Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

Largest vertebrate vomeronasal type 1 receptor gene repertoire in the semiaquatic platypus.

Vertebrate vomeronasal chemoreception plays important roles in many aspects of an organism's daily life, such as mating, territoriality, and foraging. Vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs), 2 large families of G protein-coupled receptors, serve as vomeronasal receptors to bind to various pheromones and odorants. Contrary to the previous observations of reduced olfaction in aquatic and semiaquatic mammals, we here report the surprising finding that the platypus, a semiaquatic monotreme, has the largest V1R repertoire and nearly largest combined repertoire of V1Rs and V2Rs of all vertebrates surveyed, with 270 intact genes and 579 pseudogenes in the V1R family and 15 intact genes, 55 potentially intact genes, and 57 pseudogenes in the V2R family. Phylogenetic analysis shows a remarkable expansion of the V1R repertoire and a moderate expansion of the V2R repertoire in platypus since the separation of monotremes from placentals and marsupials. Our results challenge the view that olfaction is unimportant to aquatic mammals and call for further study into the role of vomeronasal reception in platypus physiology and behavior.

Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats.

Pheromones are chemicals produced and detected by conspecifics to elicit social/sexual physiological and behavioral responses, and they are perceived primarily by the vomeronasal organ (VNO) in terrestrial vertebrates. Two large superfamilies of G protein-coupled receptors, V1rs and V2rs, have been identified as pheromone receptors in vomeronasal sensory neurons. Based on a computational analysis of the mouse and rat genome sequences, we report the first global draft of the V2r gene repertoire, composed of approximately 200 genes and pseudogenes. Rodent V2rs are subject to rapid gene births/deaths and accelerated amino acid substitutions, likely reflecting the species-specific nature of pheromones. Vertebrate V2rs appear to have originated twice prior to the emergence of the VNO in ancestral tetrapods, explaining seemingly inconsistent observations among different V2rs. The identification of the entire V2r repertoire opens the door to genomic-level studies of the structure, function, and evolution of this diverse group of sensory receptors.

Tools for anti-inflammatory drug design: in vitro models of leukocyte migration.

Inhibiting leukocyte recruitment is now a major focus in the design of novel anti-inflammatory drugs. Following the identification of lead compounds from conventional high-throughput screens using appropriate receptors or enzymes, it is important to validate the action of the compounds in a suitable in vitro model of leukocyte migration. Here, we review a range of different experimental approaches to modelling leukocyte migration, and identify the multi-well filter migration assay as the best compromise between the amount of resources required to screen multiple compounds and the amount of information gained about the effects of the compounds on cell movement behavior. However, there are pitfalls in the interpretation of data obtained using the multi-well filter migration assay, which arise from the imperfect correlation between the number of cells undergoing migration and the inhibitory activity of the test substances. We examine a number of such pitfalls and provide practical approaches to mitigate these problems as far as possible. We recommend a general strategy for screening inhibitors of cell migration using in vitro functional assays. While being more resource intensive than surrogate measures such as calcium flux, functional approaches nevertheless provide superior correlations with anti-inflammatory activity in vivo.

Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor.

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii. A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-beta. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.

Drosophila dumbfounded: a myoblast attractant essential for fusion.

Aggregation and fusion of myoblasts to form myotubes is essential for myogenesis in many organisms. In Drosophila the formation of syncytial myotubes is seeded by founder myoblasts. Founders fuse with clusters of fusion-competent myoblasts. Here we identify the gene dumbfounded (duf) and show that it is required for myoblast aggregation and fusion. duf encodes a member of the immunoglobulin superfamily of proteins that is an attractant for fusion-competent myoblasts. It is expressed by founder cells and serves to attract clusters of myoblasts from which myotubes form by fusion.

The nomenclature of chemokines.

Migration of leukocytes to injured tissues is a hallmark of inflammation. The recruitment phase of cells can be subdivided into three steps: the rolling phase, the firm adhesion phase, and the transendothelial migration phase. Each step is mediated by a complex interplay of endothelial/leukocyte surface molecule interactions (mostly of selectin and integrin families) as well as a group of small, secreted peptides, called chemokines. Chemokines activate on the one hand, the leukocytes to express the appropriate adhesion molecules and on the other hand, they lead to transendothelial migration via chemotaxis (migration along a gradient in solution) and haptotaxis (migration along a gradient bound to extracellular martrices or cell membranes). The structure, biology and pathobiology of the more than 22 known members of this group of soluble mediators, with a particular emphasis on their past and present nomenclature, is the topic of this minireview.

Adrenomedullin inhibits the secretion of cytokine-induced neutrophil chemoattractant, a member of the interleukin-8 family, from rat alveolar macrophages.

This study was undertaken to determine the effects of adrenomedullin (AM) on the secretion of cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 family, from lipopolysaccharide-stimulated rat alveolar macrophages in vitro. AM significantly increased cAMP levels in alveolar macrophages in a dose-dependent fashion. On the other hand, AM significantly inhibited CINC secretion from alveolar macrophages in a dose-dependent fashion, and 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) also significantly inhibited CINC secretion. These findings suggest that AM may play important roles in the regulation of airway inflammation via a cAMP-dependent mechanism.

Modeling the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 on the basis of the solution structure of interleukin-8.

A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

[Chemotaxis and cellular migration in respiratory pathology]. / Chimiotactisme et migrations cellulaires en pathologie respiratoire.

Numerous types of cells have a capacity for movement in physiological or pathological situations. For example, this is the case for inflammatory cells in the lung, during acute lobar pneumonia, sarcoidosis and idiopathic pulmonary fibrosis. Cellular migration is a general process which rests on the interaction between different chemotactic factors and specific receptors which are present on the target cells. On the other hand the addition of inhibitors can significantly decrease the cellular migration in the presence of chemotactic factors. In respiratory pathology, congenital chemotactic defects are exceptional (and the Chediak-Higashi syndrome and Job syndrome are examples). In contrast during the course of lung cancer, circulating monocytes often show a significant decrease in their chemotactic responsiveness.

Chemotactic subpopulation of macrophage cell line cells (M1 cells) discerned by three macrophage chemotactic factors from delayed hypersensitivity reaction sites.

The functional specificity of three types of macrophage chemotactic factors ( MCFs ), -a, -b, and -c, from purified protein derivative of tuberculin (PPD)-induced delayed hypersensitivity reaction (DHR) skin sites on guinea pigs, was analyzed using macrophage cell line cells, M1, established from myeloid leukemia cells of a SL/Am strain mouse. M1 cells yielded two subclones: Mk1 cells, which were Ia+ and migrated specifically toward MCF-c; and Mm1 cells, which were Ia- and migrated specifically toward MCF-a and -b. These differences show the heterogeneity of the biologic activities of the MCFs in the presence of cell line cells. M-1 cells, blast cells, when grown in continuous culture, migrated toward none of the MCFs . Upon differentiation to mature macrophage-like cells in conditioned medium (M+1 cells), however, they migrated to MCF-a, -b, and -c and the experimental evidence points to the possibility that M+1 cells are separated into subpopulations on the basis of their chemotactic response. Cytotoxic treatment of M+1 cells with anti-Ia antisera resulted in the specific decrease of activity toward MCF-c. Furthermore, MCF-c attracted Ia-positive M+1 cells, while MCF-a and -b attracted Ia-negative M+1 cells. Thus, our results indicate the existence of two migrating subpopulations of M+1 cells with specificities for MCF-a, and -b and for MCF-c, respectively. The data suggest that MCF-c attracts Ia-bearing accessory macrophages and MCF-a and -b attract Ia-negative macrophages in the DHR.