Biomedical subjects

David Baker

Publications and source records attributed to David Baker.

At least 163 records · Page 9Linked to original sources

PubMed2002

Inhibition of Rho GTPases with protein prenyltransferase inhibitors prevents leukocyte recruitment to the central nervous system and attenuates clinical signs of disease in an animal model of multiple sclerosis.

The ICAM-1-mediated brain endothelial cell (EC)-signaling pathway induced by adherent lymphocytes is a central element in facilitating lymphocyte migration through the tight endothelial barrier of the brain. Rho proteins, which must undergo posttranslational prenylation to be functionally active, have been shown to be an essential component of this signaling cascade. In this study, we have evaluated the effect of inhibiting protein prenylation in brain ECs on their ability to support T lymphocyte migration. ECs treated in vitro with protein prenylation inhibitors resulted in a significant reduction in transendothelial T lymphocyte migration. To determine the therapeutic potential of this approach, an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, was induced in Biozzi ABH mice. Animals treated before disease onset with protein prenylation inhibitors exhibited a dramatic and significant reduction in both leukocyte infiltration into the CNS and clinical presentation of disease compared with untreated animals. These studies demonstrate, for the first time, the potential for pharmacologically targeting CNS EC signaling responses, and particularly endothelial Rho proteins, as a means of attenuating leukocyte recruitment to the CNS.

Acute Disease↗

PubMed2002

Arvanil-induced inhibition of spasticity and persistent pain: evidence for therapeutic sites of action different from the vanilloid VR1 receptor and cannabinoid CB(1)/CB(2) receptors.

Activation of cannabinoid receptors causes inhibition of spasticity, in a mouse model of multiple sclerosis, and of persistent pain, in the rat formalin test. The endocannabinoid anandamide inhibits spasticity and persistent pain. It not only binds to cannabinoid receptors but is also a full agonist at vanilloid receptors of type 1 (VR1). We found here that vanilloid VR1 receptor agonists (capsaicin and N-N'-(3-methoxy-4-aminoethoxy-benzyl)-(4-tert-butyl-benzyl)-urea [SDZ-249-665]) exhibit a small, albeit significant, inhibition of spasticity that can be attenuated by the vanilloid VR1 receptor antagonist, capsazepine. Arvanil, a structural "hybrid" between capsaicin and anandamide, was a potent inhibitor of spasticity at doses (e.g. 0.01 mg/kg i.v.) where capsaicin and cannabinoid CB(1) receptor agonists were ineffective. The anti-spastic effect of arvanil was unchanged in cannabinoid CB(1) receptor gene-deficient mice or in wildtype mice in the presence of both cannabinoid and vanilloid receptor antagonists. Likewise, arvanil (0.1-0.25 mg/kg) exhibited a potent analgesic effect in the formalin test, which was not reversed by cannabinoid and vanilloid receptor antagonists. These findings suggest that activation by arvanil of sites of action different from cannabinoid CB(1)/CB(2) receptors and vanilloid VR1 receptors leads to anti-spastic/analgesic effects that might be exploited therapeutically.

Animals↗

PubMed2002

De novo determination of protein backbone structure from residual dipolar couplings using Rosetta.

As genome-sequencing projects rapidly increase the database of protein sequences, the gap between known sequences and known structures continues to grow exponentially, increasing the demand to accelerate structure determination methods. Residual dipolar couplings (RDCs) are an attractive source of experimental restraints for NMR structure determination, particularly rapid, high-throughput methods, because they yield both local and long-range orientational information and can be easily measured and assigned once the backbone resonances of a protein have been assigned. While very extensive RDC data sets have been used to determine the structure of ubiquitin, it is unclear to what extent such methods will generalize to larger proteins with less complete data sets. Here we incorporate experimental RDC restraints into Rosetta, an ab initio structure prediction method, and demonstrate that the combined algorithm provides a general method for de novo determination of a variety of protein folds from RDC data. Backbone structures for multiple proteins up to approximately 125 residues in length and spanning a range of topological complexities are rapidly and reproducibly generated using data sets that are insufficient in isolation to uniquely determine the protein fold de novo, although ambiguities and errors are observed for proteins with symmetry about an axis of the alignment tensor. The models generated are not high-resolution structures completely defined by experimental data but are sufficiently accurate to accelerate traditional high-resolution NMR structure determination and provide structure-based functional insights.

Algorithms↗

PubMed2002

Residues participating in the protein folding nucleus do not exhibit preferential evolutionary conservation.

To what extent does natural selection act to optimize the details of protein folding kinetics? In an effort to address this question, the relationship between an amino acid's evolutionary conservation and its role in protein folding kinetics has been investigated intensively. Despite this effort, no consensus has been reached regarding the degree to which residues involved in native-like transition state structure (the folding nucleus) are conserved. Here we report the results of an exhaustive, systematic study of sequence conservation among residues known to participate in the experimentally (Phi-value) defined folding nuclei of all of the appropriately characterized proteins reported to date. We observe no significant evidence that these residues exhibit any anomalous sequence conservation. We do observe, however, a significant bias in the existing kinetic data: the mean sequence conservation of the residues that have been the subject of kinetic characterization is greater than the mean sequence conservation of all residues in 13 of 14 proteins studied. This systematic experimental bias gives rise to the previous observation that the median conservation of residues reported to participate in the folding nucleus is greater than the median conservation of all of the residues in a protein. When this bias is corrected (by comparing, for example, the conservation of residues known to participate in the folding nucleus with that of other, kinetically characterized residues) the previously reported preferential conservation is effectively eliminated. In contrast to well-established theoretical expectations, both poorly and highly conserved residues are apparently equally likely to participate in the protein-folding nucleus.

Bias↗

PubMed2002

Towards robust regional estimates of CO2 sources and sinks using atmospheric transport models.

Information about regional carbon sources and sinks can be derived from variations in observed atmospheric CO2 concentrations via inverse modelling with atmospheric tracer transport models. A consensus has not yet been reached regarding the size and distribution of regional carbon fluxes obtained using this approach, partly owing to the use of several different atmospheric transport models. Here we report estimates of surface-atmosphere CO2 fluxes from an intercomparison of atmospheric CO2 inversion models (the TransCom 3 project), which includes 16 transport models and model variants. We find an uptake of CO2 in the southern extratropical ocean less than that estimated from ocean measurements, a result that is not sensitive to transport models or methodological approaches. We also find a northern land carbon sink that is distributed relatively evenly among the continents of the Northern Hemisphere, but these results show some sensitivity to transport differences among models, especially in how they respond to seasonal terrestrial exchange of CO2. Overall, carbon fluxes integrated over latitudinal zones are strongly constrained by observations in the middle to high latitudes. Further significant constraints to our understanding of regional carbon fluxes will therefore require improvements in transport models and expansion of the CO2 observation network within the tropics.

Journal Article↗

PubMed2002

Accurate computer-based design of a new backbone conformation in the second turn of protein L.

The rational design of loops and turns is a key step towards creating proteins with new functions. We used a computational design procedure to create new backbone conformations in the second turn of protein L. The Protein Data Bank was searched for alternative turn conformations, and sequences optimal for these turns in the context of protein L were identified using a Monte Carlo search procedure and an energy function that favors close packing. Two variants containing 12 and 14 mutations were found to be as stable as wild-type protein L. The crystal structure of one of the variants has been solved at a resolution of 1.9 A, and the backbone conformation in the second turn is remarkably close to that of the in silico model (1.1 A RMSD) while it differs significantly from that of wild-type protein L (the turn residues are displaced by an average of 7.2 A). The folding rates of the redesigned proteins are greater than that of the wild-type protein and in contrast to wild-type protein L the second beta-turn appears to be formed at the rate limiting step in folding.

Amino Acid Sequence↗

PubMed2002

A role for caspase-1 and -3 in the pathology of experimental allergic encephalomyelitis : inflammation versus degeneration.

Axonal loss, already present in the acute and first relapse phases of experimental allergic encephalomyelitis (EAE) in the ABH mouse, only becomes apparent in the third relapse in the interleukin-12 model of relapsing EAE in the Lewis rat. Caspase-1 immunostaining in the spinal cord of Lewis rats was mainly localized to inflammatory cuffs with the greatest proportion of active caspase-1-positive cells detected during the first and second relapses, correlating with enzyme activity and protein on Western blots. However, in the spinal cord of ABH mice during acute EAE, caspase-1 immunostaining was localized both on inflammatory and neuronal cells, again correlating with enzyme activity and protein production. In contrast, caspase-3 expression in the spinal cord of Lewis rats did not increase significantly until the third relapse when inflammatory and neuronal cells and axons became positive in line with a significant increase in caspase activity. In ABH mice active caspase-3 was already immunolocalized on axons and apoptotic neurons in the spinal cord during the acute stage of EAE. Because caspase-3 is a downstream cell death signal it may be possible to reduce apoptosis by selectively blocking caspase-3 and therefore provide a therapeutic target for EAE and potentially, multiple sclerosis.

Animals↗

PubMed2002

Encephalitogenic and immunogenic potential of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycoprotein (OSP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in ABH and SJL mice.

Synthetic peptides of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycoprotein (OSP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were screened for their ability to induce experimental allergic encephalomyelitis (EAE) in ABH (H-2A(g7)) and SJL (H-2(s)) mice. The use of overlapping 16mer MAG peptides identified residues 97-112 as a T-cell and encephalitogenic epitope in ABH mice which induced clinical and histological signs of acute EAE. Immunization of SJL mice with MAG peptides failed to induce disease whereas immunization of SJL mice with synthetic peptides of OSP induced major T-cell responses to OSP 73-88 and 81-96. Another epitope, OSP 57-72, that induced EAE, failed to induce T-cell responses in mice immunised with peptides based on the whole sequence supporting a role for cryptic epitopes. In comparison, whilst immunization of ABH mice with OSP revealed two immunodominant T-cell epitopes (49-64 and 137-152), an encephalitogenic epitope was not identified. Similarly, immunization of both SJL and ABH mice with CNPase peptides induced T-cell responses to several epitopes. However, these were not encephalitogenic. This study is the first to identify an encephalitogenic epitope of MAG and immunodominant epitopes of MAG, OSP and CNPase in SJL and ABH mice. The ability of both cryptic and noncryptic peptide epitopes of these myelin antigens to initiate EAE suggests that mice at least are not tolerant to some regions of MAG and OSP and that such specific autoimmune responses may play an important role in the pathogenesis of immune-mediated neurological diseases such as multiple sclerosis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2002

Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.

Neurological deficit in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) is probably a consequence of synergy between T and B cell responses to CNS antigens. During the demyelinating phase of chronic relapsing EAE in ABH mice, anti-myelin oligodendrocyte glycoprotein (MOG) responses were increased compared to the inflammatory acute phase, but such levels did not correlate with the severity of clinical disease. The pathogenicity of antibodies (Ab) to MOG, myelin basic protein (MBP), proteolipid protein (PLP) and galactocerebroside (GalC) was investigated in vivo following injection at the onset of EAE. An IgG2a monoclonal Ab (mAb), clone Z12, directed to MOG augmented clinical disease and demyelination in ABH and C57BL/6 mice, but not MOG knock-out mice. No effect was observed with F(ab(2))' fragments of Z12 or with the anti-MOG IgG1 mAbs, clones Y10 or 8-18C5. Cobra venom factor partially reduced the augmenting effect of mAb Z12 suggesting a role for complement. The pathogenic effect of anti-myelin Abs was not restricted to MOG since an anti-GalC mAb exacerbated inflammation in the CNS while an MBP mAb (clone 22) reduced clinical disease. Taken together, these data provide further evidence that myelin-reactive Abs generated during autoimmune neurological disease may play an important role not only in the pathogenesis of disease but also the regulation of myelin-targeted autoimmune disease.

Animals↗

PubMed2002

Protein structure prediction in 2002.

Central issues concerning protein structure prediction have been highlighted by the recently published summary of the fourth community-wide protein structure prediction experiment (CASP4). Although sequence/structure alignment remains the bottleneck in comparative modeling, there has been substantial progress in fully automated remote homolog detection and in de novo structure prediction. Significant further progress will probably require improvements in high-resolution modeling.

Models, Molecular↗

PubMed2002

Design, activity, and structure of a highly specific artificial endonuclease.

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.

Algorithms↗

PubMed2002

Characterization of the substrate specificity of a human 5-hydroxymethyluracil glycosylase activity.

The oxidation of pyrimidine 5-methyl groups, derived from either thymine or 5-methylcytosine, can generate 5-hydroxymethyluracil (HmU) in DNA. An activity from HeLa cells that removes 5-hydroxymethyluracil (HmU) from DNA has been partially purified and characterized using a battery of oligonucleotides containing modified bases. This partially purified activity preferentially removes HmU mispaired with guanine. The HmU repair activity also acts on uracil and fluorouracil but not 5-substituted uracil derivatives with halogens larger than fluorine. However, neither mispaired thymine nor ethenocytosine are substrates. HmU is readily removed when paired with guanine, hypoxanthine (deoxyinosine), and purine (deoxynebularine), but not from single-stranded substrates. Upon the basis of these substrate preferences, we conclude that (1) the mispaired HmU repair activity is distinct from previously reported glycosylases including UDG, TDG, MUG, and SMUG1 activities, (2) the binding pocket is highly selective for the 5-hydroxymethyl group, and (3) the preference for mispaired HmU derives from reduced thermal stability of the mispair, as opposed to selective recognition of the mispaired guanine residue in the opposing DNA strand.

DNA Glycosylases↗

PubMed2002

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.

Acid Anhydride Hydrolases↗

PubMed2002

Crystal structures and increased stabilization of the protein G variants with switched folding pathways NuG1 and NuG2.

We recently described two protein G variants (NuG1 and NuG2) with redesigned first hairpins that were almost twice as stable, folded 100-fold faster, and had a switched folding mechanism relative to the wild-type protein. To test the structural accuracy of our design algorithm and to provide insights to the dramatic changes in the kinetics and thermodynamics of folding, we have now determined the crystal structures of NuG1 and NuG2 to 1.8 A and 1.85 A, respectively. We find that they adopt hairpin structures that are closer to the computational models than to wild-type protein G; the RMSD of the NuG1 hairpin to the design model and the wild-type structure are 1.7 A and 5.1 A, respectively. The crystallographic B factor in the redesigned first hairpin of NuG1 is systematically higher than the second hairpin, suggesting that the redesigned region is somewhat less rigid. A second round of structure-based design yielded new variants of NuG1 and NuG2, which are further stabilized by 0.5 kcal/mole and 0.9 kcal/mole.

Crystallography, X-Ray↗

PubMed2002

Contact order and ab initio protein structure prediction.

Although much of the motivation for experimental studies of protein folding is to obtain insights for improving protein structure prediction, there has been relatively little connection between experimental protein folding studies and computational structural prediction work in recent years. In the present study, we show that the relationship between protein folding rates and the contact order (CO) of the native structure has implications for ab initio protein structure prediction. Rosetta ab initio folding simulations produce a dearth of high CO structures and an excess of low CO structures, as expected if the computer simulations mimic to some extent the actual folding process. Consistent with this, the majority of failures in ab initio prediction in the CASP4 (critical assessment of structure prediction) experiment involved high CO structures likely to fold much more slowly than the lower CO structures for which reasonable predictions were made. This bias against high CO structures can be partially alleviated by performing large numbers of additional simulations, selecting out the higher CO structures, and eliminating the very low CO structures; this leads to a modest improvement in prediction quality. More significant improvements in predictions for proteins with complex topologies may be possible following significant increases in high-performance computing power, which will be required for thoroughly sampling high CO conformations (high CO proteins can take six orders of magnitude longer to fold than low CO proteins). Importantly for such a strategy, simulations performed for high CO structures converge much less strongly than those for low CO structures, and hence, lack of simulation convergence can indicate the need for improved sampling of high CO conformations. The parallels between Rosetta simulations and folding in vivo may extend to misfolding: The very low CO structures that accumulate in Rosetta simulations consist primarily of local up-down beta-sheets that may resemble precursors to amyloid formation.

Algorithms↗

PubMedDate not supplied

Terrorism in France.

France has experienced two waves of major terrorist bombings since 1980. In the first wave (1985-1986), eight bombings occurred in Paris, killing 13 and injuring 281. In the second wave (1995-1996), six bombings occurred in Paris and Lyon, killing 10 and injuring 262. Based on lessons learned during these events, France has developed and improved a sophisticated national system for prehospital emergency response to conventional terrorist attacks based on its national emergency medical services (EMS) system, Service d'Aide Medicale Urgente (SAMU). According to the national plan for the emergency medical response to mass-casualty events (White Plan), the major phases of EMS response are: (1) alert; (2) search and rescue; (3) triage of victims and provision of critical care to first priority victims; (4) regulated dispatch of victims to hospitals; and (5) psychological assistance. Following the 1995 Tokyo subway sarin attack, a national plan for the emergency response to chemical and biological events (PIRATOX) was implemented. In 2002, the Ministries of Health and the Interior collaborated to produce a comprehensive national plan (BIOTOX) for the emergency response to chemical, biological, radiological, and nuclear events. Key aspects of BIOTOX are the prehospital provision of specialized advance life support for toxic injuries and the protection of responders in contaminated environments. BIOTOX was successfully used during the 2003 Severe Acute Respiratory Syndrome (SARS) outbreak in France.

Biological Warfare↗

PubMedDate not supplied

Civilian exposure to toxic agents: emergency medical response.

Civilian populations are at risk from exposure to toxic materials as a result of accidental or deliberate exposure. In addition to industrial hazards, toxic agents designed for use in warfare now are a potential hazard in everyday life through terrorist action. Civil emergency medical responders should be able to adapt their plans for dealing with casualties from hazardous materials (HazMat) to deal with the new threat. Chemical and biological warfare (CBW) and HazMat agents can be viewed as a continuous spectrum. Each of these hazards is characterized by qualities of toxicity, latency of action, persistency, and transmissibility. The incident and medical responses to release of any agent is determined by these characteristics. Chemical and biological wardare agents usually are classified as weapons of mass destruction, but strictly, they are agents of mass injury. The relationship between mass injury and major loss of life depends very much on the protection, organization, and emergency care provided. Detection of a civil toxic agent release where signs and symptoms in casualties may be the first indicator of exposure is different from the military situation where intelligence information and tuned detection systems generally will be available. It is important that emergency medical care should be given in the context of a specific action plan. Within an organized and protected perimeter, triage and decontamination (if the agent is persistent) can proceed while emergency medical care is provided at the same time. The provision of advanced life support (TOXALS) in this zone by protected and trained medical responders now is technically feasible using specially designed ventilation equipment. Leaving life support until after decontamination may have fatal consequences. Casualties from terrorist attacks also may suffer physical as well as toxic trauma and the medical response also should be capable of dealing with mixed injuries.

Biological Warfare↗

PubMedDate not supplied

An investigation into parent perceptions of the needs of siblings of children with cancer.

Although more is understood about childhood cancer's impact on the often forgotten siblings in the family, developing empirically tested interventions that support positive health outcomes is only just emerging. As family support is of key importance in sibling adjustment, further knowledge about their needs is crucial to the development of effective interventions. This investigation focused on examining parental perceptions regarding the concerns and issues for siblings of children with cancer and explored what support is helpful. Focus group methods were used to gather the data with probe questions developed from the literature and from clinical experience. Emergent topics generated were further analyzed using content analysis with 3 major topics identified: the universality of losses arising from the illness experience, behavioral challenges and adaptation, and parent-sibling communication. Parents also discussed helpful interventions. These are described and discussed in relation to the literature. The information obtained will contribute to developing interventions for siblings, specifically to produce a protocol for a therapeutic peer-support camp.

Adaptation, Psychological↗