Recent findings by medical researchers indicate that naturally occurring nanotubes may serve as tunnels that protect retroviruses and bacteria in transit from diseased to healthy cells — a fact that may explain why vaccines fare poorly against some invaders.
To better study the missions of these intercellular nanotubes, scientists have sought the means to form them quickly and easily in test tubes.
Sandia National Laboratories researchers have now learned serendipitously to form nanotubes with surprising ease.
"Our work is the first to show that the formation of nanotubes is not complicated, but can be a general effect of protein-membrane interactions alone," says Darryl Sasaki of Sandia's Bioscience and Energy Center.
Sandia is a National Nuclear Security Administration laboratory.
The tunnel-like structures have been recognized only recently as tiny but important bodily channels for the good, the bad, and the informational.
In addition to providing protected transport to certain diseases, the nanotubes also seem to help trundle bacteria to their doom in the tentacles of microphages. Lastly, the nanotubes may provide avenues to send and receive information (in the form of chemical molecules) from cell to cell far faster than their random dispersal into the bloodstream would permit.
Given the discovery of this radically different transportation system operating within human tissues, it was natural for researchers to attempt to duplicate the formation of the nanotubes. In their labs, they experimented with giant lipid vesicles that appeared to mimic key aspects of the cellular membrane.
Giant lipid vesicles resemble micron-sized spherical soap bubbles that exist in water. They are composed of a lipid bilayer membrane only five nanometers thick.
The object for experimenters was to create conditions in which the spheres would morph into cylinders of nanometer radii.
But researchers had difficulties, says Sasaki, perhaps because they used a composite lipid called egg PC that requires unnecessarily high energies to bend into a tubular shape.
Egg PC is inexpensive, readily available, and offers good, stable membrane properties. It is the usual lipid of choice in forming nanocylinders via mechanical stretching techniques.
But Sandia postdoctoral researcher Haiqing Lui instead used POPC — a single pure lipid requiring half the bending energy of egg PC.
She was trying to generate nanotubes by a completely different approach that involved the use of motor proteins to stretch naturally occurring membranes into tubes.
Working with Sandia researcher George Bachand, she serendipitously found that interaction of the POPC membrane with a high affinity protein called streptavidin alone was enough to form the nanotubes.
"Perhaps this information — linking membrane bending energy with nanotube formation — may provide some clue about the membrane structure and the cell's ability to form such intercellular connections," Sasaki says.
The formation was confirmed by Sandia researcher Carl Hayden, who characterized the nanotube formation through a confocal imaging microscope. The custom instrument allows pixel-by-pixel examination of the protein interaction with the membranes comprising the nanotubes by detecting the spectrum and lifetimes of fluorescent labels on the proteins.
Nanotube formation had been noticed previously by cell biologists, but they had dismissed the tiny outgrowths as "junk — an aberration of cells growing in culture," says Sasaki. "The reason they were only noticed recently as trafficking routes is because of labeling studies that marked organelles and proteins. This allowed a focused look at what these nanostructures might be used for."
It became clear, says Sasaki, that the organelles were being transported with "specific directionality" on the backs of motor proteins within the tubes, rather than randomly.
Three-dimensional networks of nanotubes also are found to be created by macrophages — part of the police force of the body — grown in culture, says George. The tubes in appearance and function resemble a kind of spider web, capturing bacterium and transporting them to the macrophages, which eat them.
Source : DOE/Sandia National Laboratories
Showing posts with label research. Show all posts
Showing posts with label research. Show all posts
Sunday, August 24, 2008
Worms do calculus to find meals or avoid unpleasantness
Thanks to salt and hot chili peppers, researchers have found a calculus-computing center that tells a roundworm to go forward toward dinner or turn to broaden the search. It's a computational mechanism, they say, that is similar to what drives hungry college students to a pizza.
These behavior-driving calculations, according to a paper published in the July 3 issue of the journal Nature, are done "in a tiny, specialized computer inside a primitive roundworm," says principal investigator Shawn Lockery, a University of Oregon biologist and member of the UO Institute of Neuroscience.
In their paper, the researchers documented how two related, closely located chemosensory neurons, acting in tandem, regulate behavior. The left neuron controls an on switch, while the opposing right one an off switch. These sister neurons are situated much like the two nostrils or two eyes of mammals. Together these neurons are known as ASE for antagonistic sensory cues.
It's possible, Lockery said, that the discovery someday could help research aimed at treating at least some of the 200,000 people in the United States who annually seek medical treatment, according to records of the National Institutes of Health, for problems involving taste and smell.
"This computer does some nice calculus, differentiating the rate of change of the strength of various tastes," Lockery said. "The worm uses this information to find food and to avoid poisons."
Lockery and colleagues predicted the existence of a derivative-crunching mechanism in the Journal of Neuroscience in 1999 based on findings that nematodes change directions based on taste and smell.
"In effect, they have two nostrils or two tongues but they are so close together that it is really like having one nostril or one tongue, and yet they find their way around quite effectively," Lockery said. "We knew from behavioral experiments that nematodes were doing the same thing that humans were doing, but only from the view of behavioral responses. We didn't know what was going on in the brain."
To get there, Lockery and colleagues used new imaging and molecular tools, along with some genetic engineering of their worms.
In one experiment, these chemosensory neurons carried a fluorescent protein that changed color based on neuronal activity. In another experiment, the neurons carried receptor proteins that recognize capsaicin, the active component in chili peppers.
Researchers found that when concentrations of salt were high, fluorescent proteins change from blue to yellow, showing that the left neuron (ASEL) was active as the worms continued forward movement. When salt levels were reduced, the right neuron (ASER) activated but generated a different behavior; the worms began a turning, or searching, motion.
"At this point, we wanted to know if these neurons really are controlling behavior. If ASEL really signals that things are getting better, then, if you could artificially activate ASEL the animals ought to go straight like a human going directly toward the pizza," Lockery said. "Conversely, if you activate the ASER the animals ought to turn to find their goal."
Such was the case, according to the capsaicin-receptor experiment. When the pepper ingredient was spread on turning worms with receptor proteins in the left neuron, they straightened their motion. Likewise, capsaicin applied to worms with the receptors in their right neurons caused them to change from turning motion to forward crawling.
"We have discovered a tiny, specialized computer inside a primitive round worm," Lockery said. "The computer calculates the rate of change of the strengths, or concentrations, of various tastes. The worm uses this information to find food and to avoid poisons."
Evidence for such on and off switching cells in other chemosensory networks of mammals, he added, "There are strong indications that a similar device exists in the human nervous system."
Source : University of Oregon
These behavior-driving calculations, according to a paper published in the July 3 issue of the journal Nature, are done "in a tiny, specialized computer inside a primitive roundworm," says principal investigator Shawn Lockery, a University of Oregon biologist and member of the UO Institute of Neuroscience.
In their paper, the researchers documented how two related, closely located chemosensory neurons, acting in tandem, regulate behavior. The left neuron controls an on switch, while the opposing right one an off switch. These sister neurons are situated much like the two nostrils or two eyes of mammals. Together these neurons are known as ASE for antagonistic sensory cues.
It's possible, Lockery said, that the discovery someday could help research aimed at treating at least some of the 200,000 people in the United States who annually seek medical treatment, according to records of the National Institutes of Health, for problems involving taste and smell.
"This computer does some nice calculus, differentiating the rate of change of the strength of various tastes," Lockery said. "The worm uses this information to find food and to avoid poisons."
Lockery and colleagues predicted the existence of a derivative-crunching mechanism in the Journal of Neuroscience in 1999 based on findings that nematodes change directions based on taste and smell.
"In effect, they have two nostrils or two tongues but they are so close together that it is really like having one nostril or one tongue, and yet they find their way around quite effectively," Lockery said. "We knew from behavioral experiments that nematodes were doing the same thing that humans were doing, but only from the view of behavioral responses. We didn't know what was going on in the brain."
To get there, Lockery and colleagues used new imaging and molecular tools, along with some genetic engineering of their worms.
In one experiment, these chemosensory neurons carried a fluorescent protein that changed color based on neuronal activity. In another experiment, the neurons carried receptor proteins that recognize capsaicin, the active component in chili peppers.
Researchers found that when concentrations of salt were high, fluorescent proteins change from blue to yellow, showing that the left neuron (ASEL) was active as the worms continued forward movement. When salt levels were reduced, the right neuron (ASER) activated but generated a different behavior; the worms began a turning, or searching, motion.
"At this point, we wanted to know if these neurons really are controlling behavior. If ASEL really signals that things are getting better, then, if you could artificially activate ASEL the animals ought to go straight like a human going directly toward the pizza," Lockery said. "Conversely, if you activate the ASER the animals ought to turn to find their goal."
Such was the case, according to the capsaicin-receptor experiment. When the pepper ingredient was spread on turning worms with receptor proteins in the left neuron, they straightened their motion. Likewise, capsaicin applied to worms with the receptors in their right neurons caused them to change from turning motion to forward crawling.
"We have discovered a tiny, specialized computer inside a primitive round worm," Lockery said. "The computer calculates the rate of change of the strengths, or concentrations, of various tastes. The worm uses this information to find food and to avoid poisons."
Evidence for such on and off switching cells in other chemosensory networks of mammals, he added, "There are strong indications that a similar device exists in the human nervous system."
Source : University of Oregon
New antibiotic beats superbugs at their own game
The problem with antibiotics is that, eventually, bacteria outsmart them and become resistant. But by targeting the gene that confers such resistance, a new drug may be able to finally outwit them. Rockefeller University scientists tested the new drug, called Ceftobiprole, against some of the deadliest strains of multidrug-resistant Staphylococcus aureus (MRSA) bacteria, which are responsible for the great majority of staphylococcal infections worldwide, both in hospitals and in the community.
The research, to be published in the August 2008 issue of the journal Antimicrobial Agents and Chemotherapy and available online now, looked at how well Ceftobiprole worked against bacterial clones that had already developed resistance to other drugs. In every case, Ceftobiprole won. "It just knocked out the cells 100 percent," says the study's lead investigator, Alexander Tomasz, head of the Laboratory of Microbiology at Rockefeller.
Previous research had already shown that -- in general -- Ceftobiprole was highly effective against most clinical isolates of S. aureus. "Instead, we looked more carefully at the highly resistant cells that already occur in such clinical isolates at very low frequency -- maybe in one bacterium in every 1,000," says Tomasz. Ceftobiprole was able to kill these resistant cells.
Never before has an antibiotic been tested this way. "In the history of antibiotic development, an antibiotic arrives on the scene, and sooner or later resistant bacteria emerge," Tomasz says. "We sought to test in advance which would win this particular chess game: the new drug, or the bacteria that now cause human deaths."
In an ominous new "move" in this chess game, S. aureus strains with resistance to vancomycin (VRSA), a different class of antibiotics, also began to appear in hospitals in the United States. Ceftobiprole was also able to kill these new resistant VRSA strains.
The drug is effective because the chemists who developed Ceftobiprole managed to outwit the bacteria at their own game, Tomasz says. The broad-spectrum antibiotic was discovered by Basilea Pharmaceuticals, based in Basel, Switzerland, and is being developed in the U.S. and worldwide by Johnson & Johnson. The research was supported by Johnson & Johnson along with a grant from the U.S. Public Health Service.
Source : Rockefeller University
The research, to be published in the August 2008 issue of the journal Antimicrobial Agents and Chemotherapy and available online now, looked at how well Ceftobiprole worked against bacterial clones that had already developed resistance to other drugs. In every case, Ceftobiprole won. "It just knocked out the cells 100 percent," says the study's lead investigator, Alexander Tomasz, head of the Laboratory of Microbiology at Rockefeller.
Previous research had already shown that -- in general -- Ceftobiprole was highly effective against most clinical isolates of S. aureus. "Instead, we looked more carefully at the highly resistant cells that already occur in such clinical isolates at very low frequency -- maybe in one bacterium in every 1,000," says Tomasz. Ceftobiprole was able to kill these resistant cells.
Never before has an antibiotic been tested this way. "In the history of antibiotic development, an antibiotic arrives on the scene, and sooner or later resistant bacteria emerge," Tomasz says. "We sought to test in advance which would win this particular chess game: the new drug, or the bacteria that now cause human deaths."
In an ominous new "move" in this chess game, S. aureus strains with resistance to vancomycin (VRSA), a different class of antibiotics, also began to appear in hospitals in the United States. Ceftobiprole was also able to kill these new resistant VRSA strains.
The drug is effective because the chemists who developed Ceftobiprole managed to outwit the bacteria at their own game, Tomasz says. The broad-spectrum antibiotic was discovered by Basilea Pharmaceuticals, based in Basel, Switzerland, and is being developed in the U.S. and worldwide by Johnson & Johnson. The research was supported by Johnson & Johnson along with a grant from the U.S. Public Health Service.
Source : Rockefeller University
Pandemic mutations in bird flu revealed
Scientists have discovered how bird flu adapts in patients, offering a new way to monitor the disease and prevent a pandemic, according to research published in the August issue of the Journal of General Virology. Highly pathogenic H5N1 avian influenza virus has spread through at least 45 countries in 3 continents. Despite its ability to spread, it cannot be transmitted efficiently from human to human. This indicates it is not fully adapted to its new host species, the human. However, this new research reveals mutations in the virus that may result in a pandemic.
"The mutations needed for the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues," said Professor Dr Prasert Auewarakul from Mahidol University, Thailand. "We analyzed specific molecules called haemagglutinin on viruses derived from fatal human cases. Our results suggest new candidate mutations that may allow bird flu to adapt to humans."
Viruses with a high mutation rate such as influenza virus usually exist as a swarm of variants, each slightly different from the others. These are called H5N1 bird flu quasispecies. Professor Dr Auewarakul and his colleagues found that some mutations in the quasispecies were more frequent than others, which indicates they may be adaptive changes that make the virus more efficient at infecting humans. Most of these mutations were found in the area required for the virus to bind to the host cell.
"This study shows that the H5N1 virus is adapting each time it infects a human," said Professor Dr Auewarakul. "Such adaptations may lead to the emergence of a virus that can cause a pandemic. Our research highlights the need to control infection and transmission to humans to prevent further adaptations."
The research has provided genetic markers to help scientists monitor bird flu viruses with pandemic potential. This means they will be able to detect potentially dangerous strains and prevent a pandemic. The research also gives new insights into the mechanism of the genesis of a pandemic strain.
"Our approach could be used to screen for mutations with significant functional impact," said Professor Dr Auewarakul. "It is a new method of searching for changes in H5N1 viruses that are required for the emergence of a pandemic virus. We hope it will help us to prevent a pandemic in the future."
Source : Society for General Microbiology
"The mutations needed for the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues," said Professor Dr Prasert Auewarakul from Mahidol University, Thailand. "We analyzed specific molecules called haemagglutinin on viruses derived from fatal human cases. Our results suggest new candidate mutations that may allow bird flu to adapt to humans."
Viruses with a high mutation rate such as influenza virus usually exist as a swarm of variants, each slightly different from the others. These are called H5N1 bird flu quasispecies. Professor Dr Auewarakul and his colleagues found that some mutations in the quasispecies were more frequent than others, which indicates they may be adaptive changes that make the virus more efficient at infecting humans. Most of these mutations were found in the area required for the virus to bind to the host cell.
"This study shows that the H5N1 virus is adapting each time it infects a human," said Professor Dr Auewarakul. "Such adaptations may lead to the emergence of a virus that can cause a pandemic. Our research highlights the need to control infection and transmission to humans to prevent further adaptations."
The research has provided genetic markers to help scientists monitor bird flu viruses with pandemic potential. This means they will be able to detect potentially dangerous strains and prevent a pandemic. The research also gives new insights into the mechanism of the genesis of a pandemic strain.
"Our approach could be used to screen for mutations with significant functional impact," said Professor Dr Auewarakul. "It is a new method of searching for changes in H5N1 viruses that are required for the emergence of a pandemic virus. We hope it will help us to prevent a pandemic in the future."
Source : Society for General Microbiology
A viral cloaking device
Viruses achieve their definition of success when they can thrive without killing their host. Now, biologists Pamela Bjorkman and Zhiru Yang of the California Institute of Technology have uncovered how one such virus, prevalent in humans, evolved over time to hide from the immune system.
The human immune system and the viruses hosted by our bodies are in a continual dance for survival--viruses ever seek new ways to evade detection, and our immune system devises new methods to hunt them down. Human Cytomegalovirus (HCMV), says Bjorkman, Caltech's Delbrück Professor of Biology and a Howard Hughes Medical Institute (HHMI) Investigator, "is the definition of a successful virus--it thrives but it doesn't affect the host."
HCMV is carried by eight in 10 people. Although it generally harms only those who are immunocompromised, it has also been linked with brain tumors like the one for which Ted Kennedy recently had surgery. Understanding how HCMV survives may help in the development of a vaccine, as well as in the fight against other viruses with similar evasive tactics.
"We are interested in mechanisms taken by viruses to escape our immune system," says Caltech biology postdoc and HHMI associate Zhiru Yang. She and Bjorkman published their findings on HCMV survival mechanisms in the July 15 edition of Proceedings of the National Academy of Sciences. They describe the underpinnings of a viral cloaking device, partly made of stolen goods from healthy cells, that helps HCMV to move undetected through the body.
For 20 years, Bjorkman's lab has been dedicated to understanding class 1 major histocompatibility complex (MHC) proteins and the immune response, most recently related to AIDS research. MHC proteins carry peptides, small pieces that are chopped up from the cell's internal proteins, to the cell's surface. If a cell has been infected, MHC presents viral peptides to signal T cells to kill it. So some viruses evolved to evade T cells by keeping MHC from reaching the cell surface. In turn, the immune system recruited other hunters to search for cells that don't show MHC proteins.
Sometime along its treacherous evolutionary path, HCMV stole a class 1 MHC molecule from its host and modified it for supreme stealth. "This is a decoy," Bjorkman says. She and Yang analyzed the structure of the mimic, called UL18, to compare how similar it is to the real thing. They found that despite a mere 23 percent match in genetic sequences, UL18 looks almost exactly the same as a true class 1 MHC.
The same immune cells that search for missing MHC proteins are designed to bind to them when they find them, thereby inhibiting an immune response. Yang and Bjorkman found that UL18 happens to bind 1,000 times tighter to these inhibitory receptors than real MHC molecules do. "This is exactly what the virus wants--to avoid being recognized by T cells, but to engage inhibitory receptors to turn off immune cells," Yang notes. "Only a small number of UL18 molecules are required to have the same inhibitory effect as a large number of MHC class I molecules."
"What I find astounding is that the virus stole this gene and kept it almost identical but improved upon its binding," Bjorkman says.
UL18 didn't stop there. "It also binds peptides--that's unique to this MHC mimic. We don't know why," Bjorkman adds. The peptide is obscured from killer cells by yet another shield, Yang says. In a trait it shares with HIV proteins, HCMV's UL18 covers itself with carbohydrates, which are unrecognizable to the immune system. A real class 1 MHC molecule has one site for adding carbohydrates; the fake has 13, Bjorkman notes. The only place where it's not covered is where it binds to the inhibitory receptor.
All its efforts have made UL18 virtually undetectable. "It's a good example of a viral protein that evolved from its host ancestor to block unwanted interactions," Yang says. "The more we understand that, the more effectively we can fight viruses that hide out," Bjorkman adds.
Source : California Institute of Technology
The human immune system and the viruses hosted by our bodies are in a continual dance for survival--viruses ever seek new ways to evade detection, and our immune system devises new methods to hunt them down. Human Cytomegalovirus (HCMV), says Bjorkman, Caltech's Delbrück Professor of Biology and a Howard Hughes Medical Institute (HHMI) Investigator, "is the definition of a successful virus--it thrives but it doesn't affect the host."
HCMV is carried by eight in 10 people. Although it generally harms only those who are immunocompromised, it has also been linked with brain tumors like the one for which Ted Kennedy recently had surgery. Understanding how HCMV survives may help in the development of a vaccine, as well as in the fight against other viruses with similar evasive tactics.
"We are interested in mechanisms taken by viruses to escape our immune system," says Caltech biology postdoc and HHMI associate Zhiru Yang. She and Bjorkman published their findings on HCMV survival mechanisms in the July 15 edition of Proceedings of the National Academy of Sciences. They describe the underpinnings of a viral cloaking device, partly made of stolen goods from healthy cells, that helps HCMV to move undetected through the body.
For 20 years, Bjorkman's lab has been dedicated to understanding class 1 major histocompatibility complex (MHC) proteins and the immune response, most recently related to AIDS research. MHC proteins carry peptides, small pieces that are chopped up from the cell's internal proteins, to the cell's surface. If a cell has been infected, MHC presents viral peptides to signal T cells to kill it. So some viruses evolved to evade T cells by keeping MHC from reaching the cell surface. In turn, the immune system recruited other hunters to search for cells that don't show MHC proteins.
Sometime along its treacherous evolutionary path, HCMV stole a class 1 MHC molecule from its host and modified it for supreme stealth. "This is a decoy," Bjorkman says. She and Yang analyzed the structure of the mimic, called UL18, to compare how similar it is to the real thing. They found that despite a mere 23 percent match in genetic sequences, UL18 looks almost exactly the same as a true class 1 MHC.
The same immune cells that search for missing MHC proteins are designed to bind to them when they find them, thereby inhibiting an immune response. Yang and Bjorkman found that UL18 happens to bind 1,000 times tighter to these inhibitory receptors than real MHC molecules do. "This is exactly what the virus wants--to avoid being recognized by T cells, but to engage inhibitory receptors to turn off immune cells," Yang notes. "Only a small number of UL18 molecules are required to have the same inhibitory effect as a large number of MHC class I molecules."
"What I find astounding is that the virus stole this gene and kept it almost identical but improved upon its binding," Bjorkman says.
UL18 didn't stop there. "It also binds peptides--that's unique to this MHC mimic. We don't know why," Bjorkman adds. The peptide is obscured from killer cells by yet another shield, Yang says. In a trait it shares with HIV proteins, HCMV's UL18 covers itself with carbohydrates, which are unrecognizable to the immune system. A real class 1 MHC molecule has one site for adding carbohydrates; the fake has 13, Bjorkman notes. The only place where it's not covered is where it binds to the inhibitory receptor.
All its efforts have made UL18 virtually undetectable. "It's a good example of a viral protein that evolved from its host ancestor to block unwanted interactions," Yang says. "The more we understand that, the more effectively we can fight viruses that hide out," Bjorkman adds.
Source : California Institute of Technology
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