Diversity and abundance of glycosyl hydrolase family 5 in the North Atlantic Ocean

The diversity and abundance of glycosyl hydrolase family 5 (GH5) were studied in the North Atlantic Ocean. This family was chosen because of the large number of available sequences from cultured bacteria, the variety of substrates it targets, and the high number of similar sequences in the Sargasso Sea environmental genome database. Three clone libraries of a GH5 subcluster were constructed from the Mid-Atlantic Bight and the eastern and western North Atlantic Ocean. The two North Atlantic Ocean libraries did not differ from each other but both were significantly less diverse than the Mid-Atlantic Bight library. The abundance of GH5 genes estimated by quantitative PCR was positively correlated with chlorophyll concentrations in the eastern part of a transect from Fort Pierce, Florida, to the Azores and in a depth profile, suggesting that the supply of labile organic material selects for GH5-bearing bacteria in these waters. However, the data suggest that only <1% of all bacteria harbor the GH5 subcluster. These and other data suggest that the hydrolysis of polysaccharides requires complicated multi-enzyme systems.     

Tissue development and RNA control: "HOW" is it coordinated?

The regulation of developmental processes at the RNA level enables selective and rapid modulation of gene expression. Studies in model organisms revealed the essential contribution of the signal transduction and activation of RNA (STAR) family of RNA binding proteins to developmental processes. STAR proteins coordinate the proper timing of developmental events by delaying expression or altering the mRNA or protein levels of essential genes. Recent functional analysis of the Drosophila melanogaster STAR protein, Held Out Wing (HOW), in the context of embryonic development, provided insight into its mode of activity. Here, we describe HOW's activity in the temporal repression or elevation of gene expression that is essential for coordinating the correct timing of instructive signals.     

Dimerization in vivo and inhibition of the nonreceptor form of protein tyrosine phosphatase epsilon

cyt-PTP epsilon is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTP epsilon enables analysis of phosphatase regulation in the absence of extracellular domains, which participate in dimerization and inactivation of the receptor-type phosphatases receptor-type protein tyrosine phosphatase alpha (RPTPalpha) and CD45. Using immunoprecipitation and gel filtration, we show that cyt-PTP epsilon forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTP epsilon readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTP epsilon dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTP epsilon, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTP epsilon is a major contributor to intermolecular binding and strongly interacts in a homotypic manner; the presence of D2 and the interactions that it mediates inhibit cyt-PTP epsilon activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTP epsilon lacking these regions constitutively dimerizes, and its activities in vitro towards para-nitrophenylphosphate and in vivo towards the Kv2.1 potassium channel are markedly reduced. We conclude that physiological signals can regulate dimerization and phosphorylation of cyt-PTP epsilon in the absence of direct interaction between the PTP and extracellular molecules. Furthermore, dimerization can be mediated by the D2 domain and does not strictly require the presence of PTP extracellular domains.     

GeneAnnot: interfacing GeneCards with high-throughput gene expression compendia

The interpretation of microarray expression results often includes extensive efforts to identify and annotate the gene representatives immobilised on the arrays. In this paper we describe the usage of our automatic GeneAnnot system, which links between Affymetrix arrays and the rich human gene annotations available in GeneCards. We explain GeneCards search options and results display; elaborate on the presentation of expression information in GeneCards, including both our whole-genome GeneNote project and external expression resources; describe the various parameters and displays used by GeneAnnot to assess the annotation quality and probeset specificity; and show how to search GeneAnnot and GeneNote websites directly.     

Polarization of myosin II heavy chain-protein kinase C in chemotaxing dictyostelium cells

Eukaryotic cells need morphological polarity to carry out chemotaxis (Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes, P. N. (1998) Cell 95, 81-91; Jin, T., Zhang, N., Long, Y., Parent, C., and Devreotes, P. N. (2000) Science 287, 1034-1036; Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W., and Bourne, H. R. (2000) Science 287, 1037-1040), but sensing direction does not require polarization of chemoattractant receptors. When cells are exposed to a gradient of chemoattractant, activation occurs selectively at the stimulated edge. Such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems. Here we show that in Dictyostelium discoideum cells exposed to a cAMP gradient the myosin II heavy chain kinase (MHC-PKC) and myosin II translocate to opposite ends of the cell. We further show that MHC-PKC C1 domain is responsible for the localization of MHC-PKC to the cell leading edge, but it is not sufficient to promote cell polarization. Our findings suggest a mechanism by which MHC-PKC regulates myosin II, allowing cell polarization and movement in the direction of the cAMP source.     

Temperature-sensitive mutants of the slime mould Physarum polycephalum

Summary Methods are described for the isolation and testing of temperature-sensitive plasmodial strains of Physarum polycephalum. Nineteen temperature-sensitive strains were found by screening plasmodia derived from mutagenised amoebae and the properties of these are described. A scheme is outlined for the detection of specific mitotic cycle lesions amongst temperature-sensitive strains, and the properties of a presumptive mitotic cycle mutant are described.     

Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion

In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17-18 kDa and 26-30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanced 11-ketotestosterone (11-KT) and estradiol secretion from tilapia testes and ovaries, respectively, in a dose-dependent manner (similar to tilapia pituitary extract, affinity-purified pituitary FSH, and porcine FSH). Using antibodies raised against rtFSH beta, FSH-containing cells were localized adjacent to hypothalamic nerve fibers ramifying in the proximal pars distalis (PPD), while LH cells were localized in a more peripheral region of the PPD. Moreover, FSH is under the control of hypothalamic decapeptide GnRH, an effect that was abolished through the use of specific bioneutralizing antisera, anti-rtFSH beta. It also reduced basal secretion of 11-KT.