Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole

Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.     

Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.     

Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. alpha-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure alpha-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified alpha-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both alpha-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of alpha-sarcin and made the lambda(em)m shift from 340 to 347 nm. Titration of alpha-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of alpha-sarcin molecule. This indicated that Trps were not involved in the binding of alpha-sarcin to chitin. Glycochitin in the culture medium increased the expression of alpha-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of alpha-sarcin and AFP, glycochitin increased the nuclease activity of alpha-sarcin.     

Marine phage genomics

Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.     

Protein and fatty acid composition of caveolae from apical plasmalemma of aortic endothelial cells

In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) represent a structurally and biochemically specialized membrane microdomain. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC by using cationic colloidal silica (silica-ap), or the EC were surface-radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fraction and cell homogenate followed by ultracentrifugation on a sucrose gradient gave detergent-soluble and detergent-insoluble membranous fractions. The lowest density TX-insoluble fraction appeared morphologically as distinct vesicles (caveolae; 60?nm average diameter; PVs fraction). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentration, of specific caveolar markers, viz., caveolin (both isoforms, the 24-kDa form being conspicuously more abundant) and Ca²⁺-ATPase. By contrast, angiotensin-converting enzyme and alkaline phosphodiesterase were present almost exclusively in the TX-soluble fraction. The glycoproteins in the PVs fraction were of apparent molecular weights 52, 68, 95, and 114?kDa. Analysis of the fatty acid composition revealed more palmitoleic and stearic acid in the PVs fraction then in the TX-soluble fraction. Thus, in comparison with the plasmalemma proper, the PVs fraction (1) is detergent-insoluble; (2) contains caveolin in two isoforms; (3) contains Ca²⁺-ATPase at high concentration; (4) contains a set of specific glycoproteins; and (5) is enriched in palmitoleic and stearic acids.     

The occurrence and ecology of microbial chain elongation of carboxylates in soils

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.Subject terms: Soil microbiology, Microbial ecology     

Lymphoma immunophenotyping: "borderline" lymphomas

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.