2,3-Diaminopyridine as a platform for designing structurally unique nonpeptide bradykinin B1 receptor antagonists

A novel class of 2,3-diaminopyridine bradykinin B1 receptor antagonists is disclosed. Structure-activity relationship studies (SARs) that led to compounds with significantly improved potency and pharmacokinetic properties relative to the lead compound are described.     

Population genetic structure in migratory sandhill cranes and the role of Pleistocene glaciations

Previous studies of migratory sandhill cranes (Grus canadensis) have made significant progress explaining evolution of this group at the species scale, but have been unsuccessful in explaining the geographically partitioned variation in morphology seen on the population scale. The objectives of this study were to assess the population structure and gene flow patterns among migratory sandhill cranes using microsatellite DNA genotypes and mitochondrial DNA haplotypes of a large sample of individuals across three populations. In particular, we were interested in evaluating the roles of Pleistocene glaciation events and postglaciation gene flow in shaping the present-day population structure. Our results indicate substantial gene flow across regions of the Midcontinental population that are geographically adjacent, suggesting that gene flow for most of the region follows an isolation-by-distance model. Male-mediated gene flow and strong female philopatry may explain the differing patterns of nuclear and mitochondrial variation. Taken in context with precise geographical information on breeding locations, the morphologic and microsatellite DNA variation shows a gradation from the Arctic-nesting subspecies G. c. canadensis to the nonArctic subspecies G. c. tabida. Analogous to other Arctic-nesting birds, it is probable that the population structure seen in Midcontinental sandhill cranes reflects the result of postglacial secondary contact. Our data suggest that subspecies of migratory sandhills experience significant gene flow and therefore do not represent distinct and independent genetic entities.     

Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination

Wolbachia is a group of maternally inherited endosymbiotic bacteria that infect and induce cytoplasmic incompatibility (CI) in a wide range of arthropods. In contrast to other species, the mosquito Culex pipiens displays an extremely high number of CI types suggesting differential infection by multiple Wolbachia strains. Attempts so far failed to detect Wolbachia polymorphism that might explain this high level of CI diversity found in C. pipiens populations. Here, we establish that Wolbachia infection is near to or at fixation in worldwide populations of the C. pipiens complex. Wolbachia polymorphism was addressed by sequence analysis of the Tr1 gene, a unique transposable element of the IS5 family, which allowed the identification of five C. pipiens Wolbachia strains, differing either by nucleotide substitution, presence or absence pattern, or insertion site. Sequence analysis also showed that recombination, transposition and superinfection occurred at very low frequencies. Analysis of the geographical distributions of each Wolbachia strain among C. pipiens populations indicated a strong worldwide differentiation independent from mosquito subspecies type, except in the UK. The availability of this polymorphic marker now opens the way to investigate evolution of Wolbachia populations and CI dynamics, in particular in regions where multiple crossing types coexist among C. pipiens populations.     

Design of a data model for developing laboratory information management and analysis systems for protein production

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.     

CLAMP, a novel microtubule-associated protein with EB-type calponin homology

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.     

BAliBASE 3.0: latest developments of the multiple sequence alignment benchmark

Multiple sequence alignment is one of the cornerstones of modern molecular biology. It is used to identify conserved motifs, to determine protein domains, in 2D/3D structure prediction by homology and in evolutionary studies. Recently, high-throughput technologies such as genome sequencing and structural proteomics have lead to an explosion in the amount of sequence and structure information available. In response, several new multiple alignment methods have been developed that improve both the efficiency and the quality of protein alignments. Consequently, the benchmarks used to evaluate and compare these methods must also evolve. We present here the latest release of the most widely used multiple alignment benchmark, BAliBASE, which provides high quality, manually refined, reference alignments based on 3D structural superpositions. Version 3.0 of BAliBASE includes new, more challenging test cases, representing the real problems encountered when aligning large sets of complex sequences. Using a novel, semiautomatic update protocol, the number of protein families in the benchmark has been increased and representative test cases are now available that cover most of the protein fold space. The total number of proteins in BAliBASE has also been significantly increased from 1444 to 6255 sequences. In addition, full-length sequences are now provided for all test cases, which represent difficult cases for both global and local alignment programs. Finally, the BAliBASE Web site (http://www-bio3d-igbmc.u-strasbg.fr/balibase) has been completely redesigned to provide a more user-friendly, interactive interface for the visualization of the BAliBASE reference alignments and the associated annotations.     

Distinct protective mechanisms of HO-1 and HO-2 against hydroperoxide-induced cytotoxicity

Heme oxygenases (HO-1 and HO-2) catalyze the NADPH-cytochrome P(450) reductase (CPR)-dependent degradation of heme into iron, carbon monoxide, and biliverdin, which is reduced into bilirubin. Under basal conditions, HO-1 is often undetected and can be induced by numerous stress conditions. Although HO-2 is constitutively expressed, its activity appears to be regulated by post-translational modifications. HO activity has been associated with cellular protection, by which it degrades heme, a prooxidant, into bioactive metabolites. Under given circumstances, overexpression of HO-1 can render cells more sensitive to free radicals. Here, we investigated the properties of human HO isoforms that protect against oxidative stress. Considering that CPR can be a limiting factor for optimal HO activity, we tested stable HO-1 and HO-2 cell lines that derived from the CPR cells. Results indicate that the HO-1 and HO-2 cells are more resistant than controls to hemin and to the organic tert-butyl hydroperoxide, t-BuOOH. However, HO-1 cells are less resistant than HO-2 cells to hydrogen peroxide (H(2)O(2)). The levels of oxidatively modified proteins of HO-1 and HO-2 cells in response to t-BuOOH toxicity are identical, but the level of oxidatively modified proteins of HO-2 cells is less than that of HO-1 cells in response to H(2)O(2) toxicity. Performing subcellular fractionations revealed that HO-2 and CPR are found together in the microsomal fractions, whereas HO-1 is partially present in the microsome and also found in other fractions, such as the cytosol. These same findings were observed in non-transfected primary neurons where HO-1 proteins were chemically induced with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)). The differences in subcellular localization of HO-1 and HO-2 could explain some of the discrepancies in their cellular activity and enzymatic protective mechanisms.     

Protein synthesis-independent plasticity mediates rapid and precise recovery of deprived eye responses

Monocular deprivation (MD) for a few days during a critical period of development leads to loss of cortical responses to stimulation of the deprived eye. Despite the profound effects of MD on cortical function, optical imaging of intrinsic signals and single-unit recordings revealed that deprived eye responses and orientation selectivity recovered a few hours after restoration of normal binocular vision. Moreover, recovery of deprived eye responses was not dependent upon mRNA translation, but required cortical activity. Interestingly, this fast recovery and protein synthesis independence was restricted to the hemisphere contralateral to the previously deprived eye. Collectively, these results implicate a relatively simple mechanistic process in the reactivation of a latent set of connections following restoration of binocular vision and provide new insight into how recovery of cortical function can rapidly occur in response to changes in sensory experience.     

Vav family GEFs link activated Ephs to endocytosis and axon guidance

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.