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PubMed2005

Recovery from iron deficiency in rats by the intake of recombinant yeast producing human H-ferritin.

OBJECTIVE: This study examined whether iron accumulated in ferritin-producing recombinant microbes is bioavailable to rats with iron deficiency. METHODS: Rats induced with iron deficiency were treated with iron preparations of ferrous ammonium sulfate, horse spleen ferritin, control yeast, and ferritin-producing recombinant yeast for 14 d. The bioavailability of iron was examined by measuring hemoglobin concentration, hematocrit value, and tissue iron stores. Differences between dietary groups were determined by one-way analysis of variance, and P < 0.05 was considered statistically significant. RESULTS: Based on hemoglobin concentration and hematocrit value, iron in ferrous ammonium sulfate, horse spleen ferritin, and ferritin-producing yeast were bioavailable to rats and cured iron deficiency. The efficacy of ferritin and ferritin-producing yeast was also confirmed in establishing tissue iron stores after induction of iron deficiency. CONCLUSIONS: The iron sources of ferritin and ferritin-producing yeast were as effective in recovery from iron deficiency as the iron compounds of ferric citrate and ferrous ammonium sulfate. The results suggest that the iron stored in the ferritin of recombinant yeast is bioavailable, and that recombinant yeast may contribute widely as a source of iron to resolve the global problem of iron deficiency.

Analysis of Variance↗
PubMed2005

Studying phospholipid metabolism using yeast systematic and chemical genetics.

Most phospholipid metabolic pathways in the budding yeast Saccharomyces cerevisiae are analogous to their mammalian counterparts. The biological tractability of yeast provides for an opportunity to rapidly determine functions of specific lipids or lipid metabolic pathways using both classical and chemical-genetic techniques. The recent generation of the yeast genome deletion collection revealed that approximately 75% of yeast genes are not essential for life. Coupling analysis of the yeast deletion collection with automation using high-throughput robotics enables yeast genetic screens to be more thorough and bypasses the requirement for library screens to identify genes of interest. Two high-throughput yeast genetic methods are described, systematic synthetic lethality and chemical genetics. Systematic synthetic lethality is based on the principle that inactivation of two genes separately has minimal effects on cell growth whereas inactivation of both genes simultaneously results in growth defects due to their shared requirement in a particular cellular process. Chemical genetics is the analysis of bioactive compounds to determine processes that regulate susceptibility to the compound under study, and provides powerful data regarding precise targets and mechanism of action that regulate action of the compound.

Automation↗
PubMed2001

Expression of 17beta-hydroxysteroid dehydrogenases in mesophilic and extremophilic yeast.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes responsible for reversible interconversions of biologically active 17-hydroxy and inactive 17-keto steroids. We have performed a survey of 17beta-HSD activity in yeast. Constitutive 17beta-HSD activity was found in three mesophilic yeast species: Candida tropicalis, Cryptococcus tsukubaensis, and Saccharomyces cerevisiae as well as in three extremophilic black yeast species: Hortaea werneckii, Trimmatostroma salinum, and Phaeotheca triangularis, indicating that 17beta-HSD activity is widely distributed among yeast. In extremophilic black yeast, NaCl modulated enzyme activity. Enzymes resembling 17beta-HSD from the filamentous fungus Cochliobolus lunatus were detected in Trimmatostroma salinum and Phaeotheca triangularis. Sequences with identity to the Saccharomyces cerevisiae YBR159w gene were not observed in other yeast species possessing a similar enzyme activity. The results suggest the existence of at least three different types of 17beta-HSD in yeast.

17-Hydroxysteroid Dehydrogenases↗
PubMed2003

A comparative study on the biosorption characteristics of some yeasts for Remazol Blue reactive dye.

Biosorption capacities and rates of different kinds of dried yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces marxianus, Candida sp., C. tropicalis, C. lipolytica, C. utilis, C. quilliermendii and C. membranaefaciens) for Remazol Blue reactive dye from aqueous solutions were compared under laboratory conditions as a function of initial pH and initial dye concentration. Optimum initial biosorption pH was determined as 2 for all the yeasts. All the yeast species showed comparable and very high dye sorption at 100 mg/l initial dye concentration. The equilibrium sorption capacity of the biomass increased with increasing initial dye concentration up to 400 mg/l for Candida sp. C. lipolytica and C. tropicalis; up to 300 mg/l for C. quilliermendii and C. utilis and up to 200 mg/l for S. cerevisiae, S. pombe, K. marxianus and C. membranaefaciens while the adsorption yield of dye showed the opposite trend for all the yeasts. Among the nine yeast species, C. lipolytica exhibited the highest dye uptake capacity (Q(0) = 250 mg/g). Both the Freundlich and Langmuir adsorption models were found suitable for describing the biosorption of the dye by all the Candida yeasts (except C. membranaefaciens). The results indicated that the dye uptake process followed the pseudo-second-order kinetics for each dye-yeast system.

Adsorption↗
PubMed2001

A simplified procedure to analyse mitochondrial DNA from industrial yeasts.

A rapid method based on mtDNA restriction analysis is described for yeast strain identification. The method is an adaptation of that devised by Querol et a]. [Syst. Appl. Microbiol. 15 (1992) 439] for Saccharomyces cerevisiae wine strains, and consists of the standard miniprep isolation of yeast total DNA, and the use of restriction endonucleases that recognise a large number of sites in yeast nuclear DNA, but few sites in the mitochondrial DNA. In the adapted method, the propagation of yeast cells and restriction analysis were the steps mainly affected: cell growth was reduced to 36 h by using microfuge tubes, and the restriction analysis was carried out in just 33 min using a microwave oven for DNA digestion, and minigels for restriction fragment separation. The DNA extraction procedure was performed in the same way as in the original protocol. but slightly reducing the duration of each step and scaling down the volumes of the different solutions. enzymes and reagents used. As result, a large time reduction (52.5 h) was obtained compared to the original method. The DNA obtained can be directly digested with endonucleases displaying clear restriction patterns useful for S. cerevisiae yeast strain differentiation. In addition, strains belonging to other foodborne yeast species, including spoilage yeast species, can also be identified.

DNA Restriction Enzymes↗
PubMed2003

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans.

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.

Adult↗
PubMed2005

The beetle gut: a hyperdiverse source of novel yeasts.

We isolated over 650 yeasts over a three year period from the gut of a variety of beetles and characterized them on the basis of LSU rDNA sequences and morphological and metabolic traits. Of these, at least 200 were undescribed taxa, a number equivalent to almost 30% of all currently recognized yeast species. A Bayesian analysis of species discovery rates predicts further sampling of previously sampled habitats could easily produce another 100 species. The sampled habitat is, thereby, estimated to contain well over half as many more species as are currently known worldwide. The beetle gut yeasts occur in 45 independent lineages scattered across the yeast phylogenetic tree, often in clusters. The distribution suggests that the some of the yeasts diversified by a process of horizontal transmission in the habitats and subsequent specialization in association with insect hosts. Evidence of specialization comes from consistent associations over time and broad geographical ranges of certain yeast and beetle species. The discovery of high yeast diversity in a previously unexplored habitat is a first step toward investigating the basis of the interactions and their impact in relation to ecology and evolution.

Animals↗
PubMed1999

Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

Actins↗
PubMed2000

Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention.

A recent human intervention trial showed that daily supplementation with selenized yeast (Se-yeast) led to a decrease in the overall cancer morbidity and mortality by nearly 50%; past research has also demonstrated that selenized garlic (Se-garlic) is very effective in mammary cancer chemoprevention in the rat model. The goal of this study was to compare certain biological activities of Se-garlic and Se-yeast and to elucidate the differences based on the chemical forms of selenium found in these two natural products. Characterization of organic selenium compounds in yeast (1922 microg/g Se) and garlic (296 microg/g Se) was carried out by high-performance liquid chromatography with inductively coupled plasma mass spectrometry or with electrospray mass spectrometry. Analytical speciation studies showed that the bulk of the selenium in Se-garlic and Se-yeast is in the form of gamma-glutamyl-Se-methylselenocysteine (73%) and selenomethionine (85%), respectively. The above methodology has the sensitivity and capability to account for >90% of total selenium. In the rat feeding studies, supplementation of Se-garlic in the diet at different levels consistently caused a lower total tissue selenium accumulation when compared to Se-yeast. On the other hand, Se-garlic was significantly more effective in suppressing the development of premalignant lesions and the formation of adenocarcinomas in the mammary gland of carcinogen-treated rats. Given the present finding on the identity of selenomethionine and gamma-glutamyl-Se-methylselenocysteine as the major form of selenium in Se-yeast and Se-garlic, respectively, the metabolism of these two compounds is discussed in an attempt to elucidate how their disposition in tissues might account for the differences in cancer chemopreventive activity.

Adenocarcinoma↗
PubMed2000

Release of lipids during yeast autolysis in a model wine system.

The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics. Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months. In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored. Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed. Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture. Detection was carried out with a light-scattering detector. The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days. A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability. A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall. Phospholipids were not detected in any of the autolysates. The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.

Autolysis↗
PubMed2003

The costs and benefits of killer toxin production by the yeast Pichia kluyveri.

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.

Animals↗
PubMed2001

Genetic and physiological variants of yeast selected from palm wine.

Genetic screening of 1200-palm wine yeasts lead to the selection of fourteen isolates with various genetic and physiological properties. Nine of the isolates were identified as Saccharamyces species, three as Candida species, one as Schizosaccharomyces species and one as Kluyveromyces species. Five of the isolates were wild type parents, two were respiratory deficient mutants (rho) and nine were auxotrophic mutants. Four isolates were heterozygous diploid (alphaa) and two were homozygous diploid (aa/alphaalpha) for the mating a mating types were further identified on mating with type loci. Four Mat alpha and four Mat a types were further identified on mating with standard haploid yeast strains. Forty-five percent sporulated on starvation medium producing tetrads. Fifty-two percent of the four-spored asci contained four viable spores. Maximum specific growth rate [micromax] of the fourteen isolates range from 0.13-0.26, five isolates were able to utilize exogenous nitrate for growth. Percentage alcohol production range between 5.8-8.8% for palm wine yeast, 8.5% for bakers' yeast and 10.4% for brewers yeast. The palm wine yeast were more tolerant to exogenous alcohol but had a low alcohol productivity. Hybridization enhanced alcohol productivity and tolerance in the palm wine yeasts.

Ethanol↗
PubMed2003

Assessment of yeast diversity in a marine environment in the south of Portugal by microsatellite-primed PCR.

The occurrence and diversity of yeasts in seawater was investigated in a study site located 20 Km off Faro, Portugal, above the Alvares Cabral Trench. A total of 43 water samples from different layers (above the permanent thermocline, under the thermocline and near the bottom) and directly from the surface, originated 234 isolates. All the isolates were identified using a molecular approach that included, in a first stage, MSP-PCR fingerprinting. A total of 31 MSP-PCR classes were formed, 8 for the pigmented yeasts and 23 for the non-pigmented yeasts. The pink coloured isolates were identified by direct comparison of the new fingerprints with those obtained for representative strains of the various species. For identification of the non-pigmented yeasts, a representative isolate of each MSP-PCR class was selected for sequence analysis and compared with reference sequences. The five most abundant yeast species were Sakaguchia dacryoidea, Pseudozyma aphidis, Rhodosporidium babjevae, R. diobovatum and Debaryomyces hansenii. The distribution of isolates and species in the major taxonomic groups indicated that the number of basidiomycetous yeasts and their diversity are prevalent in relation to their ascomycetous counterpart. Diversity indices were determined and superficial water and water near the bottom had the highest diversity. The sampling effort effectiveness was estimated, and found to correspond to approximately 60% of the species present. MSP-PCR identification proved suitable for pigmented basidiomycetous yeasts and, when used in conjunction with sequence analysis, was effective for the characterization of non-pigmented populations. Our results indicate that the MSP-PCR fingerprinting method is appropriate for the characterization of large groups of isolates due to its simplicity and good reproducibility.

Biodiversity↗
PubMed2002

Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis.

AIMS: To determine: (i) the relative prevalence and diversity of yeasts in salivary and root canal samples from the same patients; and (ii) the clinical factors associated with their presence in saliva and root canals. METHODOLOGY: Sixty root canal samples from teeth associated apical periodontitis and the corresponding whole unstimulated saliva samples were obtained from 55 patients. The medical history including antibiotic therapy and clinical/radiographic data on the teeth were recorded. The samples were serially diluted and cultured on yeast & fungi-selective sabouraud dextrose agar. Isolates were characterized and speciated by the germ tube formation test, hyphal morphology and a commercial biochemical test kit (Rapid ID32C(R) system). RESULTS: Twenty-three yeast isolates were recovered from 19 saliva samples and eight isolates from six root canal samples. Candida albicans (17/23 & 3/8) and Rodotorula mucilaginosa (2/23 & 4/8) were the most prevalent isolates from saliva and root canal samples. It was significantly (13.8 times) more probable that yeasts would be recovered from root canals when they were also present in the saliva (P = 0.021). The effect of coronal restoration leakage (P = 0.08) and previous root canal treatment (P = 0.123) were equivocal. The history of antibiotic therapy had no association with the presence of yeasts in saliva (OR = 1.1). CONCLUSIONS: Yeasts occurred relatively infrequently (10%) in root canals. Their presence in root canals was significantly associated with their presence in saliva. The role of yeasts in the initiation and perpetuation of periapical disease remains to be determined.

Anti-Bacterial Agents↗
PubMed2000

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Acyl Coenzyme A↗
PubMed2005

Effects of yeast stress and pH on 3-monochloropropanediol (3-MCPD)-producing reactions in model dough systems.

A major precursor of 3-monochloropropanediol (3-MCPD) in leavened cereal products is glycerol, which is formed as a natural by-product of yeast fermentation. However, yeast metabolism is affected by stresses such as low osmotic pressure from, for example, the incorporation of sugar or salt in the dough recipe. Tests with model doughs have shown that glycerol production was proportional to yeast mass and limited by available sugars, but that high levels of yeast inhibited 3-MCPD formation. The yeast fraction responsible for the inhibition of 3-MCPD in model dough was shown to be the soluble cytosol proteins, and the inhibition mechanism could be explained by the known reactions of 3-MCPD and/or its precursors with ammonia/amino acids (from yeast proteins). Added glucose did not increase the production of glycerol by yeast but it did promote the generation of 3-MCPD in cooked doughs. The latter effect was attributed to the removal of 3-MCPD inhibitors such as ammonia and amino acids by their reactions with added glucose (e.g. Maillard). The thermal generation of organic acids from added glucose also reduced the pH of cooked doughs, so the effect of pH and short-chain organic acids on 3-MCPD generation in dough was measured. There was a good correlation between initial dough pH and the level of 3-MCPD generated. The effect was weaker than that predicted by simple kinetic modelling, suggesting that the involvement of H+ and/or the organic acid was catalytic. The results showed that modifications to dough recipes involving the addition of reducing sugars and/or organic acids can have a significant impact on 3-MPCD generation in bakery products.

Amino Acids↗
PubMed2000

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import.

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

Active Transport, Cell Nucleus↗
PubMed1992

The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.

The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA. However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear. To address this question, we have expressed the SNP1 gene as a fusion protein in E. coli and developed a gel shift assay for U1 RNA binding. We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure. We therefore conclude that the SNP1 gene product is the yeast 70K homolog. This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein.

Escherichia coli↗