Description of intertidal macro- and meiobenthic assemblages in Maxwell Bay, King George Island, South Shetland Islands, Southern Ocean

The intertidal benthic fauna of the Antarctic coastal areas is largely unknown and has long been thought to be absent or, at most, to be scarce. Since climate changes cause a progressive expansion of ice-free intertidal soft-bottom areas, the fauna of these areas could serve as essential criterion to evaluate the kind and dimension of such changes. We therefore investigated the faunal composition of the intertidal soft-bottom area of Maxwell Bay (King George Island, South Shetlands) in December 2006. Samples for quantitative analyses were taken from the soft-bottom during low tide using a plastic corer. We performed detailed analyses of the soft-bottom beneath a cobble layer, while hard-bottom and macrophytes were only sporadically investigated. Approximately 5,000 specimens were collected of which polychaetes (37.3 ± 7.6 (max. 44.7) ind. × 100 cm^?³) and harpacticoids (28.9 ± 28.5 (max. 104.0) ind. × 10 cm^?³) were the most abundant macro- and meiofauna taxa of the soft-bottom, followed by oligochaetes, nematodes, mollusks, and amphipods. A total of 58 macrofauna species were registered, of which 27 were identified only to a supraspecific level. The most species-rich macrofauna taxon was polychaetes with at least 24 species, followed by amphipods, gastropods, and oligochaetes with 6 species each. The harpacticoid copepods were represented by 15 families with more than 30 species. In summary, we show that the Antarctic intertidal soft-bottom is densely populated by macro- and meiofauna and that it deserves closer attention in the future to determine whether it can indeed serve as an indicator of the effect of climate changes on the Antarctic coastal areas.     

Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Selective effects of anti-Ia serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulfate and lipopolysaccharide.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody; cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.     

The PLAC1 protein localizes to membranous compartments in the apical region of the syncytiotrophoblast

PLAC1 is a trophoblast-specific gene that maps to a locus on the X-chromosome important to placental development. We have previously shown that PLAC1 gene expression is linked to trophoblast differentiation. The objective of this study was to define the localization of the PLAC1 polypeptide as a prerequisite to understanding its function. Polyclonal antibodies specific for the putative PLAC1 polypeptide were generated. The subcellular localization of PLAC1 in the trophoblast was examined by immunohistochemical analysis of human placenta complemented by immunoblot analysis of subcellular fractions. Brightfield immunohistochemical analysis of placental tissue indicated that the PLAC1 protein localizes to the differentiated syncytiotrophoblast in the apical region of the cell. Deconvlution immunofluorescence microscopy confirmed localization to the apical region of the syncytiotrophoblast. Its distribution included both intracellular compartments as well as loci in close association with the maternal-facing, microvillous brush border membrane (MVM). These findings were supported by immunoblot analysis of subcellular fractions. A 30 kDa band was associated with the microsomal fraction of placental lysates but not the mitochondrial, nuclear, or soluble fractions, suggesting PLAC1 is targeted to a membrane location. Plasma membranes were obtained from the fetal-facing, basal surface (BM) and the maternal-facing, MVM of the syncytiotrophoblast membrane. PLAC1 immunoreactivity was only detected in membrane fractions derived from the apical MVM consistent with immunohistochemical analyses. These data demonstrate that the PLAC1 protein is restricted primarily to the differentiated trophoblast, localizing to intracellular membranous compartment(s) in the apical region of the syncytiotrophoblast and associated with its apical, microvillous membrane surface.     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Burden of rare sarcomere gene variants in the framingham and jackson heart study cohorts

Rare sarcomere protein variants cause dominant hypertrophic and dilated cardiomyopathies. To evaluate whether allelic variants in eight sarcomere genes are associated with cardiac morphology and function in the community, we sequenced 3,600 individuals from the Framingham Heart Study (FHS) and Jackson Heart Study (JHS) cohorts. Out of the total, 11.2% of individuals had one or more rare nonsynonymous sarcomere variants. The prevalence of likely pathogenic sarcomere variants was 0.6%, twice the previous estimates; however, only four of the 22 individuals had clinical manifestations of hypertrophic cardiomyopathy. Rare sarcomere variants were associated with an increased risk for adverse cardiovascular events (hazard ratio: 2.3) in the FHS cohort, suggesting that cardiovascular risk assessment in the general population can benefit from rare variant analysis.     

Characterization of a Chitinase Gene from Stenotrophomonas maltophilia Strain 34S1 and Its Involvement in Biological Control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.