Molecular characterization of the viaB locus encoding the biosynthetic machinery for Vi capsule formation in Salmonella Typhi

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi

Dexamethasone to prevent everolimus-induced stomatitis (Alliance MIST trial: A221701)

mTOR inhibitors such as everolimus may cause oral stomatitis, often a dose-limiting toxicity. Prior clinical research has suggested that a dexamethasone mouth rinse might help prevent and/or treat this. Alliance A221701 was a randomized phase III trial of patients initiating 10 mg daily oral everolimus that compared dexamethasone mouthwash taken preventively (initial dexamethasone group) versus therapeutically (initial placebo group) to assess two coprimary endpoints: the incidence of mTOR inhibitor-associated stomatitis (mIAS), and the area under the curve (AUC) of mIAS-associated pain over an 8-week treatment period. A Fisher\u27s exact test was used to compare the incidences while a Wilcoxon rank-sum test was used to compare the AUCs. In addition, we performed an exploratory analysis of the association of everolimus trough concentrations and toxicity using a Mann-Whitney U test. Due to slow accrual, this study closed after 39 patients were randomized (19 to upfront placebo and 20 to upfront dexamethasone). There were no significant differences between groups seen in either of the coprimary endpoints; furthermore, we found no association between whole blood everolimus trough concentrations and toxicity. Although limited by poor enrollment, the results of this study do not suggest that prophylactic dexamethasone mouthwash is superior to therapeutic dexamethasone mouthwash (initiated at the first sign of mouth pain) for reducing the incidence or severity of mIAS from everolimus

Floristic composition and community structure of epiphytic angiosperms in a terra firme forest in central Amazonia

This survey aimed to describe the floristic composition and structure of the epiphytic community occurring in a terra firme forest in the city of Coari, Brazil, in the Amazon region. Data collection was performed with a 1.5 ha plot method, with which upland, slope and lowland habitats were sampled. All angiosperm epiphytes and their host plants (diameter at breast height > 10 cm) were sampled. We recorded 3.528 individuals in 13 families, 48 genera and 164 species. Araceae was the most prevalent family with regard to the importance value and stood out in all related parameters, followed by Bromeliaceae, Cyclanthaceae and Orchidaceae. The species with the highest epiphytic importance values were Guzmania lingulata (L.) Mez. and Philodendron linnaei Kunth. The predominant life form was hemiepiphytic. Estimated floristic diversity was 3.2 (H'). The studied epiphytic community was distributed among 727 host plants belonging to 40 families, 123 genera and 324 species. One individual of Guarea convergens T.D. Penn. was the host with the highest richness and abundance of epiphytes. Stems/trunks of host plants were the most colonized segments, and the most favorable habitat for epiphytism was the lowlands, where 84.1% of species and 48.2% of epiphytic specimens were observed

The ins(ide) and outs(ide) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum

The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc2-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc2-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc2-P-P-Dol to Glc3Man9GlcNAc2-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough E

An alternative cis-isoprenyltransferase activity in yeast that produces polyisoprenols with chain lengths similar to mammalian dolichols

Dolichyl monophosphate (Dol-P) is a polyisoprenoid glycosyl carrier lipid essential for the assembly of a variety of glycoconjugates in the endoplasmic reticulum of eukaryotic cells. In yeast, dolichols with chain lengths of 14-17 isoprene units are predominant, whereas in mammalian cells they contain 19-22 isoprene units. In this biosynthetic pathway, t,t-farnesyl pyrophosphate is elongated to the appropriate long chain polyprenyl pyrophosphate by the sequential addition of cis-isoprene units donated by isopentenyl pyrophosphate with t,t,c-geranylgeranyl pyrophosphate being the initial intermediate formed. The condensation steps are catalyzed by cis-isoprenyltransferase (cis-IPTase). Genes encoding cis-IPTase activity have been identified in Micrococcus luteus, Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae (RER2). Yeast cells deleted for the RER2 locus display a severe growth defect, but are still viable, possibly due to the activity of an homologous locus, SRT1. The dolichol and Dol-P content of exponentially growing revertants of RER2 deleted cells (Δrer2) and of cells overexpressing SRT1 have been determined by HPLC analysis. Dolichols and Dol-Ps with 19-22 isoprene units, unusually long for yeast, were found, and shown to be utilized for the biosynthesis of lipid intermediates involved in protein N-glycosylation. In addition, cis-IPTase activity in microsomes from Δrer2 cells overexpressing SRT1 was 7- to 17-fold higher than in microsomes from Δrer2 cells. These results establish that yeast contains at least two cis-IPTases, and indicate that the chain length of dolichols is determined primarily by the enzyme catalyzing the chain elongation stage of the biosynthetic proces

Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae

Dolichol kinase (DK) catalyzes the CTP-mediated phosphorylation of dolichol in eukaryotic cells, the terminal step in dolichyl monophosphate (Dol-P) biosynthesis de novo. In S. cerevisiae, the SEC59 gene encodes a protein essential for the expression of DK, an enzyme activity that is required for cell viability and normal rates of lipid intermediate synthesis and protein N-glycosylation. This study identifies a cDNA clone from human brain that encodes the mammalian homolog of DK (hDK1p). hDK1 is capable of complementing the growth defect, elevating DK activity, and consequently increasing Dol-P levels in vivo and restoring normal N-glycosylation of carboxypeptidase Y at the restrictive temperature in the temperature-sensitive mutant sec59-1. The CTP-mediated phosphorylation of diacylglycerol (DAG) is unaffected by either the temperature-sensitive mutation in the sec59-1 strain, overexpression of the SEC59 gene, or the mammalian homolog hDK1 under conditions that produced a loss or elevation in the level of DK activity. Additionally, overexpression of hDK1p in Sf-9 cells resulted in a 15-fold increase in DK activity but not DAG kinase activity in crude microsomal fractions. The cloned cDNA contains an open reading frame that would encode a protein with 538 amino acids and a molecular weight of 59,268 kDa. Consistent with this prediction, new polypeptides were detected with an apparent molecular weight of 59-60 kDa when His6-tagged constructs of hDK1 or the SEC59 gene were expressed in Sf-9 cells or the temperature-sensitive sec59-1 mutant cells, respectively. These results identify the first cDNA clone encoding a protein required for the expression of DK activity, possibly the catalytic subunit, in a mammalian cell, and establish that the phosphorylation of dolichol and DAG are catalyzed by separate kinase activities in yeas

Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae

ISSN:0959-665

An electrospray-ionization tandem mass spectrometry method for determination of the anomeric configuration of glycosyl 1-phosphate derivatives.

A rapid, simple, and sensitive method is described for the determination of the anomeric configuration of sugar 1-phosphates, sugar nucleotides, and polyisoprenyl-phospho-sugars. Negative-ion electrospray ionization of picomole amounts of glycosyl 1-phosphate derivatives produces an intense signal of the [M-H]-deprotonated molecule which, by collision-induced dissociation, decomposes in a characteristic manner depending on cis/trans configuration of the 2-hydroxyl and phosphate groups of the glycosyl residue. A distinct feature of the product ion spectra of glycosyl 1-P and polyisoprenyl-P-sugars with cis configuration is the presence of abundant ions that correspond to the [M-H2O-H]- dehydration product and the [R-PO4-(C2H3O]- fragment arising from a cleavage across the sugar ring, where R is -H or -polyprenyl/dolichyl for glycosyl 1-P and polyisoprenyl-P-sugar, respectively. These two fragments, [M-H2O-H]- and [R-PO4-(C2H3O)]- are absent from the product ion spectra of sugar 1-P and polyisoprenyl-P-sugars with trans configuration. For sugar nucleotides, compounds with cis configuration produce, in tandem mass spectrometry, only one abundant fragment of nucleoside monophosphate, whereas those with trans configuration give nucleoside diphosphate as a major fragment ion. Accordingly, the anomeric configuration of a glycosyl 1-phosphate derivative can be easily determined by using electrospray-ionization tandem mass spectrometry provided that the glycosyl residue of known absolute configuration has a free 2-hydroxyl group and no other charge location