on the cover and bestowed VIP (very important paper) status. 

"One of the reasons that figuring out what happens when proteins fold is difficult is that we don't have the equivalent of a high-speed camera that can capture the process, " Gai said. "If the process were slow, we could take multiple 'pictures' over time and see the mechanism at work. Unfortunately, no one has this capability; the folding occurs faster than the blink of an eye." 

Gai's team uses infrared spectroscopy — a technique that measures how much light different parts of a molecule absorbs — to analyze proteins' structure and how this changes. In this case, the researchers looked at a model protein known as Trp-cage with an infrared laser setup. 

In this experiment, Gai's team used two lasers to study structural changes as a function of time. The first laser acts as the starting gun; by heating the molecule, it causes its structure to change. The second laser acts as the camera, following the motions of the protein's constituent amino acids. 

"The protein is made of different groups of atoms, and the different groups can be thought of as springs," Gai said. "Each spring has a different frequency with which it moves back and forth, which is based on the mass of the atom on either end. If the mass is bigger, the spring oscillates slower. Our 'camera' can detect the speed of that motion and we can relate it to the atoms it is made of and how that segment of the protein chain moves." 

Even in a simple protein like Trp-cage, however, there are many identical bonds, and the researchers need to be able to distinguish one from another in order to see which of them are moving while the protein folds. One strategy they used to get around this problem was to employ the molecular equivalent of a tracking device. 

"We use an amino acid with a carbon isotope marker," Culik said. "If it's incorporated into the protein correctly, we'll know where it is." 

With a single carbon atom of the Trp-cage slightly heavier than the others, the research team can use its signature to infer the position of the other atoms as they fold. The researchers could then "tune" the frequency of their laser to match different parts of the protein, allowing them to isolate them in their analyses. 

Similar isotopes could be inserted in more complicated molecules, allowing their folds to also be viewed with infrared spectroscopy. 

"This technique enhances our structural resolution. It allows us to see which part is moving," Gai said. "That would allow us to see exactly how a protein is misfolding in a disease, for example." 

Source: University of Pennsylvania [December 21, 2011]

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How the brain cell works

University of Miami biology professor Akira Chiba is leading a multidisciplinary team to develop the first systematic survey of protein interactions within brain cells. The team is aiming to reconstruct genome-wide in situ protein-protein interaction networks (isPIN) within the neurons of a multicellular organism. Preliminary data were presented at the American Society for Cell Biology annual meeting, December 3 through 7, 2011, in Denver, Colorado. 

At the core of the new imaging technology is the phenomenon known as FRET that occurs only when two fluorescently tagged molecules come within the distance of eight nanometer or less. Detecting the FRET serves as a proxy for the two proteins X and Y associating with each other within a living cell [Credit: A. Chiba/University of Miami]

"This work brings us closer to understanding the mechanics of molecules that keep us functioning," says Chiba, principal investigator of this project. "Knowing how our cells work will improve medicine. Most importantly, we will gain a better understanding of what life is at the molecular level." 

Neurons are the cells that are mainly responsible for signaling in the brain. Like all other cells, each neuron produces millions of individual proteins that associate with one another and form a complex communication network. Until recently, observing these protein-protein interactions had not been possible due to technical difficulties. Individual proteins are small and typically less than 10 nm (nanometer) in diameter. Yet, this nano-scale distance was considered to be off-limits even with super-resolution microscopy. 

Now, Chiba and his collaborators have developed a novel methodology to examine interaction of individual proteins in the fruit fly – the model organism of choice for this project. The researchers are creating genetically engineered insects that are capable of expressing over 500 fluorescently-tagged assorted proteins, two at a time. The fluorescent tags make it possible to visualize the exact spot where a given pair of proteins associates with each other. 

The team utilizes a custom- built 3D FLIM (fluorescent lifetime imaging microscopy) system to quantify this association event within the cells of a live animal. FLIM shows the location and time of such protein interaction, providing the data that allow creation of a point-by-point map of protein-protein interactions. 

The pilot phase of this multidisciplinary project is being funded by the National Institutes of Health. It employs advanced genetics, molecular imaging technology and high-performance computation, among other fields. "Collaborating fluorescent chemistry, laser optics and artificial intelligence, my team is working in the 'jungle' of the molecules of life within the living cells," Chiba says. "This is a new kind of ecology played out at the scale of nanometers—creating a sense of deja vu 80 years after the birth of modern ecology." 

At present, the researchers still need to extrapolate from data obtained in test tubes. In the future, they will begin to visualize directly how the individual proteins interact with one another in their 'native environment,' which are the cells in our body.  

Source: University of Miami [December 21, 2011]

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How Bacteria Build Homes Inside Healthy Cells

Bacteria are able to build camouflaged homes for themselves inside healthy cells -- and cause disease -- by manipulating a natural cellular process. 

Purdue University biologists led a team that revealed how a pair of proteins from the bacteria Legionella pneumophila, which causes Legionnaires disease, alters a host protein in order to divert raw materials within the cell for use in building and disguising a large structure that houses the bacteria as it replicates. 

Zhao-Qing Luo, the associate professor of biological sciences who headed the study, said the modification of the host protein creates a dam, blocking proteins that would be used as bricks in cellular construction from reaching their destination. The protein "bricks" are then diverted and incorporated into a bacterial structure called a vacuole that houses bacteria as it replicates within the cell. Because the vacuole contains materials natural to the cell, it goes unrecognized as a foreign structure. 

"The bacterial proteins use the cellular membrane proteins to build their house, which is sort of like a balloon," Luo said. "It needs to stretch and grow bigger as more bacterial replication occurs. The membrane material helps the vacuole be more rubbery and stretchy, and it also camouflages the structure. The bacteria is stealing material from the cell to build their own house and then disguising it so it blends in with the neighborhood." 

The method by which the bacteria achieve this theft is what was most surprising to Luo. 

The bacterial proteins, named AnkX and Lem3, modify the host protein through a biochemical process called phosphorylcholination that is used by healthy cells to regulate immune response. Phosphorylcholination is known to happen in many organisms and involves adding a small chemical group, called the phosphorylcholine moiety, to a target molecule, he said. 

The team discovered that AnkX adds the phosphorylcholine moiety to a host protein involved in moving proteins from the cell's endoplasmic reticulum to their cellular destinations. The modification effectively shuts down this process and creates a dam that blocks the proteins from reaching their destination. 

The bacterial protein Lem3 is positioned outside the vacuole and reverses the modification of the host protein to ensure that the protein "bricks" are free to be used in creation of the bacterial structure. 

This study was the first to identify proteins that directly add and remove the phosphorylcholine moiety, Luo said. 

"We were surprised to find that the bacterial proteins use the phosphorylcholination process and to discover that this process is reversible," he said. "This is evidence of a new way signals are relayed within cells, and we are eager to investigate it." 

The team also found that the phosphorylcholination reaction is carried out at a specific site on the protein called the Fic domain. Previous studies had shown this site induced a different reaction called AMPylation. 

It is rare for a domain to catalyze more than one reaction, and it was thought this site's only responsibility was to transfer the chemical group necessary for AMPylation, Luo said. 

"Revealing that this domain has dual roles is very important to identify or screen for compounds to inhibit its activity and fight disease," he said. "This domain has a much broader involvement in biochemical reactions than we thought and may be a promising target for effective treatments." 

During infection bacteria deliver hundreds of proteins into healthy cells that alter cellular processes to turn the hostile environment into one hospitable to bacterial replication, but the specific roles of only about 20 proteins are known, Luo said. 

"In order to pinpoint proteins that would be good targets for new antibiotics, we need to determine their roles and importance to the success of infection," he said. "We need to understand at the biochemical level exactly what these proteins do and how they take over natural cellular processes. Then we can work on finding ways to block these activities, stop the infection and save lives." 

A paper detailing their National Institutes of Health-funded work is published in the current issue of the Proceedings of National Academy of Sciences. In addition to Luo, Purdue graduate student Yunhao Tan and Randy Ronald of Indiana University co-authored the paper. 

Luo next plans to use the bacterial proteins as a tool to learn more about the complex cellular processes controlled by phosphorylcholination and to determine the biochemical processes role in cell signaling. 

Source: Purdue University [December 20, 2011]

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