A team of Washington State University scientists has devised a method that could lead to the development of vaccines against some of the most troubling infectious diseases we face diseases that have so far been difficult or impossible to vaccinate against.
The new method allows researchers to rapidly screen large numbers of pathogen proteins, called antigens, for their ability to prompt an immune response in a host. Proteins with that ability are good candidates for use in vaccines. The method will be especially valuable in the quest for vaccines against persistent diseases such as malaria, sleeping sickness and syphilis.
"It's very slick," said immunologist Wendy Brown, who led the research effort. "Now we have a high-throughput way of finding antigens from any pathogen, as long as you have the genome sequence. To me this was a huge breakthrough, because I've been spending my whole career trying to figure out ways to do this."
The research team included scientists at WSU and at the Rocky Mountain Laboratories of the National Institutes of Health.
A vaccine works by showing the body's immune system a pathogen or part of a pathogen (usually a protein) so that it can develop cellular memory and antibodies that will recognize and attack the pathogen in the future. A key step in the development of a vaccine is identifying which protein(s) to use. Until now, screening pathogen proteins to find those few that might be good candidates has been laborious, time-consuming, and in the case of persistent diseases, not very successful. Brown said prior methods required about three months to produce and purify a single protein to test. With her new method she is able to screen dozens of proteins within a few weeks.
Brown's group worked with Anaplasma, a bacterium that causes severe anemia in cattle. Anaplasma is the most common tick-borne pathogen of cattle worldwide and costs an estimated $100 million per year in lost animals and lowered productivity in the United States alone.
The new method starts with the pathogen's DNA. Previous work by WSU scientists had determined the whole genome sequence of Anaplasma. By comparing that sequence with the genome sequences of better-known microbes, Brown's team was able to pinpoint genes that code for proteins that stick out of the pathogen's cell membrane. Brown reasoned that since those proteins are exposed on the surface of the cell, they should be visible to antibodies and immune system cells, and therefore could be a good way to target the pathogen.
Once the genes were isolated, Brown's team made the proteins they coded for by using chemical 'machinery' derived from E. coli bacteria. They then purified each protein to get rid of any E. coli proteins that were present. They did that by using a chemical that would specifically bind to the Anaplasma proteins. Brown attached the chemical to tiny synthetic beads and then poured the protein mixture over the beads. Anaplasma proteins stuck to the beads, while E. coli proteins did not and were discarded. This purification step represented a big advance over other methods, which have been plagued by contamination with irrelevant proteins.
Each purified test protein was then presented to T cells from cows that had previously been exposed to Anaplasma outer membrane proteins. T cells are the immune system's "memory cells." In the body, when they recognize an antigen they have seen before, they trigger antibody production by other immune system cells. In Brown's test, if the T cells recognized a protein, they started dividing and making interferon.
Using the new procedure, Brown's team found T cells responded to about 20 proteins, including many that had never before been shown to stimulate a T cell response. The researchers are now testing whether any of these might form the basis for an effective vaccine against Anaplasma.
Brown said the new technique also will be a boon to researchers working on vaccines against pathogens that are highly contagious or especially deadly, such as the Ebola virus and the bacterium that causes anthrax. She is using it to screen proteins from Coxiella, a bacterium that causes Q fever and is considered a possible bioterrorism threat.
"If you have the genome, you don't have to touch the organism. You can just start expressing all these proteins and test them," Brown said.
Washington State University
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вторник, 3 мая 2011 г.
понедельник, 25 апреля 2011 г.
Protein Found To Predict Brain Injury In Children On "ECMO" Life Support
Johns Hopkins Children's Center scientists have discovered that high blood levels of a protein commonly found in the central nervous system can predict brain injury and death in critically ill children on a form of life support called extra-corporeal membrane oxygenation or ECMO.
ECMO, used to temporarily oxygenate the blood of patients whose heart and lungs are too weak or damaged to do so on their own, is most often used as a last resort because it can increase the risk for brain bleeding, brain swelling, stroke and death in some patients.
A detailed report of the Hopkins team's findings is published ahead of print Nov. 4 in the journal Pediatric Critical Care Medicine.
Following 22 ECMO patients, ranging from two days to 9 years of age, the researchers found that those with abnormally high levels of glial fibrillary acidic protein (GFAP) were 13 times more likely to die and 11 times more likely to suffer brain injury than children with normal GFAP levels. GFAP levels are already used as a marker of neurologic damage in adults who suffer strokes and traumatic brain injuries.
Although preliminary, the team's findings may pave the way to a much-needed way to monitor the precarious neurologic status of children on ECMO without using imaging tests like ultrasounds or CT scans. Periodic blood tests measuring GFAP levels may be one such tool to monitor brain function and help ward off brain injury and death, the researchers say.
"A simple, fast and easy-to-use test has been needed to monitor, predict and prevent brain damage in children on ECMO because these children are unresponsive or heavily sedated, and doctors cannot easily gauge their neurologic function," says study lead investigator Melania Bembea, M.D., M.P. H., a pediatric critical-care specialist at Hopkins Children's.
"Early detection of brain injury can help us prevent further harm by changing medication doses and rapidly weaning the patient from ECMO support," she adds.
The findings may have implications beyond ECMO, the researchers say, as they offer a way to monitor brain damage in other high-risk situations, including heart surgery and severely premature birth.
"Our long-term goal is to make lifesaving therapies like ECMO and heart surgery safer and more effective by improving protection of the brain, and GFAP and other biomarkers can give us a much-needed benchmark around which we can make these therapies safer," says senior investigator Allen Everett, M.D., a cardiologist at Hopkins Children's.
In the study, seven of the 22 children on ECMO developed brain bleeding or brain swelling, five of whom died subsequently. These children had much higher peak levels of GFAP than children without brain injury 5.9 nanograms per milliliter of blood compared to 0.09 in children without brain injury. GFAP levels were also markedly higher among eight of the 22 children in the study who had poor neurologic outcomes after ECMO (3.6 ng/ml) than in those children who had good neurologic outcomes (0.09 ng/ml).
Researchers also measured GFAP levels among healthy children and among newborns without neurologic injuries. Their median GFAP level was 0.055 nanograms per milliliter of blood and as high as 0.436 in some cases. By comparison, overall GFAP levels in children with neurologic injuries were 13 times greater than GFAP levels in healthy children.
The researchers caution that their findings should be replicated in a larger trial with more patients and that future studies must clarify the relationship between a rise in GFAP levels and the onset of brain injury. In the current study, GFAP levels rose sharply in some patients one or two days before their brain damage was discovered on ultrasound.
ECMO is used in about 1,000 children each year. Between 10 percent and 60 percent of children who survive ECMO suffer neurologic damage either because of their underlying disease or complications during ECMO therapy, the researchers say.
Hopkins Children's is Maryland's only hospital providing pediatric ECMO service.
The research was funded by the National Institutes of Health.
Other investigators in the study included William Savage, M.D., John Strouse, M.D., Ph.D., Jamie Schwartz, M.D., Ernest Graham, Carol Thompson, M.B.A., M.S., all of Hopkins.
Related:
Source: Johns Hopkins Medicine
ECMO, used to temporarily oxygenate the blood of patients whose heart and lungs are too weak or damaged to do so on their own, is most often used as a last resort because it can increase the risk for brain bleeding, brain swelling, stroke and death in some patients.
A detailed report of the Hopkins team's findings is published ahead of print Nov. 4 in the journal Pediatric Critical Care Medicine.
Following 22 ECMO patients, ranging from two days to 9 years of age, the researchers found that those with abnormally high levels of glial fibrillary acidic protein (GFAP) were 13 times more likely to die and 11 times more likely to suffer brain injury than children with normal GFAP levels. GFAP levels are already used as a marker of neurologic damage in adults who suffer strokes and traumatic brain injuries.
Although preliminary, the team's findings may pave the way to a much-needed way to monitor the precarious neurologic status of children on ECMO without using imaging tests like ultrasounds or CT scans. Periodic blood tests measuring GFAP levels may be one such tool to monitor brain function and help ward off brain injury and death, the researchers say.
"A simple, fast and easy-to-use test has been needed to monitor, predict and prevent brain damage in children on ECMO because these children are unresponsive or heavily sedated, and doctors cannot easily gauge their neurologic function," says study lead investigator Melania Bembea, M.D., M.P. H., a pediatric critical-care specialist at Hopkins Children's.
"Early detection of brain injury can help us prevent further harm by changing medication doses and rapidly weaning the patient from ECMO support," she adds.
The findings may have implications beyond ECMO, the researchers say, as they offer a way to monitor brain damage in other high-risk situations, including heart surgery and severely premature birth.
"Our long-term goal is to make lifesaving therapies like ECMO and heart surgery safer and more effective by improving protection of the brain, and GFAP and other biomarkers can give us a much-needed benchmark around which we can make these therapies safer," says senior investigator Allen Everett, M.D., a cardiologist at Hopkins Children's.
In the study, seven of the 22 children on ECMO developed brain bleeding or brain swelling, five of whom died subsequently. These children had much higher peak levels of GFAP than children without brain injury 5.9 nanograms per milliliter of blood compared to 0.09 in children without brain injury. GFAP levels were also markedly higher among eight of the 22 children in the study who had poor neurologic outcomes after ECMO (3.6 ng/ml) than in those children who had good neurologic outcomes (0.09 ng/ml).
Researchers also measured GFAP levels among healthy children and among newborns without neurologic injuries. Their median GFAP level was 0.055 nanograms per milliliter of blood and as high as 0.436 in some cases. By comparison, overall GFAP levels in children with neurologic injuries were 13 times greater than GFAP levels in healthy children.
The researchers caution that their findings should be replicated in a larger trial with more patients and that future studies must clarify the relationship between a rise in GFAP levels and the onset of brain injury. In the current study, GFAP levels rose sharply in some patients one or two days before their brain damage was discovered on ultrasound.
ECMO is used in about 1,000 children each year. Between 10 percent and 60 percent of children who survive ECMO suffer neurologic damage either because of their underlying disease or complications during ECMO therapy, the researchers say.
Hopkins Children's is Maryland's only hospital providing pediatric ECMO service.
The research was funded by the National Institutes of Health.
Other investigators in the study included William Savage, M.D., John Strouse, M.D., Ph.D., Jamie Schwartz, M.D., Ernest Graham, Carol Thompson, M.B.A., M.S., all of Hopkins.
Related:
Source: Johns Hopkins Medicine
Cancer In Children And Young People Caused By Rearrangements Of Multifunctional Genes
A doctoral thesis presented at the Sahlgrenska Academy, University of Gothenburg, Sweden, shows that three genes that lie behind a number of malignant tumour diseases are normally involved in several fundamental processes in the cell. This may be the reason that the tumours arise early in life and principally affect children and young people.
A family of genes known as the "FET" genes has been investigated in the work presented in the thesis. This family contains three genes that are found in modified forms in several malignant soft-tissue tumours and several forms of leukaemia. The FET genes are found in these tumours in the form of what are known as "fusion genes" in which parts of two different genes have merged to form one gene. Fusion genes are translated into abnormal fusion proteins, which can in certain cases transform normal cells to cancer cells.
The human body consists of many different types of specialised cell types such as nerve cells, fat cells and intestinal cells. These are formed when stem cells multiply and mature gradually along different developmental pathways. Cancer may arise if something goes wrong in this process. The study has shown that the activities of the genes in the FET family fall as the cells mature, and scientists therefore believe that these genes play a role during the early stages of cell maturation, when the cells are not far from the stem cell stage. The normal maturation pathway of a cell becomes blocked when fusion genes that contain FET genes arise. The result is a cancer cell with properties similar to those of stem cells, and such a cell can multiply in an uncontrolled manner.
"We found that the FET genes are also involved in the response of the cell to external and internal stress, and when cells spread. Alterations of such processes are common in cancer cells", says Mattias Andersson.
It normally requires damage to several different genes before cancer cells develop, and this usually takes a long time. However, since the FET genes are involved in several of the normal cell processes, scientists believe that in their rearranged form they can affect in parallel several of the control systems that prevent a normal cell from becoming a cancer cell. This may give rise to rapid development of cancer, and it may be the reason that tumours with FET fusion genes are often found in children and young people.
"Studying normal FET genes has increased our understanding of what may go wrong in cancer cells having rearrangements of these genes. This may in the long term lead to new methods of treatment for tumour diseases that contain FET fusion genes", says Mattias Andersson.
Source: Ulrika Lundin
University of Gothenburg
A family of genes known as the "FET" genes has been investigated in the work presented in the thesis. This family contains three genes that are found in modified forms in several malignant soft-tissue tumours and several forms of leukaemia. The FET genes are found in these tumours in the form of what are known as "fusion genes" in which parts of two different genes have merged to form one gene. Fusion genes are translated into abnormal fusion proteins, which can in certain cases transform normal cells to cancer cells.
The human body consists of many different types of specialised cell types such as nerve cells, fat cells and intestinal cells. These are formed when stem cells multiply and mature gradually along different developmental pathways. Cancer may arise if something goes wrong in this process. The study has shown that the activities of the genes in the FET family fall as the cells mature, and scientists therefore believe that these genes play a role during the early stages of cell maturation, when the cells are not far from the stem cell stage. The normal maturation pathway of a cell becomes blocked when fusion genes that contain FET genes arise. The result is a cancer cell with properties similar to those of stem cells, and such a cell can multiply in an uncontrolled manner.
"We found that the FET genes are also involved in the response of the cell to external and internal stress, and when cells spread. Alterations of such processes are common in cancer cells", says Mattias Andersson.
It normally requires damage to several different genes before cancer cells develop, and this usually takes a long time. However, since the FET genes are involved in several of the normal cell processes, scientists believe that in their rearranged form they can affect in parallel several of the control systems that prevent a normal cell from becoming a cancer cell. This may give rise to rapid development of cancer, and it may be the reason that tumours with FET fusion genes are often found in children and young people.
"Studying normal FET genes has increased our understanding of what may go wrong in cancer cells having rearrangements of these genes. This may in the long term lead to new methods of treatment for tumour diseases that contain FET fusion genes", says Mattias Andersson.
Source: Ulrika Lundin
University of Gothenburg
News From The Journal Of Neuroscience
1. Polyhedral Cages Dock Vesicles at Active Zones
Guido A. Zampighi, Nick Fain, Lorenzo M. Zampighi, Francesca Cantele, Salvatore Lanzavecchia, and Ernest M. Wright
When looking at schematic illustrations of proteins found in presynaptic active zones, it is hard to imagine how all those proteins fit together in the cell. Even with electron microscopy, the organization of vesicle docking machinery is difficult to discriminate. But this week, Zampighi et al. present images of active zone complexes that were visualized using conical electron tomography. The authors used semiautomated volume-rendering techniques that colored individual voxels based on density thresholds and/or topology. The resulting images revealed that active zones of rat cortical synapses contain several units, each of which comprised a central polyhedral cage (which the authors call a syndesome) surrounded by synaptic vesicles. Some of these vesicles were partly or fully fused to the plasma membrane, suggesting that the polyhedral cages help mediate vesicle docking and fusion. Interestingly, the polyhedral cages resemble those of clathrin coats, which are normally associated with endocytosis rather than exocytosis.
2. Neurturin and Ret Influence Retinal Circuit Formation
Milam A. Brantley Jr, Sanjay Jain, Emily E. Barr, Eugene M. Johnson Jr, and Jeffrey Milbrandt
The receptor tyrosine kinase Ret, which is activated by glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), is essential for development of many tissues, and GDNF can slow retinal degeneration in animal models. Brantley et al. have detailed the role of this signaling pathway in retinal development. Mice with reduced Ret expression showed decreased light responses, as did mice lacking the GFL neurturin, but not other GFLs. Expression of fluorescent reporters under the control of Ret or neurturin receptor promoters indicated that both of these molecules are expressed in horizontal cells and some amacrine and ganglion cells. In neurturin knock-out mice, the outer plexiform layer (where photoreceptors synapse with horizontal and bipolar cells) was disorganized, horizontal cell axons and dendrites were sparse, bipolar and horizontal cell processes were abnormally long, and synapses were mislocalized to the outer nuclear layer. Therefore, neurturin-mediated Ret signaling appears necessary for normal circuit development in the retina.
3. Disinhibition Drives OFF Cell Depolarization
Michael B. Manookin, Deborah Langrill Beaudoin, Zachary Raymond Ernst, Leigh J. Flagel, and Jonathan B. Demb
It has been assumed that depolarization of retinal ganglion cells is driven by excitation from bipolar cells. Manookin et al. now report that OFF ganglion cells are also driven by reduced inhibition. Responses to light increments and decrements were recorded in guinea pig ON and OFF ganglion cells. At all increment levels, ON cells received both excitatory and inhibitory inputs; but at each decrement level, increased excitation of OFF cells was paired with decreased inhibition. By sequentially applying receptor agonists and antagonists to test each type of synapse (including gap junctions), it was determined that OFF cell inhibition is mediated by AII amacrine cells, which are electrically coupled to ON cone bipolar cells. When the light dims, ON cone bipolar cells hyperpolarize, which hyperpolarizes AII amacrine cells, thus reducing their inhibition of OFF ganglion cells. This disinhibition is the dominant driving force for OFF ganglion cells when light decrements are small.
4. Microglia Delimit Alzheimer Plaques
Tristan Bolmont, Florent Haiss, Daniel Eicke, Rebecca Radde, Chester A. Mathis, William E. Klunk, Shinichi Kohsaka, Mathias Jucker, and Michael E. Calhoun
Microglia play roles in many neurological diseases, including Alzheimer's disease (AD). In AD, microglia surround amyloid plaques, but it is not clear whether they are harmful (e.g., promoting inflammation) or beneficial (e.g., restricting plaque growth). To gain some insight into their function, Bolmont et al. imaged interactions occurring in vivo between microglia and amyloid plaques in a mouse model of AD. Microglia extended and retracted processes in all directions, but those that were near plaques extended more processes toward the plaque. Many nearby microglia migrated to the edge of a given plaque and remained there, but there was an upper limit to the number of microglia surrounding any plaque: larger plaques were associated with larger, not more, microglia. The total volume of microglia surrounding a plaque was predictive of whether the plaque grew over time, and microglia appeared to take up amyloid particles, suggesting the microglia may limit plaque growth.
Please click here for the current table of contents.
Source: Sara Harris
Society for Neuroscience
Guido A. Zampighi, Nick Fain, Lorenzo M. Zampighi, Francesca Cantele, Salvatore Lanzavecchia, and Ernest M. Wright
When looking at schematic illustrations of proteins found in presynaptic active zones, it is hard to imagine how all those proteins fit together in the cell. Even with electron microscopy, the organization of vesicle docking machinery is difficult to discriminate. But this week, Zampighi et al. present images of active zone complexes that were visualized using conical electron tomography. The authors used semiautomated volume-rendering techniques that colored individual voxels based on density thresholds and/or topology. The resulting images revealed that active zones of rat cortical synapses contain several units, each of which comprised a central polyhedral cage (which the authors call a syndesome) surrounded by synaptic vesicles. Some of these vesicles were partly or fully fused to the plasma membrane, suggesting that the polyhedral cages help mediate vesicle docking and fusion. Interestingly, the polyhedral cages resemble those of clathrin coats, which are normally associated with endocytosis rather than exocytosis.
2. Neurturin and Ret Influence Retinal Circuit Formation
Milam A. Brantley Jr, Sanjay Jain, Emily E. Barr, Eugene M. Johnson Jr, and Jeffrey Milbrandt
The receptor tyrosine kinase Ret, which is activated by glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), is essential for development of many tissues, and GDNF can slow retinal degeneration in animal models. Brantley et al. have detailed the role of this signaling pathway in retinal development. Mice with reduced Ret expression showed decreased light responses, as did mice lacking the GFL neurturin, but not other GFLs. Expression of fluorescent reporters under the control of Ret or neurturin receptor promoters indicated that both of these molecules are expressed in horizontal cells and some amacrine and ganglion cells. In neurturin knock-out mice, the outer plexiform layer (where photoreceptors synapse with horizontal and bipolar cells) was disorganized, horizontal cell axons and dendrites were sparse, bipolar and horizontal cell processes were abnormally long, and synapses were mislocalized to the outer nuclear layer. Therefore, neurturin-mediated Ret signaling appears necessary for normal circuit development in the retina.
3. Disinhibition Drives OFF Cell Depolarization
Michael B. Manookin, Deborah Langrill Beaudoin, Zachary Raymond Ernst, Leigh J. Flagel, and Jonathan B. Demb
It has been assumed that depolarization of retinal ganglion cells is driven by excitation from bipolar cells. Manookin et al. now report that OFF ganglion cells are also driven by reduced inhibition. Responses to light increments and decrements were recorded in guinea pig ON and OFF ganglion cells. At all increment levels, ON cells received both excitatory and inhibitory inputs; but at each decrement level, increased excitation of OFF cells was paired with decreased inhibition. By sequentially applying receptor agonists and antagonists to test each type of synapse (including gap junctions), it was determined that OFF cell inhibition is mediated by AII amacrine cells, which are electrically coupled to ON cone bipolar cells. When the light dims, ON cone bipolar cells hyperpolarize, which hyperpolarizes AII amacrine cells, thus reducing their inhibition of OFF ganglion cells. This disinhibition is the dominant driving force for OFF ganglion cells when light decrements are small.
4. Microglia Delimit Alzheimer Plaques
Tristan Bolmont, Florent Haiss, Daniel Eicke, Rebecca Radde, Chester A. Mathis, William E. Klunk, Shinichi Kohsaka, Mathias Jucker, and Michael E. Calhoun
Microglia play roles in many neurological diseases, including Alzheimer's disease (AD). In AD, microglia surround amyloid plaques, but it is not clear whether they are harmful (e.g., promoting inflammation) or beneficial (e.g., restricting plaque growth). To gain some insight into their function, Bolmont et al. imaged interactions occurring in vivo between microglia and amyloid plaques in a mouse model of AD. Microglia extended and retracted processes in all directions, but those that were near plaques extended more processes toward the plaque. Many nearby microglia migrated to the edge of a given plaque and remained there, but there was an upper limit to the number of microglia surrounding any plaque: larger plaques were associated with larger, not more, microglia. The total volume of microglia surrounding a plaque was predictive of whether the plaque grew over time, and microglia appeared to take up amyloid particles, suggesting the microglia may limit plaque growth.
Please click here for the current table of contents.
Source: Sara Harris
Society for Neuroscience
Mysteries Of Vitamin A Metabolism During Embryonic Development Unlocked By Rutgers Researchers
Researchers at Rutgers have unlocked some of the mysteries of how the developing embryo reacts to fluctuations in the amount of vitamin A present in the maternal blood stream. Their results are presented in the February 28 issue of the Journal of Biological Chemistry.
The researchers studied the role of LRAT, a protein that facilitates the formation of vitamin A stores in the body, during embryonic development. In particular, they showed how LRAT protects developing tissues from potentially toxic levels of vitamin A that have been ingested by the mother. Although this function of LRAT had previously been hypothesized in adults, this is the first time that its role has been demonstrated during embryonic development.
The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids, the vitamin A metabolites produced in the body. These are essential nutrients and they control the formation of the embryo's heart, central nervous system, eyes and other important organs and tissues. Malformations of the developing embryo can occur when too little, or too much, vitamin A is consumed by the mother.
"We were looking for the mechanisms that allow the fetus to maintain adequate amount of retinoids, whether the mother has over- or under-consumed vitamin A," said Dr. Loredana Quadro, an assistant professor in the Department of Food Science and member of the Center for Lipid Research at the Rutgers School of Environmental and Biological Sciences. "We also looked at the effects of different levels of vitamin A being transferred from the mother to the fetus."
When vitamin A is ingested, it is converted into retinyl ester (RE) in the intestine from where it is secreted in the bloodstream packaged with other dietary lipids into lipoprotein particles called chylomicrons. The majority of dietary RE reaches the liver, the main body storage site of vitamin A. Under insufficient dietary vitamin A intake, the liver transforms RE into retinol (ROH), which is then secreted into the bloodstream bound to retinol-binding protein (RBP), its sole specific serum carrier, to be delivered to the target tissues. Upon intake through a specific membrane receptor named Stra6, ROH is ultimately converted to retinoic acid (RA), which is the active form of vitamin A. If tissue RA is in excess, it is transformed into inactive forms, such as 4-hydroxy retinoic acid or 4-oxo retinoic acid (OXO-RA) by the action of a specific enzyme named Cyp26A1.
"When we think about vitamin A, we think about one compound," said Quadro. "But in reality, the term vitamin A comprises a family of different compounds. Each one has a slightly different action, and plays a different role."
The Rutgers researchers took a closer look at how ROH is metabolized into RE and RA to maintain an optimal balance of retinoids during the formation of the embryo. Mutant mice lacking both RBP and LRAT were generated to perform this study, so as to interfere with the two main pathways of maternal vitamin A delivery to the fetus (ROH-RBP from the liver stores and RE of dietary origin).
"We hypothesized that the lack of ROH-RBP and LRAT would make the embryo more vulnerable to changes in maternal dietary vitamin A intake," said Quadro "and our data proved this to be correct. Indeed, a severe embryonic vitamin A deficiency is readily attainable when the mothers are deprived of dietary vitamin A during pregnancy. Therefore, this strain turned out to be a very good model to study how embryonic development is affected by fluctuations in the amount of retinoids present in the maternal diet and hence in the maternal circulation".
The researchers identified LRAT, Cyp26A1 and Stra6 as the three key molecular players that act in coordination to protect the developing tissues from potentially detrimental levels of vitamin A ingested by the mother. "Understanding vitamin A metabolism in the developing fetus could have broad implications," said Quadro. "Consumption of large doses of dietary supplements and vitamins, including vitamin A, has become a very common practice in recent years, generating the necessity to investigate the effects of high doses of vitamin A intake at different stages of the lifecycle, including pregnancy and development. These studies expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and may ultimately provide new insight into appropriate dietary practices during pregnancy."
This research was lead by Quadro and carried out primarily by her lab members, Youn-Kyung Kim, a graduate assistant, and Dr. Lesley Wassef, a post-doctoral associate. Others contributing to the study were Leora Hamberger, a former research assistant in Quadro's laboratory, Dr. William Blaner and Roseann Piantedosi from Columbia University and Dr. Krzysztof Palczewski from Case Western Reserve.
The paper was previously published on the Journal of Biological Chemistry's web site on December 19, 2007.
Source: Michele Hujber
Rutgers University
The researchers studied the role of LRAT, a protein that facilitates the formation of vitamin A stores in the body, during embryonic development. In particular, they showed how LRAT protects developing tissues from potentially toxic levels of vitamin A that have been ingested by the mother. Although this function of LRAT had previously been hypothesized in adults, this is the first time that its role has been demonstrated during embryonic development.
The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids, the vitamin A metabolites produced in the body. These are essential nutrients and they control the formation of the embryo's heart, central nervous system, eyes and other important organs and tissues. Malformations of the developing embryo can occur when too little, or too much, vitamin A is consumed by the mother.
"We were looking for the mechanisms that allow the fetus to maintain adequate amount of retinoids, whether the mother has over- or under-consumed vitamin A," said Dr. Loredana Quadro, an assistant professor in the Department of Food Science and member of the Center for Lipid Research at the Rutgers School of Environmental and Biological Sciences. "We also looked at the effects of different levels of vitamin A being transferred from the mother to the fetus."
When vitamin A is ingested, it is converted into retinyl ester (RE) in the intestine from where it is secreted in the bloodstream packaged with other dietary lipids into lipoprotein particles called chylomicrons. The majority of dietary RE reaches the liver, the main body storage site of vitamin A. Under insufficient dietary vitamin A intake, the liver transforms RE into retinol (ROH), which is then secreted into the bloodstream bound to retinol-binding protein (RBP), its sole specific serum carrier, to be delivered to the target tissues. Upon intake through a specific membrane receptor named Stra6, ROH is ultimately converted to retinoic acid (RA), which is the active form of vitamin A. If tissue RA is in excess, it is transformed into inactive forms, such as 4-hydroxy retinoic acid or 4-oxo retinoic acid (OXO-RA) by the action of a specific enzyme named Cyp26A1.
"When we think about vitamin A, we think about one compound," said Quadro. "But in reality, the term vitamin A comprises a family of different compounds. Each one has a slightly different action, and plays a different role."
The Rutgers researchers took a closer look at how ROH is metabolized into RE and RA to maintain an optimal balance of retinoids during the formation of the embryo. Mutant mice lacking both RBP and LRAT were generated to perform this study, so as to interfere with the two main pathways of maternal vitamin A delivery to the fetus (ROH-RBP from the liver stores and RE of dietary origin).
"We hypothesized that the lack of ROH-RBP and LRAT would make the embryo more vulnerable to changes in maternal dietary vitamin A intake," said Quadro "and our data proved this to be correct. Indeed, a severe embryonic vitamin A deficiency is readily attainable when the mothers are deprived of dietary vitamin A during pregnancy. Therefore, this strain turned out to be a very good model to study how embryonic development is affected by fluctuations in the amount of retinoids present in the maternal diet and hence in the maternal circulation".
The researchers identified LRAT, Cyp26A1 and Stra6 as the three key molecular players that act in coordination to protect the developing tissues from potentially detrimental levels of vitamin A ingested by the mother. "Understanding vitamin A metabolism in the developing fetus could have broad implications," said Quadro. "Consumption of large doses of dietary supplements and vitamins, including vitamin A, has become a very common practice in recent years, generating the necessity to investigate the effects of high doses of vitamin A intake at different stages of the lifecycle, including pregnancy and development. These studies expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and may ultimately provide new insight into appropriate dietary practices during pregnancy."
This research was lead by Quadro and carried out primarily by her lab members, Youn-Kyung Kim, a graduate assistant, and Dr. Lesley Wassef, a post-doctoral associate. Others contributing to the study were Leora Hamberger, a former research assistant in Quadro's laboratory, Dr. William Blaner and Roseann Piantedosi from Columbia University and Dr. Krzysztof Palczewski from Case Western Reserve.
The paper was previously published on the Journal of Biological Chemistry's web site on December 19, 2007.
Source: Michele Hujber
Rutgers University
'Kiss Of Death' For Antibiotic-Resistant Germs With The Help Of Frog Skin
Kissing a frog won't turn it into a prince - except in fairy tales - but frogs may be hopping toward a real-world transformation into princely allies in humanity's battle with antibiotic-resistant infections that threaten millions of people worldwide. Scientists today reported that frog skin contains natural substances that could be the basis for a powerful new genre of antibiotics.
In a report at the 240th National Meeting of the American Chemical Society, the team of stalwart frog-fanciers described enlisting colleagues worldwide to ship secretions from hundreds of promising frog skins to their laboratory in the United Arab Emirates. Using that amphibious treasure trove, they identified more than 100 antibiotic substances in the skins of different frog species from around the world. One even fights "Iraqibacter," the bacterium responsible for drug-resistant infections in wounded soldiers returning from Iraq.
Michael Conlon, Ph.D., who reported on the research, noted that the emergence of drug-resistant bacteria, which have the ability to shrug off conventional antibiotics, is a growing problem worldwide. As a result, patients need new types of antibiotics to replace drugs that no longer work.
"Frog skin is an excellent potential source of such antibiotic agents," said Conlon, a biochemist at the United Arab Emirates University in Al-Ain, Abu Dhabi Emirate. "They've been around 300 million years, so they've had plenty of time to learn how to defend themselves against disease-causing microbes in the environment. Their own environment includes polluted waterways where strong defenses against pathogens are a must."
Scientists have known for years that the skin of frogs is a rich source of chemicals capable of killing bacteria, viruses, and fungi. Researchers have attempted to isolate those germ-fighting chemicals and make them suitable for development into new antibiotics. Success, however, has been elusive because froggy antibiotics tend to be toxic to human cells and certain chemicals in the bloodstream easily destroy them.
Conlon and colleagues described an approach to overcome these problems. They discovered a way to tweak the molecular structure of frog skin antibiotic substances, making them less toxic to human cells but more powerful germ killers. Similarly, the scientists also discovered other tweaks that enabled the frog skin secretions to shrug off attack by destructive enzymes in the blood. The result was antibiotics that last longer in the bloodstream and are more likely to be effective as infection fighters, Conlon noted.
The antibiotic substances work in an unusual way that makes it very difficult for disease-causing microbes to develop resistance, Conlon said.
The scientists are currently screening skin secretions from more than 6,000 species of frogs for antibiotic activity. So far, they have purified and determined the chemical structure of barely 200, leaving a potential bonanza of antibiotic substances awaiting discovery.
"Many people are working with me, giving me samples of frog skin secretions," said Conlon, who has a dozen research collaborators in Japan, France, the United States, and other countries. "We only actually use the frogs to get the chemical structure of the antibiotic, and then we make it in the lab. We take great care not to harm these delicate creatures, and scientists return them to the wild after swabbing their skin for the precious secretions."
One substance isolated from the skin secretions of the Foothill Yellow-legged Frog - a species once common in California and Oregon but now facing extinction - shows promise for killing methicillin-resistant Staphylococcus aureus (MRSA) bacteria. MRSA is a "superbug," infamous for causing deadly outbreaks of infection among hospitalized patients. Now it is occurring in settings outside hospitals, including schools, nursing homes, and day care centers.
The skin of the mink frog, likewise, contains secretions that show promise for fighting "Iraqibacter," caused by multidrug-resistant Acinetobacter baumanni.
Some of the substances could make their way into clinical trials within the next five years, Conlon predicted. He envisions that pharmaceutical companies could develop the chemicals as creams or ointments for treating skin infections or as injectable drugs for treating drug-resistant infections throughout the body. The United Arab Emirates University provided funding for the study.
"The research also is important because it underscores the importance of preserving biodiversity," Conlon pointed out. "Some frog species - including those that may contain potentially valuable medicinal substances - are in jeopardy worldwide due to loss of habitat, water pollution, and other problems."
The Skinny on Frog Skin
Frogs and toads have a "Lycra" type skin that protects them from injury and disease. It comes in a rainbow of color and patterns.
Frog skin is water permeable, letting water in and out. Frogs seldom drink with their mouths. Rather, they absorb water through their skin. A "seat pouch" on their bellies absorbs water.
Many species have skin glands that produce toxins and other substances to repel predators. Scientists are studying some as potential pain medications.
Skin colors and patterns are protective devices that warn predators that the frog may be poisonous. Some frogs change the color of their skin to absorb or reflect heat and thus control their body temperature. Patterned skin can help camouflage the frog, so it sinks into the background, hidden from predators.
Frogs regularly shed their skin. Most eat the shed skin.
Source:
Michael Bernstein
Michael Woods
American Chemical Society
In a report at the 240th National Meeting of the American Chemical Society, the team of stalwart frog-fanciers described enlisting colleagues worldwide to ship secretions from hundreds of promising frog skins to their laboratory in the United Arab Emirates. Using that amphibious treasure trove, they identified more than 100 antibiotic substances in the skins of different frog species from around the world. One even fights "Iraqibacter," the bacterium responsible for drug-resistant infections in wounded soldiers returning from Iraq.
Michael Conlon, Ph.D., who reported on the research, noted that the emergence of drug-resistant bacteria, which have the ability to shrug off conventional antibiotics, is a growing problem worldwide. As a result, patients need new types of antibiotics to replace drugs that no longer work.
"Frog skin is an excellent potential source of such antibiotic agents," said Conlon, a biochemist at the United Arab Emirates University in Al-Ain, Abu Dhabi Emirate. "They've been around 300 million years, so they've had plenty of time to learn how to defend themselves against disease-causing microbes in the environment. Their own environment includes polluted waterways where strong defenses against pathogens are a must."
Scientists have known for years that the skin of frogs is a rich source of chemicals capable of killing bacteria, viruses, and fungi. Researchers have attempted to isolate those germ-fighting chemicals and make them suitable for development into new antibiotics. Success, however, has been elusive because froggy antibiotics tend to be toxic to human cells and certain chemicals in the bloodstream easily destroy them.
Conlon and colleagues described an approach to overcome these problems. They discovered a way to tweak the molecular structure of frog skin antibiotic substances, making them less toxic to human cells but more powerful germ killers. Similarly, the scientists also discovered other tweaks that enabled the frog skin secretions to shrug off attack by destructive enzymes in the blood. The result was antibiotics that last longer in the bloodstream and are more likely to be effective as infection fighters, Conlon noted.
The antibiotic substances work in an unusual way that makes it very difficult for disease-causing microbes to develop resistance, Conlon said.
The scientists are currently screening skin secretions from more than 6,000 species of frogs for antibiotic activity. So far, they have purified and determined the chemical structure of barely 200, leaving a potential bonanza of antibiotic substances awaiting discovery.
"Many people are working with me, giving me samples of frog skin secretions," said Conlon, who has a dozen research collaborators in Japan, France, the United States, and other countries. "We only actually use the frogs to get the chemical structure of the antibiotic, and then we make it in the lab. We take great care not to harm these delicate creatures, and scientists return them to the wild after swabbing their skin for the precious secretions."
One substance isolated from the skin secretions of the Foothill Yellow-legged Frog - a species once common in California and Oregon but now facing extinction - shows promise for killing methicillin-resistant Staphylococcus aureus (MRSA) bacteria. MRSA is a "superbug," infamous for causing deadly outbreaks of infection among hospitalized patients. Now it is occurring in settings outside hospitals, including schools, nursing homes, and day care centers.
The skin of the mink frog, likewise, contains secretions that show promise for fighting "Iraqibacter," caused by multidrug-resistant Acinetobacter baumanni.
Some of the substances could make their way into clinical trials within the next five years, Conlon predicted. He envisions that pharmaceutical companies could develop the chemicals as creams or ointments for treating skin infections or as injectable drugs for treating drug-resistant infections throughout the body. The United Arab Emirates University provided funding for the study.
"The research also is important because it underscores the importance of preserving biodiversity," Conlon pointed out. "Some frog species - including those that may contain potentially valuable medicinal substances - are in jeopardy worldwide due to loss of habitat, water pollution, and other problems."
The Skinny on Frog Skin
Frogs and toads have a "Lycra" type skin that protects them from injury and disease. It comes in a rainbow of color and patterns.
Frog skin is water permeable, letting water in and out. Frogs seldom drink with their mouths. Rather, they absorb water through their skin. A "seat pouch" on their bellies absorbs water.
Many species have skin glands that produce toxins and other substances to repel predators. Scientists are studying some as potential pain medications.
Skin colors and patterns are protective devices that warn predators that the frog may be poisonous. Some frogs change the color of their skin to absorb or reflect heat and thus control their body temperature. Patterned skin can help camouflage the frog, so it sinks into the background, hidden from predators.
Frogs regularly shed their skin. Most eat the shed skin.
Source:
Michael Bernstein
Michael Woods
American Chemical Society
Discovery Of Circadian Rhythm-Metabolism Link
UC Irvine researchers have found a molecular link between circadian rhythms - our own body clock - and metabolism. The discovery reveals new possibilities for the treatment of diabetes, obesity and other related diseases.
Paolo Sassone-Corsi, Distinguished Professor and Chair of Pharmacology, and his colleagues have identified that an essential protein called CLOCK that regulates the body's circadian rhythms, works in balance with another protein called SIRT1 that modulates how much energy a cell uses.
"This interplay has far-reaching implications for human illness and aging, and it is likely vital for proper metabolism," said Sassone-Corsi, one of the world's leading researchers on circadian rhythms. The study appears in the July 25 issue of Cell.
Circadian rhythms of 24 hours govern fundamental physiological functions in almost all organisms. The circadian clocks are intrinsic time-tracking systems in our bodies that anticipate environmental changes and adapt themselves to the appropriate time of day.
Disruption of these rhythms can profoundly influence human health and has been linked to metabolic disorders, insomnia, depression, coronary heart diseases and cancer.
It is estimated that up to 15 percent of our genes are regulated by these circadian clocks. Sassone-Corsi identified in 2006 that the protein CLOCK is an essential molecular gear of the circadian machinery.
Now, he and his colleagues have shown that the protein SIRT1 counterbalances the function of CLOCK. Even though SIRT1's function differs from CLOCK's, the two proteins interact, creating a bond that is finely regulated in the cell.
SIRT1 senses energy levels in the cell; its activity is modulated by how many nutrients a cell is consuming. It also helps cells resist oxidative and radiation-induced stress, and for this reason SIRT1 is known to help control the process of aging.
CLOCK and SIRT1 are both part of the epigenome, which consists of proteins existing in connection with a cell's DNA that take external environmental factors and make the cell's genes behave differently, even though those genes do not structurally change.
"When this balance between these two vital proteins is upset, normal cellular function can be disrupted," Sassone-Corsi said. "Because of the role these two enzymes play, changes in our sleep patterns or our diets can directly be translated into how our cells act."
The findings also suggest that proper sleep and diet could help maintain or rebuild the CLOCK-SIRT1 equilibrium and may help explain why lack of proper rest or disruption in our normal sleep patterns is known to increase hunger, which can lead to obesity and related illnesses and can accelerate the aging process.
The specific interaction between CLOCK and SIRT1 also could lead to the development of drugs aimed at facilitating healthy metabolism, thereby helping to solve major social and medical problems such as diabetes and obesity.
Yasukazu Nakahata, Milota Kaluzova, Benedetto Grimaldi, Saurabh Sahar and Jun Hirayama of UCI, and Danica Chen and Leonard P. Guarente of the Massachusetts Institute of Technology participated in the study, which was supported by the Cancer Research Coordinating Committee of the University of California and the National Institutes of Health.
About the University of California, Irvine:
The University of California, Irvine is a top-ranked university dedicated to research, scholarship and community service. Founded in 1965, UCI is among the fastest-growing University of California campuses, with more than 27,000 undergraduate and graduate students and nearly 2,000 faculty members. The third-largest employer in dynamic Orange County, UCI contributes an annual economic impact of $3.6 billion. For more UCI news, visit today.uci/.
Source: Tom Vasich
University of California - Irvine
Paolo Sassone-Corsi, Distinguished Professor and Chair of Pharmacology, and his colleagues have identified that an essential protein called CLOCK that regulates the body's circadian rhythms, works in balance with another protein called SIRT1 that modulates how much energy a cell uses.
"This interplay has far-reaching implications for human illness and aging, and it is likely vital for proper metabolism," said Sassone-Corsi, one of the world's leading researchers on circadian rhythms. The study appears in the July 25 issue of Cell.
Circadian rhythms of 24 hours govern fundamental physiological functions in almost all organisms. The circadian clocks are intrinsic time-tracking systems in our bodies that anticipate environmental changes and adapt themselves to the appropriate time of day.
Disruption of these rhythms can profoundly influence human health and has been linked to metabolic disorders, insomnia, depression, coronary heart diseases and cancer.
It is estimated that up to 15 percent of our genes are regulated by these circadian clocks. Sassone-Corsi identified in 2006 that the protein CLOCK is an essential molecular gear of the circadian machinery.
Now, he and his colleagues have shown that the protein SIRT1 counterbalances the function of CLOCK. Even though SIRT1's function differs from CLOCK's, the two proteins interact, creating a bond that is finely regulated in the cell.
SIRT1 senses energy levels in the cell; its activity is modulated by how many nutrients a cell is consuming. It also helps cells resist oxidative and radiation-induced stress, and for this reason SIRT1 is known to help control the process of aging.
CLOCK and SIRT1 are both part of the epigenome, which consists of proteins existing in connection with a cell's DNA that take external environmental factors and make the cell's genes behave differently, even though those genes do not structurally change.
"When this balance between these two vital proteins is upset, normal cellular function can be disrupted," Sassone-Corsi said. "Because of the role these two enzymes play, changes in our sleep patterns or our diets can directly be translated into how our cells act."
The findings also suggest that proper sleep and diet could help maintain or rebuild the CLOCK-SIRT1 equilibrium and may help explain why lack of proper rest or disruption in our normal sleep patterns is known to increase hunger, which can lead to obesity and related illnesses and can accelerate the aging process.
The specific interaction between CLOCK and SIRT1 also could lead to the development of drugs aimed at facilitating healthy metabolism, thereby helping to solve major social and medical problems such as diabetes and obesity.
Yasukazu Nakahata, Milota Kaluzova, Benedetto Grimaldi, Saurabh Sahar and Jun Hirayama of UCI, and Danica Chen and Leonard P. Guarente of the Massachusetts Institute of Technology participated in the study, which was supported by the Cancer Research Coordinating Committee of the University of California and the National Institutes of Health.
About the University of California, Irvine:
The University of California, Irvine is a top-ranked university dedicated to research, scholarship and community service. Founded in 1965, UCI is among the fastest-growing University of California campuses, with more than 27,000 undergraduate and graduate students and nearly 2,000 faculty members. The third-largest employer in dynamic Orange County, UCI contributes an annual economic impact of $3.6 billion. For more UCI news, visit today.uci/.
Source: Tom Vasich
University of California - Irvine
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