Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.     

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.     

Correction to: Managing biological invasions: the cost of inaction

Biological Invasions -     

Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods

Among Polystomatidae (Monogenea), the genus Polystoma, which mainly infests neobatrachian hosts, is the most diverse and occurs principally in Africa, from where half the species have been reported. Previous molecular phylogenetic studies have shown that this genus originated in South America, and later colonised Eurasia and Africa. No mention was made on dispersal corridors between Europe and Africa or of the origin of the African Polystoma radiation. Therefore, a molecular phylogeny was inferred from ITS1 sequences of 21 taxa comprising two species from America, seven representatives from Europe and 12 from Africa. The topology of the phylogenetic tree reveals that a single event of colonisation took place from Europe to Africa and that the putative host carrying along the ancestral polystome is to be found among ancestral pelobatids. Percentage divergences estimates suggest that some presumably distinct vesicular species in unrelated South African anurans and some neotenic forms found in several distinct hosts in Ivory Coast, could, in fact, belong to two single polystome species parasitising divergent hosts. Two main factors are identified that may explain the diversity of African polystomes: (i), we propose that following some degree of generalism, at least during the juvenile stages of both hosts and parasites, distinctive larval behaviour of polystomes engenders isolation between parasite populations that precludes sympatric speciations; (ii), cospeciation events between Ptychadena hosts and their parasites are another factor of diversification of Polystoma on the African continent. Finally, we discuss the systematic status of the Madagascan parasite Metapolystoma, as well as the colonisation of Madagascar by the host Ptychadena mascareniensis.     

Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology

Repetitive DNA sequences may adopt unusual pairing arrangements. At acid to neutral pH, cytidine-rich DNA oligodeoxynucleotides can form the i-motif structure in which two parallel-stranded duplexes with C.C(+) pairs are intercalated head-to-tail. The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytidine tracts, the nucleotide sequences between them, and the experimental conditions.We have found that a natural fragment of the human centromeric satellite III, d(CCATTCCATTCCTTTCC), can form two monomeric i-motif structures that differ in their intercalation topology and that are favored at pH values higher (the eta-form) and lower (the lambda-form) than 4.6. The change in intercalation may be related to adenine protonation in the loops.We studied the uridine derivative methylated on the first cytidine base, d(5mCCATTCCAUTCCUTTCC), whose proton spectrum is better resolved. The intercalation topologies are (C7.C17)/(5mC1.C11)/(C6.C16)/(C2.C12) for form lambda and (5mC1.C11)/(C7.C17)/(C2.C12)/(C6.C16) for form eta. We have solved the structure of the eta-form, and we present a model for the lambda-form. The switch from eta to lambda involves disruption of the i-motif. In both forms, the central AUT linker crosses the wide groove, and the first and the third linkers loop across the minor grooves. The i-motif core is extended in the eta-form by the inter-loop reverse Watson-Crick A3.U13 pair, whose dissociation constant is around 10(-2) at 0 degrees C, and in the lambda-form by the interloop T5.T15 pair.In contrast, d(5mCCATTCCTTACCTTTCC) folds into a pH-independent structure that has the same intercalation topology as the lambda-form. The i-motif core is extended below by the interloop T5.T15 pair and closed on top by the T8.A10 pair.Thus, the C-rich strand of the human satellite III tandem repeats, like the G-rich strand, can fold into various compact structures. The relevance of these features to centromeric function remains unknown.     

Quantitative cleavage of the N-glycosidic bond under the normal conditions of methanolysis used for the analysis of glycoprotein monosaccharides

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.     

The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.     

Delineation of two distinct 6p deletion syndromes

Deletions of the short arm of chromosome 6 are relatively rare, the main features being developmental delay, craniofacial malformations, hypotonia, and defects of the heart and kidney, with hydrocephalus and eye abnormalities occurring in some instances. We present the molecular cytogenetic investigation of six cases with 6p deletions and two cases with unbalanced translocations resulting in monosomy of the distal part of 6p. The breakpoints of the deletions have been determined accurately by using 55 well-mapped probes and fluorescence in situ hybridization (FISH). The cases can be grouped into two distinct categories: interstitial deletions within the 6p22–p24 segment and terminal deletions within the 6p24–pter segment. Characteristics correlating with specific regions are: short neck, clinodactyly or syndactyly, brain, heart and kidney defects with deletions within 6p23–p24; and corneal opacities/iris coloboma/Rieger anomaly, hypertelorism and deafness with deletions of 6p25. The two cases with unbalanced translocations presented with a Larsen-like syndrome including some characteristics of the 6p deletion syndrome, which can be explained by the deletion of 6p25. Such investigation of cytogenetic abnormalities of 6p using FISH techniques and a defined set of probes will allow a direct comparison of reported cases and enable more accurate diagnosis as well as prognosis in patients with 6p deletions. Received: 29 July 1998 / Accepted: 28 October 1998