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PubMed2000

Epidemiology of yeast colonization in the intensive care unit.

In order to investigate the epidemiology of colonization and possible transmission of yeasts among patients and healthcare workers in adult intensive care units (ICUs), 194 patients were followed for a mean of 9 +/- 11 days and 63 healthcare workers were followed for a mean of 132 +/- 52 days. Among the patients, 142 (73%) were colonized by yeast, with Candida albicans being the species most commonly recovered. Most patients (65%) were already colonized with yeast upon admission to the intensive care unit; only 17% became colonized after admission. Persistent colonization occurred in 51 (55%) of 92 patients who had more than three cultures performed; in 75% of them, colonization persisted with the same strain of Candida albicans or Candida glabrata. Bacterial infection in the month preceding entry into the ICU was the only risk factor significantly associated with yeast colonization. Among the healthcare workers, yeasts were isolated from 42 (67%). Candida albicans was most frequently recovered from the oropharynx (19% of occasions), and Candida parapsilosis was most frequently found on hands (8% of occasions). Persistent colonization of the oropharynx occurred in only six healthcare workers, and none had persistence of yeasts on hands. In this non-outbreak setting, 5 (4%) of 123 patient/healthcare worker interactions that were linked epidemiologically yielded the same strain of Candida albicans, providing evidence for possible cross-transmission. No similar link was found between healthcare worker-patient interactions and colonization with Candida glabrata or Candida parapsilosis.

Adult↗
PubMed2006

Electrical stimulation of the energy metabolism in yeast cells using a planar Ti-Au-electrode interface.

We report on the influence of dielectric pulse injection on the energy metabolism of yeast cells with a planar interdigitated electrode interface. The energy metabolism was measured via NADH fluorescence. The application of dielectric pulses results in a distinct decrease of the fluorescence, indicating a response of the energy metabolism of the yeast cells. The reduction of the NADH signal significantly depends on the pulse parameters, i.e., amplitude and width. Furthermore, the interface is used to detect electrical changes in the cell-electrolyte system, arising from glucose-induced oscillations in yeast cells and yeast extract, by dielectric spectroscopy at 10 kHz. These dielectric investigations revealed a beta(1)-dispersion for the system electrolyte/yeast cells as well as for the system electrolyte/yeast extract. In agreement with control measurements we obtained a glycolytic period of 45 s for yeast cells and of 11 min for yeast extract.

Electric Conductivity↗
PubMed1992

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole.

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.

Animals↗
PubMed1978

Comparison of a nitrate reductase-inactivating enzyme from the maize root with a protease form yeast which inactivates tryptophan synthase.

A maize root fraction which inactivates nitrate reductase has been shown to have protease activity which can be measured by the hydrolysis of azocasein. This inactivating enzyme was also found to inactivate yeast tryptophan synthase. Yeast proteases A and B, which inactivate this latter enzyme, also gave a specific inactivation of the maize nitrate reductase. The maize root inactivating enzyme, like yeast protease B, degraded casein, and was inhibited by phenylmethylsulphonyl fluoride. A partially-purified yeast inhibitor prevented catalysis by the yeast proteases and maize root inactivating enzyme, but purified yeast inhibitors were without effect on the latter protein. The level of nitrate reductase-inactivating activity, and associated azocasein-degrading activity, increased with age of the maize root. Evidence was obtained for a heat stable inhibitor which maintained them in an inactive state, especially in the young root tip cells.

Kinetics↗
PubMed1986

Yeast centromeric plasmids as shuttle vectors between Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae.

A number of hybrid plasmids which can autonomously replicate in E. coli, B. subtilis and S. cerevisiae was constructed. Replication of these plasmids both in yeast and in B. subtilis starts on a sequences originating from Staphylococcus aureus plasmids pC194 and pE194. In yeast these hybrids are unstable like those yeast vectors which contain eukaryotic ARSs, but their stability has been increased by addition of yeast centromeric sequence. Both pC194 and pE194 DNAs contain sequences which reveal strong similarities with the yeast ARS consensus. Nevertheless the replication efficiences of these plasmids in yeast are different.

Bacillus subtilis↗
PubMed1985

The isolation of glucose tolerance factors from brewer's yeast and their relationship to chromium.

Growth of brewer's yeast in the presence of Cr3+ led to increased yields of substances with GTF activity in assays performed with isolated adipocytes. The formation of these substances may be a means of diminishing the toxic effects of Cr on yeast. During fractionation of extracts of brewer's yeast the Cr was easily dissociated from any complexes that may have been present. The GTF activity for both yeast and adipocytes was isolated in cationic and anionic small amino-acid or peptide-like molecules. These substances caused increased glycolysis in yeast and increased glycolysis and fatty acid synthesis in adipocytes. No evidence was found that GTF aided the binding of insulin to its receptor. The GTF activity could only be demonstrated with adipocytes from rats fed a torula yeast-high sucrose diet, which may have caused the rats to have a decreased sensitivity to insulin.

Adipose Tissue↗
PubMed1993

Evaluation of liquid yeast-derived sucrase enzyme replacement in patients with sucrase-isomaltase deficiency.

BACKGROUND: No enzyme replacement therapy exists for patients with congenital sucrase-isomaltase deficiency (CSID). A by-product of the manufacture of baker's yeast is a liquid preparation containing high sucrase activity. The aim of the present study was to investigate the activity and stability of this preparation and its effect on breath hydrogen excretion and gastrointestinal symptoms after sucrose ingestion in 14 patients with CSID. METHODS: The homogeneity of yeast sucrase was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its activity was measured. Stability at various temperatures and pH ranges and in the presence of gastric aspirate, pepsin, and bovine serum albumin was assessed. Fourteen patients with CSID underwent double-blind placebo-controlled breath tests with yeast sucrase. They then completed an 8-week dose response study that used different enzyme concentrations while consuming a sucrose-containing diet. RESULTS: Liquid yeast sucrase is highly glycosylated, contains no lactase activity, and is stable at 4 degrees C and over a wide range of pH. Pepsin digestion of the enzyme in vitro can be blunted by bovine serum albumin and by increasing the pH. Yeast sucrase reduces breath hydrogen excretion in patients with CSID who are given a sucrose load (P < 0.001) and allows most patients to consume a sucrose-containing diet. CONCLUSIONS: Liquid yeast sucrase offers effective enzyme replacement therapy for patients with CSID.

Administration, Oral↗
PubMed1985

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal.

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Anticodon↗
PubMed1985

Crystallographic refinement of yeast aspartic acid transfer RNA.

The structure of yeast transfer RNA aspartic acid has been refined in one crystal form to 3 A resolution using the restrained least-squares method of Hendrickson and Konnert and real-space fitting using the FRODO program of Jones. The final crystallographic discrepancy index R is 23.5% for 4585 reflections with magnitudes twice their standard deviations between 10 and 3 A. With lower occupancies for some residues of the D-loop, the phosphate U1, and the base U33, the R-factor is 22.3%. The adaptation of the restrained least-squares program for nucleic acids and the progress of the refinement are described. The conformations are analysed with respect to stereochemistry and folding of the backbone. The contacts and hydrogen bonds of the secondary structure are compared with those of yeast tRNAPhe. The presence of only four bases in the variable loop, instead of five as in yeast tRNAPhe, leads to a rotation of residue 48 and a lateral movement of residue 46. These two rearrangements induce different environments for [U8 . . . A14] . . . A21 as well as for A9 and G45. Otherwise, all tertiary contacts observed in yeast tRNAPhe are present in yeast tRNAAsp, except for the absence of hydrogen-bonding between G18 of the D-loop and C56 of the T-loop. The presence of anticodon triplet pairing leads to a distribution of temperature factors different from that observed in yeast tRNAPhe with a stabilization of the AC stem-and-loop and a destabilization of the T and D-loops. We are inclined to suggest that the labilization of the interactions between the T and D-loops is a consequence of the interaction of the anticodon triplets of symmetry-related molecules through hydrogen bonding, which mimics the interaction between the anticodon and its cognate codon on the messenger RNA.

Anticodon↗
PubMed1987

Ribosomal proteins EL11 from Escherichia coli and L15 from Saccharomyces cerevisiae bind to the same site in both yeast 26 S and mouse 28 S rRNA.

The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T1. In both cases, the position of this fragment in the L-rRNA sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts. Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation. Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.

Animals↗
PubMed1992

Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions.

As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.

Amino Acid Sequence↗
PubMed1979

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.

Aneuploidy↗
PubMed1987

Yeast species and biotypes associated with oral leukoplakia and lichen planus.

Of 36 patients, 17 had oral leukoplakia, including homogeneous and nonhomogeneous types, and 19 had reticular lesions of oral lichen planus. A sample of yeast flora in each patient was taken from the pathologic lesion as well as from normal-appearing mucosa. The isolated yeasts were identified according to species level, and identification was extended beyond the species level for one species, Candida albicans, to reveal the biotype by means of the Odds and Abbott procedure comprising tests for acid and salt tolerance, proteinase production, resistance to 5-fluorocytosine and safranine, and assimilation of urea, sorbose, and citrate. Yeasts were present in the lesions of 82% of leukoplakia patients, compared to 37% of lichen planus patients, a frequency of yeasts corresponding to that in healthy adults. C. albicans was the dominating species in lesions of both diseases, constituting 82% of all yeasts in the leukoplakia lesions. In addition, the following species were identified: Candida tropicalis, Candida pintolopesii, Torulopsis glabrata, and Saccharomyces cerevisiae. Eighteen biotypes of C. albicans were encountered, the most frequently occurring biotypes being 355 and 177. Differences between C. albicans biotypes isolated from pathologic and normal mucosa were encountered in five of eleven leukoplakia patients and in one of three lichen planus patients. This indicates that the oral cavity comprises several ecologic niches for yeasts. As nonhomogeneous leukoplakias are more likely to develop into carcinoma than are homogeneous leukoplakias, it is interesting to note that the C. albicans biotypes isolated from nodular lesions (one type of nonhomogeneous leukoplakia)--biotypes 145, 175, and 575--rarely occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae.

The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.

Antibodies, Viral↗
PubMed1990

Manipulating yeast genome using plasmid vectors.

The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

Cloning, Molecular↗
PubMed1986

Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system.

A homologous cell-free system has been derived from the yeast Saccharomyces cerevisiae that allows the translation, translocation, and glycosylation of the precursors of yeast mating factor alpha and invertase. The precursors were translated in a yeast lysate from mRNA obtained by in vitro transcription of the MF alpha 1 and SUC2 genes. Inclusion of yeast microsomes resulted in the glycosylation of the alpha-factor precursor, which was demonstrated to be sequestered within the membrane vesicles. Similar results, including signal sequence cleavage, were observed for invertase. Processing of secretory proteins translated in a yeast lysate could not be achieved using microsomes derived from canine pancreas, nor were yeast microsomes active in a wheat germ translation system.

Biological Transport↗
PubMed1988

Expression of the SV40 promoter in fission yeast: identification and characterization of an AP-1-like factor.

The SV40 promoter is expressed well in the fission yeast S. pombe, and it initiates transcription at the same site as in mammalian cells. The majority of the enhancer sequences, however, do not contribute to this activity. DNAase I footprint analysis of the promoter revealed the presence of an AP-1-like factor in S. pombe cells that protects a region of the promoter almost identical to that protected by human AP-1. The specificity of binding of the yeast and mammalian AP-1 proteins was found to be similar. We have found two AP-1-like binding activities in budding yeast cells, one of which appears quite distinct from the binding activity of the product of the budding yeast GCN4 gene. We also demonstrate that in fission yeast the AP-1 binding site can act as an upstream activating sequence. The DNA-protein complexes containing the mammalian AP-1 and fission yeast AP-1-like factors are sensitive to phosphatase treatment, indicating that they may be phosphorylated.

Deoxyribonuclease I↗
PubMed1983

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast.

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).

Kinetics↗