Patterns of fungal diversity and composition along a salinity gradient

Estuarine salinity gradients are known to influence plant, bacterial and archaeal community structure. We sequenced 18S rRNA genes to investigate patterns in sediment fungal diversity (richness and evenness of taxa) and composition (taxonomic and phylogenetic) along an estuarine salinity gradient. We sampled three marshes—a salt, brackish and freshwater marsh—in Rhode Island. To compare the relative effect of the salinity gradient with that of plants, we sampled fungi in plots with Spartina patens and in plots from which plants were removed 2 years prior to sampling. The fungal sediment community was unique compared with previously sampled fungal communities; we detected more Ascomycota (78%), fewer Basidiomycota (6%) and more fungi from basal lineages (16%) (Chytridiomycota, Glomeromycota and four additional groups) than typically found in soil. Across marshes, fungal composition changed substantially, whereas fungal diversity differed only at the finest level of genetic resolution, and was highest in the intermediate, brackish marsh. In contrast, the presence of plants had a highly significant effect on fungal diversity at all levels of genetic resolution, but less of an effect on fungal composition. These results suggest that salinity (or other covarying parameters) selects for a distinctive fungal composition, and plants provide additional niches upon which taxa within these communities can specialize and coexist. Given the number of sequences from basal fungal lineages, the study also suggests that further sampling of estuarine sediments may help in understanding early fungal evolution.     

In vitro and in vivo profile of 5-[(4'-trifluoromethyl-biphenyl-2-carbonyl)-amino]-1H-indole-2-carboxylic acid benzylmethyl carbamoylamide (dirlotapide), a novel potent MTP inhibitor for obesity

The synthesis of a novel gut selective MTP inhibitor, 5-[(4'-trifluoromethyl-biphenyl-2-carbonyl)-amino]-1H-indole-2-carboxylic acid benzylmethyl carbamoylamide (dirlotapide), and its in vitro and in vivo profile are described. Dirlotapide (3) demonstrated excellent potency against MTP enzyme in HepG2 cells and canine hepatocytes. This novel MTP inhibitor also showed excellent efficacy when tested in a canine food intake model.     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.     

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.      

Analysis of glycosyltransferase expression in metastatic prostate cancer cells capable of rolling activity on microvascular endothelial (E)-selectin

Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLe(X))-bearing glycoproteins. To explore the molecular basis of sLe(X) synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLe(X) and ESL synthesis, in ESL(+) MDA cells among other ESL(-) metastatic PCa cell lines. We also created and examined ESL(hi) and ESL(lo) variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL(+) MDA cells expressed a 31- and 10-fold higher level of alpha1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL(-) PCa cell lines. Consistent with these findings, ESL(hi) MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESL(lo) MDA cells. We also noted that alpha1,3 FT7 was expressed at a 5-fold greater level in ESL(hi) MDA cells. Furthermore, ESL(lo) MDA cells did not display sLe(X) on glycoproteins capable of bearing sLe(X), notably P-selectin glycoprotein ligand-1. These results implicate the importance of alpha1,3 FT3, FT6, and/or FT7 in sLe(X) and ESL synthesis on metastatic PCa cells.     

In vitro and in vivo profile of 2-(3-di-fluoromethyl-5-phenylpyrazol-1-yl)-5-methanesulfonylpyridine, a potent, selective, and orally active canine COX-2 inhibitor

The synthesis of a novel canine COX-2 selective inhibitor, 2-(3-difluoromethyl-5-phenylpyrazol-1-yl)-5-methanesulfonylpyridine, and its in vitro and in vivo profile are described. Pyrazole 8 demonstrated excellent potency and selectivity for canine COX-2 in both in vitro and ex vivo whole blood assays. This novel COX-2 inhibitor also showed a good pharmacokinetic profile (pk) following oral (po), intravenous (iv), and subcutaneous (sc) dosing and demonstrated excellent in vivo efficacy in a canine synovitis model.