Site of the day: http://uberaffiliate.com/
ScienceDaily (Nov. 5, 2009) — New insight into how nature handles some fundamental processes is guiding researchers in the design of tailor-made proteins for applications such as artificial photosynthetic centers, long-range electron transfers, and fuel-cell catalysts for energy conversion.
From rusting iron to forest fires to the beating of a human heart, oxidation-reduction reactions, which transfer electrons from one atom to another, are at the heart of many chemical and biological processes. Each process requires a particular redox potential, just as different electronic devices can require their own special battery.
How nature fine-tunes these potentials over a broad range with little change to the protein's electron-transfer properties or efficiency has largely remained a mystery.
Now, a team led by University of Illinois chemistry professor Yi Lu has unearthed nature's secret, and has utilized it to their advantage. The researchers describe their work in a paper to appear in the Nov. 5 issue of the journal Nature. "We show that two important interactions, hydrophobicity (water repelling) and hydrogen bonding, are capable of fine-tuning the reduction potential of a particular class of copper-containing proteins called cupredoxins," Lu said. "We extended the range both above and below what had previously been found in nature."
Lu, graduate student and lead author Nicholas M. Marshall, and their collaborators also show that the effects of hydrophobicity and hydrogen bonding are additive, which offers additional control and extends the range of redox (short for oxidation-reduction reaction) potentials beyond what nature, by itself, provides.
Previously, to cover a wide potential range, scientists had to use several different redox agents in conjunction. This made it difficult, if not impossible, to tune the redox potentials without changing other electron transfer properties or the efficiency.
Also, stable, water-soluble redox agents are rare, Lu said, and those that are available have a limited potential range. "Consequently, there is a huge demand for efficient, water-soluble redox agents with a wide potential range for environmentally friendly aqueous or biochemical studies," he said.
To unlock nature's secret, Lu's team studied the behavior of the cupredoxin, azurin. Cupredoxins are redox-active copper proteins that play crucial roles in many important processes, such as photosynthesis and cell signaling. Cupredoxins use a single redox-active center, whose reduction potential is tunable without compromising the structure and electron transfer properties of the protein.
The researchers found that two interactions -- hydrophobicity and hydrogen bonding -- can selectively raise or lower azurin's redox potential. The interactions occur not in the metalloprotein's innermost, primary core, but in a secondary sphere that surrounds the primary core.
Increasing the hydrophobicity in the secondary sphere can significantly increase the redox potential, the researchers report. The more this secondary region repels water, the more the overall charge on the copper ion becomes destabilized and the higher the potential becomes.
The effect of the hydrogen bonding interaction is subtler than the effect of hydrophobicity, Lu said. Hydrogen bonding can either increase or decrease electron densities around a residue that binds the copper ion in azurin, making the copper ion either easier or harder to reduce and thus slightly changing the redox potential.
"This was nature's secret," Lu said. "That by adjusting the hydrophobicity and the hydrogen bonding, we can raise or lower the redox potential, without changing the protein's electron-transfer properties or decreasing the protein's efficiency."
The result is a tailor-made redox agent that can be set with a very high potential, a very low potential, or with a potential somewhere in between.
"This unprecedented level of control over an electron-transfer protein was achieved by mapping out the major interactions," Lu said, "an approach that may apply to other redox proteins of interest, as well."
Lu is affiliated with the university's Beckman Institute, the departments of biochemistry, bioengineering, and materials science and engineering, the Frederick Seitz Materials Research Laboratory, and the Center of Biophysics and Computational Biology. The National Science Foundation and the National Institutes of Health funded the work.
(http://www.sciencedaily.com/releases/2009/11/091104132702.htm)
Sunday, June 20, 2010
Intracellular Express: Why Transport Protein Molecules Have Brakes
Site of the day: http://uberaffiliate.com/
ScienceDaily (May 21, 2010) — Every single one of our cells contains so-called motor proteins that transport important substances from one location to another. However, very little is known about how exactly these transport processes occur. Biophysicists at the Technische Universitaet Muenchen (TUM) and Ludwig Maximilians Universitaet Muenchen (LMU) have now succeeded in explaining fundamental functions of a particularly interesting motor protein.
They report their findings in the current issue of the Proceedings of the National Academy of Sciences (USA).
Motorized transport proteins are one of the keys to the development of higher organisms. It is they that enable the cell to transport important substances directly and quickly to a specific location in the cell. As bacteria cannot do this, they are not able to form larger cells or even large organisms with many cells. Particularly important are fast transport proteins in the primary cilia, the cell's antennas, with which they channel information from the surroundings into the cell.
Like trucks on a highway, kinesins transport cellular cargoes to their destinations. They do this by crawling along protein fibers, so-called microtubules, which extend through the entire cell. Kinesins consist of two long intertwined protein chains. At one end of every protein there is a head that can attach itself to certain structures on the surface of the microtubules; the freight is attached to the other end.
Very special kinesins are at work in the cilia of the Caenorhabditis elegans nematode: They consist of two different protein chains and are therefore especially suitable for investigating the transport mechanisms. As freight, the researchers attached small plastic beads to the ends of these motor proteins. They can manipulate these beads with "optical tweezers," a specially formed laser beam.
One end of the protein molecule was held with the optical tweezers; the other was able to walk on microtubules. This enabled the scientists to measure the force with which the motor protein can pull. In this experimental setup, the kinesin-2 with its freight walks as far as 1,500 nanometers in tiny steps measuring a mere eight nanometers. "If we didn't hold it back, it might still go a lot further," says Zeynep Okten from the Institute for Cell Biology at LMU.
The kinesin-2 investigated consists of one KLP11 and one KLP20 protein. By exchanging the heads of the chains, the researchers were able to show that KLP11 is a non-processive motor protein. It only becomes a transport protein in combination with KLP20. In further experiments they were able to explain why nature chooses this unusual combination: KLP20 proteins have no "brakes." A transport protein made of two KLP20 units would be permanently on the go and would waste energy. The KLP11, in contrast, has a mechanism called autoinhibition, which makes sure that the transport protein is at a standstill if no freight is attached.
"Our results show that a molecular motor must take on a large number of functions over and above simple transport, if it wants to operate successfully in a cell," says Professor Matthias Rief from the Physics Department of the TU Muenchen. It must be possible to switch the motor on and off, and it must be able to accept a load needed at a specific location and hand it over at the destination. "It is impressive how nature manages to combine all of these functions in one molecule," Rief says. "In this respect it is still far superior to all the efforts of modern nanotechnology and serves as a great example to us all."
This work was supported by funds from the Cluster of Excellence Center for Integrated Protein Science Munich (CIPSM), a long-term European Molecular Biology Organization fellowship, and grants from the Deutsche Forschungsgemeinschaft (DFG) and the Friedrich-Baur-Stiftung.
Journal Reference:
Melanie Brunnbauer, Felix Mueller-Planitz, Suleyman Kosem, Thi-Hieu Hoa, Renate Dombi, J. Christof M. Gebhardt, Matthias Rief, and Zeynep Okten. Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner. PNAS, May 17, 2010 DOI: 10.1073/pnas.1005177107
(http://www.sciencedaily.com/releases/2010/05/100521191233.htm)
ScienceDaily (May 21, 2010) — Every single one of our cells contains so-called motor proteins that transport important substances from one location to another. However, very little is known about how exactly these transport processes occur. Biophysicists at the Technische Universitaet Muenchen (TUM) and Ludwig Maximilians Universitaet Muenchen (LMU) have now succeeded in explaining fundamental functions of a particularly interesting motor protein.
They report their findings in the current issue of the Proceedings of the National Academy of Sciences (USA).
Motorized transport proteins are one of the keys to the development of higher organisms. It is they that enable the cell to transport important substances directly and quickly to a specific location in the cell. As bacteria cannot do this, they are not able to form larger cells or even large organisms with many cells. Particularly important are fast transport proteins in the primary cilia, the cell's antennas, with which they channel information from the surroundings into the cell.
Like trucks on a highway, kinesins transport cellular cargoes to their destinations. They do this by crawling along protein fibers, so-called microtubules, which extend through the entire cell. Kinesins consist of two long intertwined protein chains. At one end of every protein there is a head that can attach itself to certain structures on the surface of the microtubules; the freight is attached to the other end.
Very special kinesins are at work in the cilia of the Caenorhabditis elegans nematode: They consist of two different protein chains and are therefore especially suitable for investigating the transport mechanisms. As freight, the researchers attached small plastic beads to the ends of these motor proteins. They can manipulate these beads with "optical tweezers," a specially formed laser beam.
One end of the protein molecule was held with the optical tweezers; the other was able to walk on microtubules. This enabled the scientists to measure the force with which the motor protein can pull. In this experimental setup, the kinesin-2 with its freight walks as far as 1,500 nanometers in tiny steps measuring a mere eight nanometers. "If we didn't hold it back, it might still go a lot further," says Zeynep Okten from the Institute for Cell Biology at LMU.
The kinesin-2 investigated consists of one KLP11 and one KLP20 protein. By exchanging the heads of the chains, the researchers were able to show that KLP11 is a non-processive motor protein. It only becomes a transport protein in combination with KLP20. In further experiments they were able to explain why nature chooses this unusual combination: KLP20 proteins have no "brakes." A transport protein made of two KLP20 units would be permanently on the go and would waste energy. The KLP11, in contrast, has a mechanism called autoinhibition, which makes sure that the transport protein is at a standstill if no freight is attached.
"Our results show that a molecular motor must take on a large number of functions over and above simple transport, if it wants to operate successfully in a cell," says Professor Matthias Rief from the Physics Department of the TU Muenchen. It must be possible to switch the motor on and off, and it must be able to accept a load needed at a specific location and hand it over at the destination. "It is impressive how nature manages to combine all of these functions in one molecule," Rief says. "In this respect it is still far superior to all the efforts of modern nanotechnology and serves as a great example to us all."
This work was supported by funds from the Cluster of Excellence Center for Integrated Protein Science Munich (CIPSM), a long-term European Molecular Biology Organization fellowship, and grants from the Deutsche Forschungsgemeinschaft (DFG) and the Friedrich-Baur-Stiftung.
Journal Reference:
Melanie Brunnbauer, Felix Mueller-Planitz, Suleyman Kosem, Thi-Hieu Hoa, Renate Dombi, J. Christof M. Gebhardt, Matthias Rief, and Zeynep Okten. Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner. PNAS, May 17, 2010 DOI: 10.1073/pnas.1005177107
(http://www.sciencedaily.com/releases/2010/05/100521191233.htm)
Artificial Antibodies Hold Biomedical Promise
Site of the day: http://uberaffiliate.com/
ScienceDaily (May 20, 2010) — Antibodies are the immune system's warriors. Their role is to pinpoint disease pathogens, attaching to them and neutralizing their effects. Though antibodies are of great value for biomedical research, the process of creating them has been time-consuming and tedious. Researchers at the Biodesign Institute at Arizona State University have developed a much faster and simpler way of making synthetic antibodies, by carrying out the usual steps in reverse.
Stephen Albert Johnston and Chris Diehnelt of Biodesign's Center for Innovations in Medicine at Arizona State University, along with their colleagues, have developed a technique for constructing amino acid sequences, then linking them together to form a synthetic antibody, or synbody, that can bind with one or more protein molecules contained in the vast repository of human proteins -- the proteome.
The group has developed a high affinity synbody capable of binding with AKT-1, a critical protein believed to play a role in aging, obesity, and cancer. In addition to the potential of synbodies to directly target proteins associated with disease-causing microbes, they also show great potential as a research tool and building block for novel diagnostics and treatments. The team's findings appear in the current issue of the journal PLoS ONE.
As Johnston notes, traditional antibodies are already in wide use for biological research, but the existing procedures for producing them are laborious and costly. "Traditional antibodies are made by taking the protein you want to bind," Johnston says, "and injecting it into an animal, which responds by making antibodies." These antibodies, or the cells that produce them, are then extracted.
Rather than beginning with a protein in order to produce an antibody, the new technique involves building an antibody first. "We turn the whole process on its head, making the antibody chemically, then finding out what it's an antibody to," Johnston says.
To accomplish this, a 20-unit random sequence of amino acids are joined together like beads on a necklace to form a peptide. By uniting two of these peptide chains, linked together by means of a chemical scaffold, a binding molecule or ligand is created, which can attach to a specific protein with high affinity. The resulting synbody may then be screened against a multitude of human proteins, to find its mate.
The strategy relies on the fact that the binding affinity of two such amino acid sequences is the product of their combined affinity, allowing two peptides with weak attraction to a given protein to be joined to produce a synbody with strong binding properties. Remarkably, the assemblage of both the individual peptides and the synbody are carried out randomly.
The raw material for the synbody comes from a library of 10,000 peptides, with each amino acid sequence randomly composed. As Johnston explains, "the randomness turned out to be the key to all of this, because a random sequence has more flexibility and degrees of freedom than life sequences do." Each resulting linear peptide chain is able to find 2 or 3 points of contact with virtually any protein. When two such peptides are combined to form a synbody, a high-affinity ligand is produced, displaying specificity for a given protein.
Currently, the only limiting technological consideration is the number of proteins that can be placed on an array slide and that capability, as Johnston notes, is rapidly improving. Another key advantage in the use of synbodies is that they remain stable over time, unlike their biological counterparts, making them far more suitable for diagnostic assays.
Exposing random synbodies to multiple proteins helps build a library of effective ligands over time. To create a synbody to a particular disease protein on the other hand, the protein is exposed to multiple peptides. Once two are identified that link to the protein, they may be combined into a disease-specific synbody -- an effective, though much slower process.
The ability to produce ligands to all 30,000 proteins in the human proteome would be a boon to science, offering the ability to study any protein in the body with fine-grained specificity and to develop a suite of new diagnostic tools. Proposals exist to complete such a daunting task by traditional means, at an estimated cost of $1 billion over the course of ten years.
"I'm too impatient," Johnston says. "And it's too much money." The synbody approach, in which a Lego-like peptide kit is used to produce high-affinity ligands offers a plausible route to addressing the problem by high throughput means at substantially lower cost.
Journal Reference:
Chris W. Diehnelt, Miti Shah, Nidhi Gupta, Paul E. Belcher, Matthew P. Greving, Phillip Stafford, Stephen Albert Johnston. Discovery of High-Affinity Protein Binding Ligands - Backwards. PLoS ONE, 2010; 5 (5): e10728 DOI: 10.1371/journal.pone.0010728
(http://www.sciencedaily.com/releases/2010/05/100519173100.htm)
ScienceDaily (May 20, 2010) — Antibodies are the immune system's warriors. Their role is to pinpoint disease pathogens, attaching to them and neutralizing their effects. Though antibodies are of great value for biomedical research, the process of creating them has been time-consuming and tedious. Researchers at the Biodesign Institute at Arizona State University have developed a much faster and simpler way of making synthetic antibodies, by carrying out the usual steps in reverse.
Stephen Albert Johnston and Chris Diehnelt of Biodesign's Center for Innovations in Medicine at Arizona State University, along with their colleagues, have developed a technique for constructing amino acid sequences, then linking them together to form a synthetic antibody, or synbody, that can bind with one or more protein molecules contained in the vast repository of human proteins -- the proteome.
The group has developed a high affinity synbody capable of binding with AKT-1, a critical protein believed to play a role in aging, obesity, and cancer. In addition to the potential of synbodies to directly target proteins associated with disease-causing microbes, they also show great potential as a research tool and building block for novel diagnostics and treatments. The team's findings appear in the current issue of the journal PLoS ONE.
As Johnston notes, traditional antibodies are already in wide use for biological research, but the existing procedures for producing them are laborious and costly. "Traditional antibodies are made by taking the protein you want to bind," Johnston says, "and injecting it into an animal, which responds by making antibodies." These antibodies, or the cells that produce them, are then extracted.
Rather than beginning with a protein in order to produce an antibody, the new technique involves building an antibody first. "We turn the whole process on its head, making the antibody chemically, then finding out what it's an antibody to," Johnston says.
To accomplish this, a 20-unit random sequence of amino acids are joined together like beads on a necklace to form a peptide. By uniting two of these peptide chains, linked together by means of a chemical scaffold, a binding molecule or ligand is created, which can attach to a specific protein with high affinity. The resulting synbody may then be screened against a multitude of human proteins, to find its mate.
The strategy relies on the fact that the binding affinity of two such amino acid sequences is the product of their combined affinity, allowing two peptides with weak attraction to a given protein to be joined to produce a synbody with strong binding properties. Remarkably, the assemblage of both the individual peptides and the synbody are carried out randomly.
The raw material for the synbody comes from a library of 10,000 peptides, with each amino acid sequence randomly composed. As Johnston explains, "the randomness turned out to be the key to all of this, because a random sequence has more flexibility and degrees of freedom than life sequences do." Each resulting linear peptide chain is able to find 2 or 3 points of contact with virtually any protein. When two such peptides are combined to form a synbody, a high-affinity ligand is produced, displaying specificity for a given protein.
Currently, the only limiting technological consideration is the number of proteins that can be placed on an array slide and that capability, as Johnston notes, is rapidly improving. Another key advantage in the use of synbodies is that they remain stable over time, unlike their biological counterparts, making them far more suitable for diagnostic assays.
Exposing random synbodies to multiple proteins helps build a library of effective ligands over time. To create a synbody to a particular disease protein on the other hand, the protein is exposed to multiple peptides. Once two are identified that link to the protein, they may be combined into a disease-specific synbody -- an effective, though much slower process.
The ability to produce ligands to all 30,000 proteins in the human proteome would be a boon to science, offering the ability to study any protein in the body with fine-grained specificity and to develop a suite of new diagnostic tools. Proposals exist to complete such a daunting task by traditional means, at an estimated cost of $1 billion over the course of ten years.
"I'm too impatient," Johnston says. "And it's too much money." The synbody approach, in which a Lego-like peptide kit is used to produce high-affinity ligands offers a plausible route to addressing the problem by high throughput means at substantially lower cost.
Journal Reference:
Chris W. Diehnelt, Miti Shah, Nidhi Gupta, Paul E. Belcher, Matthew P. Greving, Phillip Stafford, Stephen Albert Johnston. Discovery of High-Affinity Protein Binding Ligands - Backwards. PLoS ONE, 2010; 5 (5): e10728 DOI: 10.1371/journal.pone.0010728
(http://www.sciencedaily.com/releases/2010/05/100519173100.htm)
Tuesday, June 15, 2010
Key Regulator of Fat Cell Development Identified
Site of the day: http://johnchow.com/blog/
ScienceDaily (June 6, 2010) — New research led by UCD Conway Fellow, Professor Johan Ericsson has identified a key regulator of fat cell differentiation that may be a novel target for obesity drugs.
The results of the research are published in the current edition of the scientific journal, Proceedings of the National Academy of Sciences (PNAS).
Adipose tissue plays an important role in controlling the balance of insulin in our bodies as well as energy production and consumption. White adipose tissue is a storage depot for excess energy in the form of fat. Excessive adipocyte (fat cell) size and/or number is a hallmark of obesity, which is a major risk factor for developing type-II diabetes, cardiovascular disease and hypertension.
Professor Ericsson and his team have demonstrated that the protein Fbxw7 regulates the cascade of events that control the development of fat cells. They found that when Fbxw7 was inactive, the development of fat cells was enhanced. In fact, in the case of mouse precursor or immature fat cells, the inactivation of this protein alone was enough to convert them to mature fat cells. The team also demonstrated that Fbxw7 blocks the development of fat cells because it tags other proteins in the regulatory cascade for destruction.
Commenting on the results, Professor Ericsson said; 'The picture we have built of the role of Fbxw7 in energy and lipid metabolism suggests that it is a key regulator of this process. As such, it may impact on two very important health problems, type-II diabetes and obesity'.
In the future, the group would like to determine exactly how Fbxw7 regulates adipocyte differentiation, and identify the factors and signals that control the amount of this protein in fat cells. It will also be important to determine if the levels of Fbxw7 are changed during the development of obesity.
In Ireland, an estimated 200,000 people are suffering from type-II diabetes and it is anticipated that this will rise by 37% in the coming years. This places an enormous burden on the national healthcare budget as people are treated for the disease and its complications.
Johan Ericsson is a Science Foundation Ireland funded Stokes professor. Prior to joining UCD Conway Institute in 2009, he worked at the Ludwig Institute for Cancer Research in Sweden.
Journal Reference:
M. T. Bengoechea-Alonso, J. Ericsson. The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBP for degradation. Proceedings of the National Academy of Sciences, 2010; DOI: 10.1073/pnas.0913367107
(http://www.sciencedaily.com/releases/2010/06/100603091817.htm)
ScienceDaily (June 6, 2010) — New research led by UCD Conway Fellow, Professor Johan Ericsson has identified a key regulator of fat cell differentiation that may be a novel target for obesity drugs.
The results of the research are published in the current edition of the scientific journal, Proceedings of the National Academy of Sciences (PNAS).
Adipose tissue plays an important role in controlling the balance of insulin in our bodies as well as energy production and consumption. White adipose tissue is a storage depot for excess energy in the form of fat. Excessive adipocyte (fat cell) size and/or number is a hallmark of obesity, which is a major risk factor for developing type-II diabetes, cardiovascular disease and hypertension.
Professor Ericsson and his team have demonstrated that the protein Fbxw7 regulates the cascade of events that control the development of fat cells. They found that when Fbxw7 was inactive, the development of fat cells was enhanced. In fact, in the case of mouse precursor or immature fat cells, the inactivation of this protein alone was enough to convert them to mature fat cells. The team also demonstrated that Fbxw7 blocks the development of fat cells because it tags other proteins in the regulatory cascade for destruction.
Commenting on the results, Professor Ericsson said; 'The picture we have built of the role of Fbxw7 in energy and lipid metabolism suggests that it is a key regulator of this process. As such, it may impact on two very important health problems, type-II diabetes and obesity'.
In the future, the group would like to determine exactly how Fbxw7 regulates adipocyte differentiation, and identify the factors and signals that control the amount of this protein in fat cells. It will also be important to determine if the levels of Fbxw7 are changed during the development of obesity.
In Ireland, an estimated 200,000 people are suffering from type-II diabetes and it is anticipated that this will rise by 37% in the coming years. This places an enormous burden on the national healthcare budget as people are treated for the disease and its complications.
Johan Ericsson is a Science Foundation Ireland funded Stokes professor. Prior to joining UCD Conway Institute in 2009, he worked at the Ludwig Institute for Cancer Research in Sweden.
Journal Reference:
M. T. Bengoechea-Alonso, J. Ericsson. The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBP for degradation. Proceedings of the National Academy of Sciences, 2010; DOI: 10.1073/pnas.0913367107
(http://www.sciencedaily.com/releases/2010/06/100603091817.htm)
Breakthrough in Stem Cell Culturing
Site of the day: http://johnchow.com/blog/
ScienceDaily (June 1, 2010) — For the first time, human embryonic stem cells have been cultured under chemically controlled conditions without the use of animal substances, which is essential for future clinical uses.
The method has been developed by researchers at Karolinska Institutet and is presented in the journal Nature Biotechnology.
Embryonic stem cells can be turned into any other type of cell in the body and have potential uses in treatments where sick cells need to be replaced. One problem, however, is that it is difficult to culture and develop human embryonic stem cells without simultaneously contaminating them. They are currently cultured with the help of proteins from animals, which rules out subsequent use in the treatment of humans. Alternatively the stem cells can be cultured on other human cells, known as feeder cells, but these release thousands of uncontrolled proteins and therefore lead to unreliable research results.
A research team at Karolinska Institutet has now managed to produce human stem cells entirely without the use of other cells or substances from animals. Instead they are cultured on a matrix of a single human protein: laminin-511.
"Now, for the first time, we can produce large quantities of human embryonic stem cells in an environment that is completely chemically defined," says professor Karl Tryggvason, who led the study. "This opens up new opportunities for developing different types of cell which can then be tested for the treatment of disease."
Together with researchers at the Harvard Stem Cell Institute, the researchers have also shown that in the same way they can culture what are known as reprogrammed stem cells, which have been converted "back" from tissue cells to stem cells.
Laminin-511 is part of our connective tissue and acts in the body as a matrix to which cells can attach. In the newly formed embryo, the protein is also needed to keep stem cells as stem cells. Once the embryo begins to develop different types of tissue, other types of laminin are needed.
Until now, different types of laminin have not been available to researchers, because they are almost impossible to extract from tissues and difficult to produce. Over the last couple of decades, Karl Tryggvason's research group has cloned the genes for most human laminins, studied their biological role, described two genetic laminin diseases and, in recent years, even managed to produce several types of laminin using gene technology. In this latest experiment, the researchers produced the laminin-511 using recombinant techniques.
Journal Reference:
Sergey Rodin, Anna Domogatskaya, Susanne Ström, Emil M Hansson, Kenneth R Chien, José Inzunza, Outi Hovatta, Karl Tryggvason. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nature Biotechnology, 2010; DOI: 10.1038/nbt.1620
(http://www.sciencedaily.com/releases/2010/05/100531082905.htm)
ScienceDaily (June 1, 2010) — For the first time, human embryonic stem cells have been cultured under chemically controlled conditions without the use of animal substances, which is essential for future clinical uses.
The method has been developed by researchers at Karolinska Institutet and is presented in the journal Nature Biotechnology.
Embryonic stem cells can be turned into any other type of cell in the body and have potential uses in treatments where sick cells need to be replaced. One problem, however, is that it is difficult to culture and develop human embryonic stem cells without simultaneously contaminating them. They are currently cultured with the help of proteins from animals, which rules out subsequent use in the treatment of humans. Alternatively the stem cells can be cultured on other human cells, known as feeder cells, but these release thousands of uncontrolled proteins and therefore lead to unreliable research results.
A research team at Karolinska Institutet has now managed to produce human stem cells entirely without the use of other cells or substances from animals. Instead they are cultured on a matrix of a single human protein: laminin-511.
"Now, for the first time, we can produce large quantities of human embryonic stem cells in an environment that is completely chemically defined," says professor Karl Tryggvason, who led the study. "This opens up new opportunities for developing different types of cell which can then be tested for the treatment of disease."
Together with researchers at the Harvard Stem Cell Institute, the researchers have also shown that in the same way they can culture what are known as reprogrammed stem cells, which have been converted "back" from tissue cells to stem cells.
Laminin-511 is part of our connective tissue and acts in the body as a matrix to which cells can attach. In the newly formed embryo, the protein is also needed to keep stem cells as stem cells. Once the embryo begins to develop different types of tissue, other types of laminin are needed.
Until now, different types of laminin have not been available to researchers, because they are almost impossible to extract from tissues and difficult to produce. Over the last couple of decades, Karl Tryggvason's research group has cloned the genes for most human laminins, studied their biological role, described two genetic laminin diseases and, in recent years, even managed to produce several types of laminin using gene technology. In this latest experiment, the researchers produced the laminin-511 using recombinant techniques.
Journal Reference:
Sergey Rodin, Anna Domogatskaya, Susanne Ström, Emil M Hansson, Kenneth R Chien, José Inzunza, Outi Hovatta, Karl Tryggvason. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nature Biotechnology, 2010; DOI: 10.1038/nbt.1620
(http://www.sciencedaily.com/releases/2010/05/100531082905.htm)
Scaffold Gradients: Finding the Right Environment for Developing Cells
Site of the day: http://johnchow.com/blog/
ScienceDaily (May 28, 2010) — People often have strong opinions on the "right" firmness of mattresses for themselves, and, as it turns out, some cell types have similar preferences for their support structures. Now a research team from the National Institute of Standards and Technology (NIST) and the National Institutes of Health (NIH) has developed a way to offer cells a three-dimensional scaffold that varies over a broad range of degrees of stiffness to determine where they develop best.
Their recently published technique is a way to rapidly optimize 3D cell growth media to meet the developmental needs of specific cell types for a wide variety of potential tissue-replacement therapies.
Tissue engineering is a relatively new field that is developing methods to grow or regenerate bodily tissues -- skin, bone, cartilage, blood vessels, perhaps one day even whole organs -- to replace those damaged by injury or disease. One of the key challenges in the field is developing appropriate three-dimensional "scaffolds," artificial materials that can hold tissue progenitor cells and allow them to be nurtured and supported while they multiply and develop into desired tissues. Research has shown that cells often need to develop in a 3D environment if they are to mature and differentiate properly.
Hydrogels -- most familiar for their use in soft contact lenses -- are a promising material for tissue scaffolds. They consist of a loose network of polymer chains that is swollen with water; in fact, like the majority of the body's tissues, they are mostly water.
But, says NIST materials scientist Kaushik Chatterjee, deciding on a hydrogel is just the beginning. "Now you've got these gels, what sort of properties do you want? What gets you the best kind of whatever tissue you're after -- in our case, bone? We focused on stiffness because cells are known to sense and respond to changes in the stiffness of their environment."
To test this, the research team developed a method to create samples of a typical hydrogel used in biomedical research, PEGDM (poly(ethylene glycol) dimethacrylate), where the stiffness of the gel increases smoothly from one end of the sample to the other. This approach, using smoothly varying gradients of compounds to test many possible combinations simultaneously, is called combinatorial screening. NIST has pioneered such techniques for a variety of materials problems, but this research is one of the first applications of combinatorial screening to 3D scaffolds for tissue engineering. The team tested the technique on mouse osteoblasts -- cells responsible for building bone -- mixed in with the PEGDM gel. Interestingly, although cell survival rates were higher at the softer end of the test strips and got progressively worse towards the stiffer ends, cell differentiation and mineralization, which are measures of how well the cells actually develop into bone tissue, did the reverse. Fewer cells survive in a stiff gel, but those that do are much more active in building bone. That result, of course, is specific to osteoblasts, says Chatterjee, "These are bone cells and they seem to like the stiffer environment more than softer ones, but you could apply something similar to, say, nerve cells, and they might like the softer ones more."
In addition, the researchers note, the gel stiffness gradient induced a matching gradient in the tissue mineralization. This is potentially important, they say, because tissue gradients often occur naturally at the interfaces of, for example, teeth or ligaments, so 3D scaffold gradients could be a valuable tool for engineering graded tissues for regenerative medicine.
The research was supported by NIST and NIH.
Journal Reference:
Kaushik Chatterjee, Sheng Lin-Gibson, William E. Wallace, Sapun H. Parekh, Young Jong Lee, Marcus T. Cicerone, Marian F. Young, Carl G. Simon Jr. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials, 2010; 31 (19): 5051 DOI: 10.1016/j.biomaterials.2010.03.024
(http://www.sciencedaily.com/releases/2010/05/100527122158.htm)
ScienceDaily (May 28, 2010) — People often have strong opinions on the "right" firmness of mattresses for themselves, and, as it turns out, some cell types have similar preferences for their support structures. Now a research team from the National Institute of Standards and Technology (NIST) and the National Institutes of Health (NIH) has developed a way to offer cells a three-dimensional scaffold that varies over a broad range of degrees of stiffness to determine where they develop best.
Their recently published technique is a way to rapidly optimize 3D cell growth media to meet the developmental needs of specific cell types for a wide variety of potential tissue-replacement therapies.
Tissue engineering is a relatively new field that is developing methods to grow or regenerate bodily tissues -- skin, bone, cartilage, blood vessels, perhaps one day even whole organs -- to replace those damaged by injury or disease. One of the key challenges in the field is developing appropriate three-dimensional "scaffolds," artificial materials that can hold tissue progenitor cells and allow them to be nurtured and supported while they multiply and develop into desired tissues. Research has shown that cells often need to develop in a 3D environment if they are to mature and differentiate properly.
Hydrogels -- most familiar for their use in soft contact lenses -- are a promising material for tissue scaffolds. They consist of a loose network of polymer chains that is swollen with water; in fact, like the majority of the body's tissues, they are mostly water.
But, says NIST materials scientist Kaushik Chatterjee, deciding on a hydrogel is just the beginning. "Now you've got these gels, what sort of properties do you want? What gets you the best kind of whatever tissue you're after -- in our case, bone? We focused on stiffness because cells are known to sense and respond to changes in the stiffness of their environment."
To test this, the research team developed a method to create samples of a typical hydrogel used in biomedical research, PEGDM (poly(ethylene glycol) dimethacrylate), where the stiffness of the gel increases smoothly from one end of the sample to the other. This approach, using smoothly varying gradients of compounds to test many possible combinations simultaneously, is called combinatorial screening. NIST has pioneered such techniques for a variety of materials problems, but this research is one of the first applications of combinatorial screening to 3D scaffolds for tissue engineering. The team tested the technique on mouse osteoblasts -- cells responsible for building bone -- mixed in with the PEGDM gel. Interestingly, although cell survival rates were higher at the softer end of the test strips and got progressively worse towards the stiffer ends, cell differentiation and mineralization, which are measures of how well the cells actually develop into bone tissue, did the reverse. Fewer cells survive in a stiff gel, but those that do are much more active in building bone. That result, of course, is specific to osteoblasts, says Chatterjee, "These are bone cells and they seem to like the stiffer environment more than softer ones, but you could apply something similar to, say, nerve cells, and they might like the softer ones more."
In addition, the researchers note, the gel stiffness gradient induced a matching gradient in the tissue mineralization. This is potentially important, they say, because tissue gradients often occur naturally at the interfaces of, for example, teeth or ligaments, so 3D scaffold gradients could be a valuable tool for engineering graded tissues for regenerative medicine.
The research was supported by NIST and NIH.
Journal Reference:
Kaushik Chatterjee, Sheng Lin-Gibson, William E. Wallace, Sapun H. Parekh, Young Jong Lee, Marcus T. Cicerone, Marian F. Young, Carl G. Simon Jr. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials, 2010; 31 (19): 5051 DOI: 10.1016/j.biomaterials.2010.03.024
(http://www.sciencedaily.com/releases/2010/05/100527122158.htm)
Monday, June 14, 2010
Genetic 'Parts' List Now Available for Hypothalamus -- Key Part of the Mammalian Brain
Site of the day: http://forums.digitalpoint.com/
ScienceDaily (June 10, 2010) — A Johns Hopkins and Japanese research team has generated the first comprehensive genetic "parts" list of a mouse hypothalamus, an enigmatic region of the brain -- roughly cherry-sized, in humans -- that controls hunger, thirst, fatigue, body temperature, wake-sleep cycles and links the central nervous system to control of hormone levels.
Flaws in hypothalamus development may underlie both inborn and acquired metabolic balance problems that can lead to obesity, diabetes, mood disorders and high blood pressure, according to a report on the study published May 2 in the advance online publication of Nature Neuroscience.
"Knowing how cells develop in this part of the brain will help us understand how they regulate behavior, mood and metabolism," says Seth Blackshaw, Ph.D., an assistant professor in the Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine.
The hypothalamus is one of the most diverse and complex parts of the brain, and having an index of the genes involved in producing its many cell types is a toolbox that researchers can use to manipulate the activity of brain cells by turning them on and off, or tracing their connections. This may ultimately lead to better diagnostic and treatment options for a variety of disorders.
"The study of hypothalamic development, particularly of cell specification, will help us to understand how hypothalamic neurons function to regulate behavior and physiology," says Blackshaw. "Because of when and where we saw certain genes turn on, we now have identified a set of candidate players that guide the assembly of the different parts of the hypothalamus and that specify the many individual cell types within it."
The hypothalamus is composed of at least dozens of types of neurons -- and more likely hundreds -- each of which corresponds to a gene that has remained unidentified until now. Its cellular arrangement is more akin to a bowl of spaghetti than a neatly organized club sandwich, according to Blackshaw. The catalog of molecular markers identified here helps unravel this complexity.
The team's first challenge was to dissect away, at the very start of neural development, the part of the mouse brain which develops into the hypothalamus, and then cut tiny slices of this region for use in microarray analysis, a technology that reveals multiple gene activity. By analyzing all the roughly 20,000 genes in the mouse genome, the team identified 1200 as strongly activated in developing hypothalamus and characterized the cells within the hypothalamus in which they were activated. The team then characterized the expression of the most interesting 350 genes in detail using another gene called Shh, for sonic hedgehog, as a landmark to identify the precise region of the hypothalamus in which these genes were turned on. This involved processing close to 20,000 tissue sections -- painstakingly sliced at one-fiftieth of a millimeter thickness and then individually examined.
"We were able to use this data to find genes whose expression matched every individual hypothalamic nucleus and essentially assemble a jigsaw puzzle of gene expression patterns that completely covered the developing hypothalamus," Blackshaw says. "Now that we have a complete set of molecular landmarks, along with an extensive molecular parts list, we can begin to learn how all these parts fit together to create this essential and highly complex brain region."
Authors of the paper, in addition to Blackshaw, are Daniel A. Lee, Ana Miranda-Angulo, Yangqin Yang, Aya C. Yoshida, Hong Wang, Hiromi Mashiko, Lizhi Jiang, Marina Avetisyan, Lixin Qi, and Jiang Qian, all of Johns Hopkins; Ayane Kataoka and Tomomi Shimogori of RIKEN-BSI, 2-1 Hirosawa, Wako-shi, Saitama, Japan.
This research was supported by March of Dimes, the Klingenstein Fund, the W.M. Keck Foundation, and the Japan Society for Promotion of Science.
Journal Reference:
Tomomi Shimogori, Daniel A Lee, Ana Miranda-Angulo, Yanqin Yang, Hong Wang, Lizhi Jiang, Aya C Yoshida, Ayane Kataoka, Hiromi Mashiko, Marina Avetisyan, Lixin Qi, Jiang Qian, Seth Blackshaw. A genomic atlas of mouse hypothalamic development. Nature Neuroscience, 2010; 13 (6): 767 DOI: 10.1038/nn.2545
(http://www.sciencedaily.com/releases/2010/06/100609160542.htm)
ScienceDaily (June 10, 2010) — A Johns Hopkins and Japanese research team has generated the first comprehensive genetic "parts" list of a mouse hypothalamus, an enigmatic region of the brain -- roughly cherry-sized, in humans -- that controls hunger, thirst, fatigue, body temperature, wake-sleep cycles and links the central nervous system to control of hormone levels.
Flaws in hypothalamus development may underlie both inborn and acquired metabolic balance problems that can lead to obesity, diabetes, mood disorders and high blood pressure, according to a report on the study published May 2 in the advance online publication of Nature Neuroscience.
"Knowing how cells develop in this part of the brain will help us understand how they regulate behavior, mood and metabolism," says Seth Blackshaw, Ph.D., an assistant professor in the Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine.
The hypothalamus is one of the most diverse and complex parts of the brain, and having an index of the genes involved in producing its many cell types is a toolbox that researchers can use to manipulate the activity of brain cells by turning them on and off, or tracing their connections. This may ultimately lead to better diagnostic and treatment options for a variety of disorders.
"The study of hypothalamic development, particularly of cell specification, will help us to understand how hypothalamic neurons function to regulate behavior and physiology," says Blackshaw. "Because of when and where we saw certain genes turn on, we now have identified a set of candidate players that guide the assembly of the different parts of the hypothalamus and that specify the many individual cell types within it."
The hypothalamus is composed of at least dozens of types of neurons -- and more likely hundreds -- each of which corresponds to a gene that has remained unidentified until now. Its cellular arrangement is more akin to a bowl of spaghetti than a neatly organized club sandwich, according to Blackshaw. The catalog of molecular markers identified here helps unravel this complexity.
The team's first challenge was to dissect away, at the very start of neural development, the part of the mouse brain which develops into the hypothalamus, and then cut tiny slices of this region for use in microarray analysis, a technology that reveals multiple gene activity. By analyzing all the roughly 20,000 genes in the mouse genome, the team identified 1200 as strongly activated in developing hypothalamus and characterized the cells within the hypothalamus in which they were activated. The team then characterized the expression of the most interesting 350 genes in detail using another gene called Shh, for sonic hedgehog, as a landmark to identify the precise region of the hypothalamus in which these genes were turned on. This involved processing close to 20,000 tissue sections -- painstakingly sliced at one-fiftieth of a millimeter thickness and then individually examined.
"We were able to use this data to find genes whose expression matched every individual hypothalamic nucleus and essentially assemble a jigsaw puzzle of gene expression patterns that completely covered the developing hypothalamus," Blackshaw says. "Now that we have a complete set of molecular landmarks, along with an extensive molecular parts list, we can begin to learn how all these parts fit together to create this essential and highly complex brain region."
Authors of the paper, in addition to Blackshaw, are Daniel A. Lee, Ana Miranda-Angulo, Yangqin Yang, Aya C. Yoshida, Hong Wang, Hiromi Mashiko, Lizhi Jiang, Marina Avetisyan, Lixin Qi, and Jiang Qian, all of Johns Hopkins; Ayane Kataoka and Tomomi Shimogori of RIKEN-BSI, 2-1 Hirosawa, Wako-shi, Saitama, Japan.
This research was supported by March of Dimes, the Klingenstein Fund, the W.M. Keck Foundation, and the Japan Society for Promotion of Science.
Journal Reference:
Tomomi Shimogori, Daniel A Lee, Ana Miranda-Angulo, Yanqin Yang, Hong Wang, Lizhi Jiang, Aya C Yoshida, Ayane Kataoka, Hiromi Mashiko, Marina Avetisyan, Lixin Qi, Jiang Qian, Seth Blackshaw. A genomic atlas of mouse hypothalamic development. Nature Neuroscience, 2010; 13 (6): 767 DOI: 10.1038/nn.2545
(http://www.sciencedaily.com/releases/2010/06/100609160542.htm)
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