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Sample records for active yeast cells

  1. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  2. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    PubMed Central

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    Summary We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence activated cell sorting. PMID:26060080

  3. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

  4. [Detection of viable metabolically active yeast cells using a colorimetric assay].

    PubMed

    Růzicka, F; Holá, V

    2008-02-01

    The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

  5. Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells.

    PubMed

    Cruz, Aline H da Silva; Brock, Matthias; Zambuzzi-Carvalho, Patrícia F; Santos-Silva, Ludier K; Troian, Rogério F; Góes, Alfredo M; Soares, Célia M de Almeida; Pereira, Maristela

    2011-07-01

    The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

  6. Synchronous activation of cell division by light or temperature stimuli in the dimorphic yeast Schizosaccharomyces japonicus.

    PubMed

    Okamoto, Sho; Furuya, Kanji; Nozaki, Shingo; Aoki, Keita; Niki, Hironori

    2013-09-01

    Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.

  7. Detecting estrogenic activity in water samples withestrogen-sensitive yeast cells using spectrophotometry and fluorescencemicroscopy

    SciTech Connect

    Wozei, E.; Holman, H-Y.N.; Hermanowicz, S.W.; Borglin S.

    2006-03-15

    Environmental estrogens are environmental contaminants that can mimic the biological activities of the female hormone estrogen in the endocrine system, i.e. they act as endocrine disrupters. Several substances are reported to have estrogen-like activity or estrogenic activity. These include steroid hormones, synthetic estrogens (xenoestrogens), environmental pollutants and phytoestrogens (plant estrogens). Using the chromogenic substrate ortho-nitrophenyl-{beta}-D-galactopyranoside (ONPG) we show that an estrogen-sensitive yeast strain RMY/ER-ERE, with human estrogen receptor (hER{alpha}) gene and the lacZ gene which encodes the enzyme {beta}-galactosidase, is able to detect estrogenic activity in water samples over a wide range of spiked concentrations of the hormonal estrogen 17{beta}-estradiol (E2). Ortho-nitrophenol (ONP), the yellow product of this assay can be detected using spectrophotometry but requires cell lysis to release the enzyme and allow product formation. We improved this aspect in a fluorogenic assay by using fluorescein di-{beta}-D-galactopyranoside (FDG) as a substrate. The product was visualized using fluorescence microscopy without the need to kill, fix or lyse the cells. We show that in live yeast cells, the uptake of E2 and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximum enzyme-catalyzed fluorescent product formation evident after about 30 minutes of exposure to E2. The fluorogenic assay was applied to a selection of estrogenic compounds and the Synchrotron-based Fourier transform infrared (SR-FTIR) spectra of the cells obtained to better understand the yeast whole cell response to the compounds. The fluorogenic assay is most sensitive to E2, but the SR-FTIR spectra suggest that the cells respond to all the estrogenic compounds tested even when no fluorescent response was detected. These findings are promising and may shorten the duration of environmental water screening and monitoring regimes using

  8. Protein aggregation activates erratic stress response in dietary restricted yeast cells

    PubMed Central

    Bhadra, Ankan Kumar; Das, Eshita; Roy, Ipsita

    2016-01-01

    Chronic stress and prolonged activation of defence pathways have deleterious consequences for the cell. Dietary restriction is believed to be beneficial as it induces the cellular stress response machinery. We report here that although the phenomenon is beneficial in a wild-type cell, dietary restriction leads to an inconsistent response in a cell that is already under proteotoxicity-induced stress. Using a yeast model of Huntington’s disease, we show that contrary to expectation, aggregation of mutant huntingtin is exacerbated and activation of the unfolded protein response pathway is dampened under dietary restriction. Global proteomic analysis shows that when exposed to a single stress, either protein aggregation or dietary restriction, the expression of foldases like peptidyl-prolyl isomerase, is strongly upregulated. However, under combinatorial stress, this lead is lost, which results in enhanced protein aggregation and reduced cell survival. Successful designing of aggregation-targeted therapeutics will need to take additional stressors into account. PMID:27633120

  9. Ethanol Effects Involve Non-canonical Unfolded Protein Response Activation in Yeast Cells

    PubMed Central

    Navarro-Tapia, Elisabet; Pérez-Torrado, Roberto; Querol, Amparo

    2017-01-01

    The unfolded protein response (UPR) is a conserved intracellular signaling pathway that controls transcription of endoplasmic reticulum (ER) homeostasis related genes. Ethanol stress has been recently described as an activator of the UPR response in yeast Saccharomyces cerevisiae, but very little is known about the causes of this activation. Although some authors ensure that the UPR is triggered by the unfolded proteins generated by ethanol in the cell, there are studies which demonstrate that protein denaturation occurs at higher ethanol concentrations than those used to trigger the UPR. Here, we studied UPR after ethanol stress by three different approaches and we concluded that unfolded proteins do not accumulate in the ER under. We also ruled out inositol depletion as an alternative mechanism to activate the UPR under ethanol stress discarding that ethanol effects on the cell decreased inositol levels by different methods. All these data suggest that ethanol, at relatively low concentrations, does not cause unfolded proteins in the yeasts and UPR activation is likely due to other unknown mechanism related with a restructuring of ER membrane due to the effect of ethanol. PMID:28326077

  10. Optimization of permeabilization process of yeast cells for catalase activity using response surface methodology

    PubMed Central

    Trawczyńska, Ilona; Wójcik, Marek

    2015-01-01

    Biotransformation processes accompanied by whole yeast cells as biocatalyst are a promising area of food industry. Among the chemical sanitizers currently used in food technology, hydrogen peroxide is a very effective microbicidal and bleaching agent. In this paper, permeabilization has been applied to Saccharomyces cerevisiae yeast cells aiming at increased intracellular catalase activity for decomposed H2O2. Ethanol, which is non-toxic, biodegradable and easily available, has been used as permeabilization factor. Response surface methodology (RSM) has been applied in determining the influence of different parameters on permeabilization process. The aim of the study was to find such values of the process parameters that would yield maximum activity of catalase during decomposition of hydrogen peroxide. The optimum operating conditions for permeabilization process obtained by RSM were as follows: 53% (v/v) of ethanol concentration, temperature of 14.8 °C and treatment time of 40 min. After permeabilization, the activity of catalase increased ca. 40 times and its maximum value equalled to 4711 U/g. PMID:26019618

  11. Detection of hormone active chemicals using genetically engineered yeast cells and microfluidic devices with interdigitated array electrodes.

    PubMed

    Ino, Kosuke; Kitagawa, Yusuke; Watanabe, Tsuyoshi; Shiku, Hitoshi; Koide, Masahiro; Itayama, Tomoaki; Yasukawa, Tomoyuki; Matsue, Tomokazu

    2009-10-01

    Endocrine disruptors that act like hormones in the endocrine system might have toxic effects. Therefore, it is important to develop a portable device that can detect hormone active chemicals in samples rapidly and easily. In this study, a microfluidic device was developed for the detection of hormone active chemicals using genetically engineered yeast cells. The yeast cells were used as biosensors since they were genetically engineered to respond to the presence of hormone active chemicals by synthesizing beta-galactosidase (beta-gal). For achieving further sensitivity, we incorporated interdigitated array (IDA) electrodes (width, 1.2 microm; gap, 0.8 microm) with 40 electrode fingers into the analytical chamber of the microfluidic device. The yeast cells precultured with a hormone active chemical, 17beta-estradiol (E2), were trapped from the main channel of the device to the analytical camber by electrophoresis. After trapping in the analytical chamber, we performed electrochemical detection of beta-gal induced in the yeast cells with the IDA electrodes. Actually, electrochemical detection was performed on p-aminophenol that was converted from p-aminophenyl-beta-D-galactopyranoside with beta-gal. The electrochemical signals from the yeast cells precultured with 17beta-estradiol were successfully detected with the device. Furthermore, the inhibitory effects of antagonists such as tamoxifen were also detected electrochemically by using the device. Thus, the present microfluidic device can be used for highly sensitive detection of hormone active chemicals.

  12. Expanding the yeast prion world: Active prion conversion of non-glutamine/asparagine-rich Mod5 for cell survival.

    PubMed

    Suzuki, Genjiro; Tanaka, Motomasa

    2013-01-01

    Mammalian and fungal prion proteins form self-perpetuating β-sheet-rich fibrillar aggregates called amyloid. Prion inheritance is based on propagation of the regularly oriented amyloid structures of the prion proteins. All yeast prion proteins identified thus far contain aggregation-prone glutamine/asparagine (Gln/Asn)-rich domains, although the mammalian prion protein and fungal prion protein HET-s do not contain such sequences. In order to fill this gap, we searched for novel yeast prion proteins lacking Gln/Asn-rich domains via a genome-wide screen based on cross-seeding between two heterologous proteins and identified Mod5, a yeast tRNA isopentenyltransferase, as a novel non-Gln/Asn-rich yeast prion protein. Mod5 formed self-propagating amyloid fibers in vitro and the introduction of Mod5 amyloids into non-prion yeast induced dominantly and cytoplasmically heritable prion state [MOD (+) ], which harbors aggregates of endogenous Mod5. [MOD (+) ] yeast showed an increased level of membrane lipid ergosterol and acquired resistance to antifungal agents. Importantly, enhanced de novo formation of [MOD (+) ] was observed when non-prion yeast was grown under selective pressures from antifungal drugs. Our findings expand the family of yeast prions to non-Gln/Asn-rich proteins and reveal the acquisition of a fitness advantage for cell survival through active prion conversion.

  13. Activation of the cell integrity pathway is channelled through diverse signalling elements in fission yeast.

    PubMed

    Barba, Gregorio; Soto, Teresa; Madrid, Marisa; Núñez, Andrés; Vicente, Jeronima; Gacto, Mariano; Cansado, José

    2008-04-01

    MAPK Pmk1p is the central element of a cascade involved in the maintenance of cell integrity and other functions in Schizosaccharomyces pombe. Pmk1p becomes activated by multiple stressing situations and also during cell separation. GTPase Rho2p acts upstream of the protein kinase C homolog Pck2p to activate the Pmk1 signalling pathway through direct interaction with MAPKKK Mkh1p. In this work we analyzed the functional significance of both Rho2p and Pck2p in the transduction of various stress signals by the cell integrity pathway. The results indicate that basal Pmk1p activity can be positively regulated by alternative mechanisms which are independent on the control by Rho2p and/or Pck2p. Unexpectedly, Pck1p, another protein kinase C homolog, negatively modulates Pmk1p basal activity by an unknown mechanism. Moreover, different elements appear to regulate the stress-induced activation of Pmk1p depending on the nature of the triggering stimuli. Whereas Pmk1p activation induced by hyper- or hypotonic stresses is channeled through Rho2p-Pck2p, other stressors, like glucose deprivation or cell wall disturbance, are transduced via other pathways in addition to that of Rho2p-Pck2p. On the contrary, Pmk1p activation observed during cell separation or after treatment with hydrogen peroxide does not involve Rho2p-Pck2p. Finally, Pck2p function is critical to maintain a Pmk1p basal activity that allows Pmk1p activation induced by heat stress. These data demonstrate the existence of a complex signalling network modulating Pmk1p activation in response to a variety of stresses in fission yeast.

  14. In vivo yeast cell morphogenesis is regulated by a p21-activated kinase in the human pathogen Penicillium marneffei.

    PubMed

    Boyce, Kylie J; Schreider, Lena; Andrianopoulos, Alex

    2009-11-01

    Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37 degrees C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37 degrees C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37 degrees C. Loss of pakB also leads to the inappropriate production of yeast cells at 25 degrees C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.

  15. Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.

    PubMed

    Epstein, Sharon; Kirkpatrick, Clare L; Castillon, Guillaume A; Muñiz, Manuel; Riezman, Isabelle; David, Fabrice P A; Wollheim, Claes B; Riezman, Howard

    2012-03-01

    Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

  16. Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

    PubMed

    González, Iveth J; Desponds, Chantal; Schaff, Cédric; Mottram, Jeremy C; Fasel, Nicolas

    2007-02-01

    The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

  17. The SLT2(MPK1) MAP kinase is activated during periods of polarized cell growth in yeast.

    PubMed Central

    Zarzov, P; Mazzoni, C; Mann, C

    1996-01-01

    The SLT2(MPK1) mitogen-activated protein kinase signal transduction pa thway has been implicated in several biological processes in Saccharomyces cerevisiae, including the regulation of cytoskeletal and cell wall structure, polarized cell growth, and response to nutrient availability, hypo-osmotic shock and heat shock. We examined the conditions under which the SLT2 pathway is activated. We found that the SLT2 kinase is tyrosine phosphorylated and activated during periods in which yeast cells are undergoing polarized cell growth, namely during bud formation of vegetative cell division and during projection formation upon treatment with mating pheromone. BCK1(SLK1), a MEK kinase, is required for SLT2 activation in both of these situations. Upstream of BCK1(SLK1), we found that the STE20 kinase was required for SLT2 activation by mating pheromone, but was unnecessary for its activation during the vegetative cell cycle. Finally, SLT2 activation during vegetative growth was partially dependent on CDC28 in that the stimulation of SLT2 tyrosine phosphorylation was significantly reduced directly after a temperature shift in cdc28 ts mutants. Our data are consistent with a role for SLT2 in promoting polarized cell growth. Images PMID:8598209

  18. Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

    PubMed Central

    Ortega, Francisco G; Fernández-Baldo, Martín A; Fernández, Jorge G; Serrano, María J; Sanz, María I; Diaz-Mochón, Juan J; Lorente, José A; Raba, Julio

    2015-01-01

    In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors. PMID:25844035

  19. Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast.

    PubMed

    Ortega, Francisco G; Fernández-Baldo, Martín A; Fernández, Jorge G; Serrano, María J; Sanz, María I; Diaz-Mochón, Juan J; Lorente, José A; Raba, Julio

    2015-01-01

    In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

  20. Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis.

    PubMed

    Čáp, Michal; Váchová, Libuše; Palková, Zdena

    2015-01-01

    Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.

  1. The antioxidant activities of six (1→3)-β-d-glucan derivatives prepared from yeast cell wall.

    PubMed

    Tang, Qilin; Huang, Gangliang; Zhao, Fengying; Zhou, Lu; Huang, Shiqi; Li, Hui

    2017-02-02

    The alkali-insoluble (1→3)-β-d-glucan (PJ) was extracted from yeast cell wall by acid-base method. The sulfated glucan (S-PJ), carboxymethylated glucan (CM-PJ), phosphorylated glucan (P-PJ), carboxymethylated-phosphorylated glucan (CP-PJ), carboxymethylated-sulfated glucan (CS-PJ), and sulfated-phosphorylated glucan (SP-PJ) were prepared and characterized, respectively. The reduction ability, hydroxyl radical/superoxide anion scavenging activities, and anti-lipid peroxidation effect of above-mentioned derivatives were assayed. It indicated that S-PJ had the significant reduction capacity, P-PJ had the obvious hydroxyl radical/superoxide anion scavenging activities and anti-lipid peroxidation effect.

  2. Regulation of Cdc28 Cyclin-Dependent Protein Kinase Activity during the Cell Cycle of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Mendenhall, Michael D.; Hodge, Amy E.

    1998-01-01

    The cyclin-dependent protein kinase (CDK) encoded by CDC28 is the master regulator of cell division in the budding yeast Saccharomyces cerevisiae. By mechanisms that, for the most part, remain to be delineated, Cdc28 activity controls the timing of mitotic commitment, bud initiation, DNA replication, spindle formation, and chromosome separation. Environmental stimuli and progress through the cell cycle are monitored through checkpoint mechanisms that influence Cdc28 activity at key cell cycle stages. A vast body of information concerning how Cdc28 activity is timed and coordinated with various mitotic events has accrued. This article reviews that literature. Following an introduction to the properties of CDKs common to many eukaryotic species, the key influences on Cdc28 activity—cyclin-CKI binding and phosphorylation-dephosphorylation events—are examined. The processes controlling the abundance and activity of key Cdc28 regulators, especially transcriptional and proteolytic mechanisms, are then discussed in detail. Finally, the mechanisms by which environmental stimuli influence Cdc28 activity are summarized. PMID:9841670

  3. Purification of soluble beta-glucan with immune-enhancing activity from the cell wall of yeast.

    PubMed

    Lee, J N; Lee, D Y; Ji, I H; Kim, G E; Kim, H N; Sohn, J; Kim, S; Kim, C W

    2001-04-01

    Beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions, especially by activating macrophages. However, a major obstacle to the clinical application of beta-(1-->3)-glucan is its low solubility in aqueous media. In this study, soluble beta-glucan, free of mannoprotein, was prepared, and its effects on TNF-alpha secretion and phagocytosis by macrophages were evaluated. Beta-glucan was first rendered soluble from the yeast cell wall by alkaline extraction (glucan-p1). The extract contained 2.8% of protein which was subsequently removed by successive DEAE-cellulose and ConA chromatography. Beta-glucan thus prepared was completely free of mannoprotein and was soluble at neutral pH (glucan-p3). The effects of beta-glucan on phagocytosis and TNF-alpha release activity were investigated. While glucan-p1 moderately induced TNF-alpha secretion at 200 microg/ml (550 pg of TNF-alpha/5 x 10(5) cells), glucan-p3 markedly stimulated macrophages at 200 microg/ml (2,860 pg of TNF-alpha/5 x 10(5) cells). Furthermore, glucan-p3 stimulated phagocytosis about 20% more than glucan-p1 did. In conclusion, we purified water-soluble beta-glucan which was completely devoid of mannoprotein and effectively stimulated the macrophage function, enabling it to be used as an intravenous injection for sepsis.

  4. High-throughput screening of improved protease inhibitors using a yeast cell surface display system and a yeast cell chip.

    PubMed

    Aoki, Wataru; Yoshino, Yuichi; Morisaka, Hironobu; Tsunetomo, Keiji; Koyo, Hirotaka; Kamiya, Shinji; Kawata, Noriyuki; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2011-01-01

    Protease-targeted inhibitors have been promising pharmaceuticals. Here, we combined a yeast cell surface display system with a yeast cell chip for the high-throughput screening of protease inhibitors, and succeeded in improving the activity of a protease inhibitor.

  5. Rho1 GTPase and PKC ortholog Pck1 are upstream activators of the cell integrity MAPK pathway in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.

  6. Blad-Containing Oligomer Fungicidal Activity on Human Pathogenic Yeasts. From the Outside to the Inside of the Target Cell

    PubMed Central

    Pinheiro, Ana M.; Carreira, Alexandra; Rollo, Filipe; Fernandes, Rui; Ferreira, Ricardo B.; Monteiro, Sara A.

    2016-01-01

    Blad polypeptide comprises residues 109–281 of Lupinus albus β-conglutin precursor. It occurs naturally as a major subunit of an edible, 210 kDa oligomer which accumulates to high levels, exclusively in the cotyledons of Lupinus seedlings between the 4th and 14th day after the onset of germination. Blad-containing oligomer (BCO) exhibits a potent and broad spectrum fungicide activity toward plant pathogens and is now on sale in the US under the tradename FractureTM. In this work we demonstrate its antifungal activity toward human pathogens and provide some insights on its mode of action. BCO bioactivity was evaluated in eight yeast species and compared to that of amphotericin B (AMB). BCO behaved similarly to AMB in what concerns both cellular inhibition and cellular death. As a lectin, BCO binds strongly to chitin. In addition, BCO is known to possess ‘exochitinase’ and ‘endochitosanase’ activities. However, no clear disruption was visualized at the cell wall after exposure to a lethal BCO concentration, except in cell buds. Immunofluorescent and immunogold labeling clearly indicate that BCO enters the cell, and membrane destabilization was also demonstrated. The absence of haemolytic activity, its biological origin, and its extraordinary antifungal activity are the major outcomes of this work, and provide a solid background for a future application as a new antifungal therapeutic drug. Furthermore, its predictable multisite mode of action suggests a low risk of inducing resistance mechanisms, which are now a major problem with other currently available antifungal drugs. PMID:27933037

  7. Rga4 modulates the activity of the fission yeast cell integrity MAPK pathway by acting as a Rho2 GTPase-activating protein.

    PubMed

    Soto, Teresa; Villar-Tajadura, Maria Antonia; Madrid, Marisa; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2010-04-09

    Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in a specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, down-regulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

  8. A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

    PubMed Central

    1996-01-01

    A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants. PMID:8978817

  9. Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

    PubMed

    Palková, Zdena; Váchová, Libuše

    2016-09-01

    Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned.

  10. Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

    The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.

  11. Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

    PubMed

    Ramanavicius, A; Morkvenaite-Vilkonciene, I; Kisieliute, A; Petroniene, J; Ramanaviciene, A

    2017-01-01

    In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals.

  12. Yeast growth in raffinose results in resistance to acetic-acid induced programmed cell death mostly due to the activation of the mitochondrial retrograde pathway.

    PubMed

    Guaragnella, Nicoletta; Zdralević, Maša; Lattanzio, Paolo; Marzulli, Domenico; Pracheil, Tammy; Liu, Zhengchang; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2013-12-01

    In order to investigate whether and how a modification of mitochondrial metabolism can affect yeast sensitivity to programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the up-regulation of the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathway target genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4, encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential was measured, capable to consume oxygen in a manner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.

  13. Recovery from rapamycin: drug-insensitive activity of yeast target of rapamycin complex 1 (TORC1) supports residual proliferation that dilutes rapamycin among progeny cells.

    PubMed

    Evans, Stephanie K; Burgess, Karl E V; Gray, Joseph V

    2014-09-19

    The target of rapamycin complex 1 (TORC1) is a key conserved regulator of eukaryotic cell growth. The xenobiotic rapamycin is a potent inhibitor of the yeast complex. Surprisingly, the EGO complex, a nonessential in vivo activator of TORC1, is somehow required for yeast cells to recover efficiently from a period of treatment with rapamycin. Why? Here, we found that rapamycin is only a partial inhibitor of TORC1. We confirmed that saturating amounts of rapamycin do not fully inhibit proliferation of wild-type cells, and we found that the residual proliferation in the presence of the drug is dependent on the EGO complex and on the activity of TORC1. We found that this residual TORC1-dependent proliferation is key to recovery from rapamycin treatment. First, the residual proliferation rate correlates with the ability of cells to recover from treatment. Second, the residual proliferation rate persists long after washout of the drug and until cells recover. Third, the total observable pool of cell-associated rapamycin is extremely stable and decreases only with increasing cell number after washout of the drug. Finally, consideration of the residual proliferation rate alone accurately and quantitatively accounts for the kinetics of recovery of wild-type cells and for the nature and severity of the ego- mutant defect. Overall, our results revealed that rapamycin is a partial inhibitor of yeast TORC1, that persistence of the drug limits recovery, and that rapamycin is not detoxified by yeast but is passively diluted among progeny cells because of residual proliferation.

  14. Yeast cell factories for fine chemical and API production

    PubMed Central

    Pscheidt, Beate; Glieder, Anton

    2008-01-01

    This review gives an overview of different yeast strains and enzyme classes involved in yeast whole-cell biotransformations. A focus was put on the synthesis of compounds for fine chemical and API (= active pharmaceutical ingredient) production employing single or only few-step enzymatic reactions. Accounting for recent success stories in metabolic engineering, the construction and use of synthetic pathways was also highlighted. Examples from academia and industry and advances in the field of designed yeast strain construction demonstrate the broad significance of yeast whole-cell applications. In addition to Saccharomyces cerevisiae, alternative yeast whole-cell biocatalysts are discussed such as Candida sp., Cryptococcus sp., Geotrichum sp., Issatchenkia sp., Kloeckera sp., Kluyveromyces sp., Pichia sp. (including Hansenula polymorpha = P. angusta), Rhodotorula sp., Rhodosporidium sp., alternative Saccharomyces sp., Schizosaccharomyces pombe, Torulopsis sp., Trichosporon sp., Trigonopsis variabilis, Yarrowia lipolytica and Zygosaccharomyces rouxii. PMID:18684335

  15. Detection of estrogenic activity in Flemish surface waters using an in vitro recombinant assay with yeast cells.

    PubMed

    Witters, H E; Vangenechten, C; Berckmans, P

    2001-01-01

    Numerous environmental chemicals possess estrogen-like properties. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects on humans and wildlife. Sources of potential exposure to endocrine disrupting compounds have to be identified for risk and hazard assessment. Extracts prepared from 16 selected water samples taken in Flemish rivers, effluents of municipal wastewater treatment plants and reservoirs for drinking water production were analysed for estrogenic activity with a cellular bioassay. Yeast cells, which are stably transfected with the DNA sequence of hER and which contain expression plasmids with the reporter gene lac-Z, encoding the enzyme beta-galactosidase, were used to measure receptor binding. Flemish rivers showed the highest estrogenic potency, compared to effluents of waste water treatment plants and reservoirs which showed low induction factors (beta-galactosidase production) relative to solvent control conditions. By comparison with a standard curve for 17 beta-estradiol (E2), estrogenic potency in water samples was calculated as E2-equivalents and ranged from below detection limit (approximately 2.75 ng E2/l) up to 81.4 ng/l E2-equivalents. About 7 water samples had more than 10 ng/l E2-equivalents. These elevated levels of E2-equivalents are likely to exert significant adverse effects on reproduction success of wildlife, which should be verified with in vivo studies.

  16. Glucuronidation does not suppress the estrogenic activity of quercetin in yeast and human breast cancer cell model systems.

    PubMed

    Ruotolo, Roberta; Calani, Luca; Brighenti, Furio; Crozier, Alan; Ottonello, Simone; Del Rio, Daniele

    2014-10-01

    Several plant-derived molecules, referred to as phytoestrogens, are thought to mimic the actions of endogenous estrogens. Among these, quercetin, one of the most widespread flavonoids in the plant kingdom, has been reported as estrogenic in some occasions. However, quercetin occurs in substantial amounts as glycosides such as quercetin-3-O-glucoside (isoquercitrin) and quercetin-3-O-rutinoside (rutin) in dietary sources. It is now well established that quercetin undergoes substantial phase II metabolism after ingestion by humans, with plasma metabolites after a normal dietary intake rarely exceeding nmol/L concentrations. Therefore, attributing phytoestrogenic activity to flavonoids without taking into account the fact that it is their phase II metabolites that enter the circulatory system, will almost certainly lead to misleading conclusions. With the aim of clarifying the above issue, the goal of the present study was to determine if plant-associated quercetin glycosides and human phase II quercetin metabolites, actually found in human biological fluids after intake of quercetin containing foods, are capable of interacting with the estrogen receptors (ER). To this end, we used a yeast-based two-hybrid system and an estrogen response element-luciferase reporter assay in an ER-positive human cell line (MCF-7) to probe the ER interaction capacities of quercetin and its derivatives. Our results show that quercetin-3-O-glucuronide, one of the main human phase II metabolites produced after intake of dietary quercetin, displays ERα- and ERβ-dependent estrogenic activity, the functional consequences of which might be related to the protective activity of diets rich in quercetin glycosides.

  17. Training of yeast cell dynamics.

    PubMed

    Reijenga, Karin A; Bakker, Barbara M; van der Weijden, Coen C; Westerhoff, Hans V

    2005-04-01

    In both industrial fermenters and in their natural habitats, microorganisms often experience an inhomogeneous and fluctuating environment. In this paper we mimicked one aspect of this nonideal behaviour by imposing a low and oscillating extracellular glucose concentration on nonoscillating suspensions of yeast cells. The extracellular dynamics changed the intracellular dynamics--which was monitored through NADH fluorescence--from steady to equally dynamic; the latter followed the extracellular dynamics at the frequency of glucose pulsing. Interestingly, the amplitude of the oscillation of the NADH fluorescence increased with time. This increase in amplitude was sensitive to inhibition of protein synthesis, and was due to a change in the cells rather than in the medium; the cell population was 'trained' to respond to the extracellular dynamics. To examine the mechanism behind this 'training', we subjected the cells to a low and constant extracellular glucose concentration. Seventy-five minutes of adaptation to a low and constant glucose concentration induced the same increase of the amplitude of the forced NADH oscillations as did the train of glucose pulses. Furthermore, 75 min of adaptation to a low (oscillating or continuous) glucose concentration decreased the K(M) of the glucose transporter from 26 mm to 3.5 mm. When subsequently the apparent K(M) was increased by addition of maltose, the amplitude of the forced oscillations dropped to its original value. This demonstrated that the increased affinity of glucose transport was essential for the training of the cells' dynamics.

  18. An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeast Saccharomyces cerevisiae.

    PubMed

    Almeida, Bruno; Sampaio-Marques, Belém; Carvalho, Joana; Silva, Manuel T; Leão, Cecília; Rodrigues, Fernando; Ludovico, Paula

    2007-05-01

    Ciclopirox olamine (CPO), a fungicidal agent widely used in clinical practice, induced in Saccharomyces cerevisiae an active cell death (ACD) process characterized by changes in nuclear morphology and chromatin condensation associated with the appearance of a population in the sub-G(0)/G(1) cell cycle phase and an arrest delay in the G(2)/M phases. This ACD was associated neither with intracellular reactive oxygen species (ROS) signaling, as revealed by the use of different classes of ROS scavengers, nor with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive phenotype. Furthermore, CPO-induced cell death seems to be dependent on unknown protease activity but independent of the apoptotic regulators Aif1p and Yca1p and of autophagic pathways involving Apg5p, Apg8p and Uth1p. Our results show that CPO triggers in S. cerevisiae an atypical nonapoptotic, nonautophagic ACD with as yet unknown regulators.

  19. Stabilization and encapsulation of photosensitive resveratrol within yeast cell.

    PubMed

    Shi, Guorong; Rao, Liqun; Yu, Huazhong; Xiang, Hua; Yang, Hua; Ji, Runa

    2008-02-12

    The photosensitive resveratrol was successfully encapsulated in yeast cells for the first time, as characterized by FT-IR spectra, fluorescence and confocal micrographs of the yeast cells, resveratrol and microcapsules. The release characteristic of the obtained yeast-encapsulated resveratrol in simulated gastric fluid was evaluated, and its storage stability as a powder was investigated at 25 degrees C/75% relative humidity (RH), 25 degrees C/90% RH and 60 degrees C under the laboratory fluorescent lighting conditions (ca. 300 lx) or in the dark. Also, the scavenging capacity of yeast-encapsulated resveratrol on DPPH radical was compared with that of non-encapsulated resveratrol. It could be demonstrated clearly that no chemical changes occurred during the encapsulation. Besides, the DPPH radical-scavenging activity increased after the encapsulation. In addition, the yeast-encapsulated resveratrol exhibited good stability, and its bioavailability was enhanced as a result of increased solubility of resveratrol and sustained releasing.

  20. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    PubMed

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH.

  1. Chromatographic purification of highly active yeast ribosomes.

    PubMed

    Meskauskas, Arturas; Leshin, Jonathan A; Dinman, Jonathan D

    2011-10-24

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

  2. Biodiversity of brewery yeast strains and their fermentative activities.

    PubMed

    Berlowska, Joanna; Kregiel, Dorota; Rajkowska, Katarzyna

    2015-01-01

    We investigated the genetic, biochemical, fermentative and physiological characteristics of brewery yeast strains and performed a hierarchical cluster analysis to evaluate their similarity. We used five different ale and lager yeast strains, originating from different European breweries and deposited at the National Collection of Yeast Cultures (UK). Ale and lager strains exhibited different genomic properties, but their assimilation profiles and pyruvate decarboxylase activities corresponded to their species classifications. The activity of another enzyme, succinate dehydrogenase, varied between different brewing strains. Our results confirmed that ATP and glycogen content, and the activity of the key metabolic enzymes succinate dehydrogenase and pyruvate decarboxylase, may be good general indicators of cell viability. However, the genetic properties, physiology and fermentation capacity of different brewery yeasts are unique to individual strains.

  3. Immobilisation increases yeast cells' resistance to dehydration-rehydration treatment.

    PubMed

    Borovikova, Diana; Rozenfelde, Linda; Pavlovska, Ilona; Rapoport, Alexander

    2014-08-20

    This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains.

  4. Oxidative Stress and Programmed Cell Death in Yeast

    PubMed Central

    Farrugia, Gianluca; Balzan, Rena

    2012-01-01

    Yeasts, such as Saccharomyces cerevisiae, have long served as useful models for the study of oxidative stress, an event associated with cell death and severe human pathologies. This review will discuss oxidative stress in yeast, in terms of sources of reactive oxygen species (ROS), their molecular targets, and the metabolic responses elicited by cellular ROS accumulation. Responses of yeast to accumulated ROS include upregulation of antioxidants mediated by complex transcriptional changes, activation of pro-survival pathways such as mitophagy, and programmed cell death (PCD) which, apart from apoptosis, includes pathways such as autophagy and necrosis, a form of cell death long considered accidental and uncoordinated. The role of ROS in yeast aging will also be discussed. PMID:22737670

  5. Remodeling of the Fission Yeast Cdc42 Cell-Polarity Module via the Sty1 p38 Stress-Activated Protein Kinase Pathway.

    PubMed

    Mutavchiev, Delyan R; Leda, Marcin; Sawin, Kenneth E

    2016-11-07

    The Rho family GTPase Cdc42 is a key regulator of eukaryotic cellular organization and cell polarity [1]. In the fission yeast Schizosaccharomyces pombe, active Cdc42 and associated effectors and regulators (the "Cdc42 polarity module") coordinate polarized growth at cell tips by controlling the actin cytoskeleton and exocytosis [2-4]. Localization of the Cdc42 polarity module to cell tips is thus critical for its function. Here we show that the fission yeast stress-activated protein kinase Sty1, a homolog of mammalian p38 MAP kinase, regulates localization of the Cdc42 polarity module. In wild-type cells, treatment with latrunculin A, a drug that leads to actin depolymerization, induces dispersal of the Cdc42 module from cell tips and cessation of polarized growth [5, 6]. We show that latrunculin A treatment also activates the Sty1 MAP kinase pathway and, strikingly, we find that loss of Sty1 MAP kinase signaling prevents latrunculin A-induced dispersal of the Cdc42 module, allowing polarized growth even in complete absence of the actin cytoskeleton. Regulation of the Cdc42 module by Sty1 is independent of Sty1's role in stress-induced gene expression. We also describe a system for activation of Sty1 kinase "on demand" in the absence of any external stress, and use this to show that Sty1 activation alone is sufficient to disperse the Cdc42 module from cell tips in otherwise unperturbed cells. During nitrogen-starvation-induced quiescence, inhibition of Sty1 converts non-growing, depolarized cells into growing, polarized cells. Our results place MAP kinase Sty1 as an important physiological regulator of the Cdc42 polarity module.

  6. Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site

    PubMed Central

    Park, Hay-Oak; Sanson, Anthony; Herskowitz, Ira

    1999-01-01

    Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G1 cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G1 cells by recognizing cell type-specific landmarks at the presumptive bud site. PMID:10444589

  7. The transcription factors ADR1 or CAT8 are required for RTG pathway activation and evasion from yeast acetic acid-induced programmed cell death in raffinose

    PubMed Central

    Laera, Luna; Guaragnella, Nicoletta; Ždralević, Maša; Marzulli, Domenico; Liu, Zhengchang; Giannattasio, Sergio

    2016-01-01

    Yeast Saccharomyces cerevisiae grown on glucose undergoes programmed cell death (PCD) induced by acetic acid (AA-PCD), but evades PCD when grown in raffinose. This is due to concomitant relief of carbon catabolite repression (CCR) and activation of mitochondrial retrograde signaling, a mitochondria-to-nucleus communication pathway causing up-regulation of various nuclear target genes, such as CIT2, encoding peroxisomal citrate synthase, dependent on the positive regulator RTG2 in response to mitochondrial dysfunction. CCR down-regulates genes mainly involved in mitochondrial respiratory metabolism. In this work, we investigated the relationships between the RTG and CCR pathways in the modulation of AA-PCD sensitivity under glucose repression or de-repression conditions. Yeast single and double mutants lacking RTG2 and/or certain factors regulating carbon source utilization, including MIG1, HXK2, ADR1, CAT8, and HAP4, have been analyzed for their survival and CIT2 expression after acetic acid treatment. ADR1 and CAT8 were identified as positive regulators of RTG-dependent gene transcription. ADR1 and CAT8 interact with RTG2 and with each other in inducing cell resistance to AA-PCD in raffinose and controlling the nature of cell death. In the absence of ADR1 and CAT8, AA-PCD evasion is acquired through activation of an alternative factor/pathway repressed by RTG2, suggesting that RTG2 may play a function in promoting necrotic cell death in repressing conditions when RTG pathway is inactive. Moreover, our data show that simultaneous mitochondrial retrograde pathway activation and SNF1-dependent relief of CCR have a key role in central carbon metabolism reprogramming which modulates the yeast acetic acid-stress response. PMID:28357334

  8. Ceramide triggers metacaspase-independent mitochondrial cell death in yeast.

    PubMed

    Carmona-Gutierrez, Didac; Reisenbichler, Angela; Heimbucher, Petra; Bauer, Maria A; Braun, Ralf J; Ruckenstuhl, Christoph; Büttner, Sabrina; Eisenberg, Tobias; Rockenfeller, Patrick; Fröhlich, Kai-Uwe; Kroemer, Guido; Madeo, Frank

    2011-11-15

    The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.

  9. Yeast fuel cell: Application for desalination

    NASA Astrophysics Data System (ADS)

    Mardiana, Ummy; Innocent, Christophe; Cretin, Marc; Buchari, Buchari; Gandasasmita, Suryo

    2016-02-01

    Yeasts have been implicated in microbial fuel cells as biocatalysts because they are non-pathogenic organisms, easily handled and robust with a good tolerance in different environmental conditions. Here we investigated baker's yeast Saccharomyces cerevisiae through the oxidation of glucose. Yeast was used in the anolyte, to transfer electrons to the anode in the presence of methylene blue as mediator whereas K3Fe(CN)6 was used as an electron acceptor for the reduction reaction in the catholyte. Power production with biofuel cell was coupled with a desalination process. The maximum current density produced by the cell was 88 mA.m-2. In those conditions, it was found that concentration of salt was removed 64% from initial 0.6 M after 1-month operation. This result proves that yeast fuel cells can be used to remove salt through electrically driven membrane processes and demonstrated that could be applied for energy production and desalination. Further developments are in progress to improve power output to make yeast fuel cells applicable for water treatment.

  10. Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Stueland, C S; Lew, D J; Cismowski, M J; Reed, S I

    1993-01-01

    In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation. Images PMID:8388545

  11. X-ray irradiation of yeast cells

    NASA Astrophysics Data System (ADS)

    Masini, Alessandra; Batani, Dimitri; Previdi, Fabio; Conti, Aldo; Pisani, Francesca; Botto, Cesare; Bortolotto, Fulvia; Torsiello, Flavia; Turcu, I. C. Edmond; Allott, Ric M.; Lisi, Nicola; Milani, Marziale; Costato, Michele; Pozzi, Achille; Koenig, Michel

    1997-10-01

    Saccharomyces Cerevisiae yeast cells were irradiated using the soft X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterized by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency.

  12. Preservation of frozen yeast cells by trehalose.

    PubMed

    Diniz-Mendes, L; Bernardes, E; de Araujo, P S; Panek, A D; Paschoalin, V M

    1999-12-05

    Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.

  13. SLipid-induced cell dysfunction and cell death: lessons from yeast.

    PubMed

    Kohlwein, Sepp D; Petschnigg, Julia

    2007-12-01

    The 2001 Nobel Prize in Medicine, awarded to two yeast researchers for contributions to understanding the eukaryotic cell cycle, spotlighted yeast as an experimental model system in biomedical research. Major discoveries of molecular processes underlying lipid and biomembrane biogenesis were first made in yeast: secretory pathways, vesicle and membrane fusion, and the unfolded protein response. The discovery of programmed cell death that is conserved at multiple levels (quite intriguing for a unicellular organism), and energy metabolism controlled by adenosine monophosphate-activated protein kinases, mitogen-activated protein kinase signaling pathways, and the target of rapamycin (TOR) pathway (originally discovered in yeast)-all refer to functional and structural similarities with mammalian cells beyond the mere metabolic level. This article reviews recently uncovered aspects of fatty acid-associated malfunctions and lipotoxicity in yeast that may aid in understanding the molecular basis of lipid-associated disorders in mammals.

  14. The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle

    PubMed Central

    2010-01-01

    Background The G1-to-S transition of the cell cycle in the yeast Saccharomyces cerevisiae involves an extensive transcriptional program driven by transcription factors SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). Activation of these factors ultimately depends on the G1 cyclin Cln3. Results To determine the transcriptional targets of Cln3 and their dependence on SBF or MBF, we first have used DNA microarrays to interrogate gene expression upon Cln3 overexpression in synchronized cultures of strains lacking components of SBF and/or MBF. Secondly, we have integrated this expression dataset together with other heterogeneous data sources into a single probabilistic model based on Bayesian statistics. Our analysis has produced more than 200 transcription factor-target assignments, validated by ChIP assays and by functional enrichment. Our predictions show higher internal coherence and predictive power than previous classifications. Our results support a model whereby SBF and MBF may be differentially activated by Cln3. Conclusions Integration of heterogeneous genome-wide datasets is key to building accurate transcriptional networks. By such integration, we provide here a reliable transcriptional network at the G1-to-S transition in the budding yeast cell cycle. Our results suggest that to improve the reliability of predictions we need to feed our models with more informative experimental data. PMID:20573214

  15. Assembly of the Yeast Cell Wall

    PubMed Central

    Cabib, Enrico; Farkas, Vladimir; Kosík, Ondrej; Blanco, Noelia; Arroyo, Javier; McPhie, Peter

    2008-01-01

    The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to β(1–3)glucose branches of β(1–6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to β(1–6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established. PMID:18694928

  16. Anaerobic digestion of distillery spent wash: Influence of enzymatic pre-treatment of intact yeast cells.

    PubMed

    Mallick, P; Akunna, J C; Walker, G M

    2010-03-01

    The potential benefits of enzymatic digestion of intact yeast cells on anaerobic digestion of Scotch whisky distillery spent wash and pot ale were investigated. Various yeast cell wall hydrolytic enzymes were studied based on their effect on dissolution of cell wall glucan and mannoprotein. The synergistic activity of beta-glucanase and protease showed greater than 90% yeast cell digestion at 37 degrees C in 24h. The widely-used industrial enzyme papain showed 95% yeast cell digestion in spent wash at 1% enzyme concentration within 22h at 50 degrees C. Anaerobic digestion of pot ale residues containing intact yeast cells pre-treated with lytic enzymes showed COD reductions of 87%, compared with only 13% without enzymes. Similar results were observed with distillery spent wash centrate. The hydrolysis of intact yeast cells in distillery liquid residues was found to be a rate-limiting step in anaerobic treatment of such residues.

  17. Gamma radiation effects on Sporothrix schenckii yeast cells.

    PubMed

    Lacerda, Camila Maria de Souza; Martins, Estefânia Mara do Nascimento; de Resende, Maria Aparecida; de Andrade, Antero Silva Ribeiro

    2011-06-01

    Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

  18. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

    PubMed

    Mak, P; Cruz, F D; Chen, S

    1999-11-01

    Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast

  19. Vitality enhancement of the rehydrated active dry wine yeast.

    PubMed

    Rodríguez-Porrata, B; Novo, M; Guillamón, J; Rozès, N; Mas, A; Otero, R Cordero

    2008-08-15

    In winemaking, spontaneous grape must fermentations have been replaced by inoculation of commercial active dry wine yeast (ADWY). Yeast rehydration is the key to avoiding stuck and sluggish fermentations. Despite the importance of this step, not enough is known about what this process implies for winemaking as a whole or about what kind of practices could help to improve it. The main aim of this study is to determine the best yeast rehydration conditions for ensuring good cell viability and vitality before inoculation into the must. The experimental rehydration media in this study can be divided into four groups: carbon and nitrogen compounds, metallic ions, oxidant and antioxidant agents, and membrane fluidity agents. We studied the biochemical and biophysical behaviour of ADWY after rehydration in the various media under oenological conditions, i.e. incubation at 37 degrees C for 30 min. The viability of rehydrated yeast cells was evaluated by plating, and assessed by fluorescence microscopy and flow cytometry. The vitality of rehydrated cells was estimated by indirect impedance. The rehydrating solution complemented with magnesium provided the best vitality rate because the time taken to reach the activity threshold was cut by two thirds. This improvement was also illustrated by the less time needed to stop the leakage of intracellular compounds during the rehydration process.

  20. Generation, modulation and maintenance of the plasma membrane asymmetric phospholipid composition in yeast cells during growth: their relation to surface potential and membrane protein activity.

    PubMed

    Cerbón, J; Calderón, V

    1995-04-12

    During growth a cyclic exposure of anionic phospholipids to the external surface of the plasma membrane was found. The surface charge density (sigma) increased gradually reaching a maximum in the first 5 h of growth and returned gradually to their initial value at the end of the logarithmic phase of growth (10-12 h). Phosphatidylinositol, that determines to a large extent the magnitude of the sigma, increased 83% in the yeast cells during the first 4 h of growth and returned gradually to their initial level at 10-12 h. During the stationary phase (12-24 h), both sigma and the anionic/zwitterionic phospholipid ratio, remained without any significant variation. The high-affinity H-linked glutamate transport system that behaves as a sensor of the changes in the membrane surface potential (phi) increased its activity in the first 5 h and then decreased it, following with great accuracy the sigma variations and remained without changes during the stationary phase of growth. The phosphatidylserine (PS) relative concentration in the cells (9.0%) did not significantly change during the whole growth curve, but their asymmetric distribution varied, contributing to the changes in sigma. PS facing the outer membrane surface increased 2.45-times during the first 5 h of growth and then returned to their original value at the end of the log phase (12 h). Phosphatidylcholine (PC) remained constant during the whole growth curve (50%), while phosphatidylethanolamine (PE) decreased 3-fold in the first 4 h and then increased to its original value at 10 h. Interestingly, PE at the outer membrane surface remained constant (3% of the total phospholipids) during the whole growth curve. During growth yeast cells change their phospholipid composition originating altered patterns of the plasma membrane phospholipid composition and IN-OUT distribution. This dynamic asymmetry is involved in the regulation of the surface potential and membrane protein activity.

  1. Yeast and cancer cells - common principles in lipid metabolism.

    PubMed

    Natter, Klaus; Kohlwein, Sepp D

    2013-02-01

    One of the paradigms in cancer pathogenesis is the requirement of a cell to undergo transformation from respiration to aerobic glycolysis - the Warburg effect - to become malignant. The demands of a rapidly proliferating cell for carbon metabolites for the synthesis of biomass, energy and redox equivalents, are fundamentally different from the requirements of a differentiated, quiescent cell, but it remains open whether this metabolic switch is a cause or a consequence of malignant transformation. One of the major requirements is the synthesis of lipids for membrane formation to allow for cell proliferation, cell cycle progression and cytokinesis. Enzymes involved in lipid metabolism were indeed found to play a major role in cancer cell proliferation, and most of these enzymes are conserved in the yeast, Saccharomyces cerevisiae. Most notably, cancer cell physiology and metabolic fluxes are very similar to those in the fermenting and rapidly proliferating yeast. Both types of cells display highly active pathways for the synthesis of fatty acids and their incorporation into complex lipids, and imbalances in synthesis or turnover of lipids affect growth and viability of both yeast and cancer cells. Thus, understanding lipid metabolism in S. cerevisiae during cell cycle progression and cell proliferation may complement recent efforts to understand the importance and fundamental regulatory mechanisms of these pathways in cancer.

  2. Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

    PubMed Central

    Herman, P K; Rine, J

    1997-01-01

    Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression. PMID:9321396

  3. Neutron activation analysis for the demonstration of amphibolite rock-weathering activity of a yeast.

    PubMed

    Rades-Rohkohl, E; Hirsch, P; Fränzle, O

    1979-12-01

    Neutron activation analysis was employed in a survey of weathering abilities of rock surface microorganisms. A yeast isolated from an amphibolite of a megalithic grave was found actively to concentrate, in media and in or on cells, iron and other elements when grown in the presence of ground rock. This was demonstrated by comparing a spectrum of neutron-activated amphibolite powder (particle size, 50 to 100 mum) with the spectra of neutron-activated, lyophilized yeast cells which had grown with or without amphibolite powder added to different media. The most active yeast (IFAM 1171) did not only solubilize Fe from the rock powder, but significant amounts of Co, Eu, Yb, Ca, Ba, Sc, Lu, Cr, Th, and U were also mobilized. The latter two elements occurred as natural radioactive isotopes in this amphibolite. When the yeast cells were grown with neutron-activated amphibolite, the cells contained the same elements. Furthermore, the growth medium contained Fe, Co, and Eu which had been solubilized from the amphibolite. This indicates the presence, in this yeast strain, of active rockweathering abilities as well as of uptake mechanisms for solubilized rock components.

  4. Neutron Activation Analysis for the Demonstration of Amphibolite Rock-Weathering Activity of a Yeast

    PubMed Central

    Rades-Rohkohl, E.; Hirsch, P.; Fränzle, O.

    1979-01-01

    Neutron activation analysis was employed in a survey of weathering abilities of rock surface microorganisms. A yeast isolated from an amphibolite of a megalithic grave was found actively to concentrate, in media and in or on cells, iron and other elements when grown in the presence of ground rock. This was demonstrated by comparing a spectrum of neutron-activated amphibolite powder (particle size, 50 to 100 μm) with the spectra of neutron-activated, lyophilized yeast cells which had grown with or without amphibolite powder added to different media. The most active yeast (IFAM 1171) did not only solubilize Fe from the rock powder, but significant amounts of Co, Eu, Yb, Ca, Ba, Sc, Lu, Cr, Th, and U were also mobilized. The latter two elements occurred as natural radioactive isotopes in this amphibolite. When the yeast cells were grown with neutron-activated amphibolite, the cells contained the same elements. Furthermore, the growth medium contained Fe, Co, and Eu which had been solubilized from the amphibolite. This indicates the presence, in this yeast strain, of active rockweathering abilities as well as of uptake mechanisms for solubilized rock components. PMID:16345472

  5. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  6. IQGAP Family Members in Yeast, Dictyostelium, and Mammalian Cells

    PubMed Central

    Shannon, Katie B.

    2012-01-01

    IQGAPs are a family of scaffolding proteins with multiple domains, named for the IQ motifs and GTPase activating protein (GAP) related domains. Despite their GAP homology, IQGAP proteins act as effectors for GTP-bound GTPases of the Ras superfamily and do not stimulate GTP hydrolysis. IQGAPs are found in eukaryotic cells from yeast to human, and localize to actin-containing structures such as lamellipodia, membrane ruffles, cell-cell adhesions, phagocytic cups, and the actomyosin ring formed during cytokinesis. Mammalian IQGAPs also act as scaffolds for signaling pathways. IQGAPs perform their myriad functions through association with a large number of proteins including filamentous actin (F-actin), GTPases, calcium-binding proteins, microtubule binding proteins, kinases, and receptors. The focus of this paper is on recent studies describing new binding partners, mechanisms of regulation, and biochemical and physiological functions of IQGAPs in yeast, amoeba, and mammalian cells. PMID:22505937

  7. Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast.

    PubMed

    Angermayr, Michaela; Hochleitner, Elisabeth; Lottspeich, Friedrich; Bandlow, Wolfhard

    2007-09-01

    Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A)(6) mutant proteins. Both mutant alleles (S > A)(6) or (S > D)(6) complement in vivo, however, growth of the RIO1 (S > A)(6) mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D)(6) mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G(1)/S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.

  8. Active yeast ribosome preparation using monolithic anion exchange chromatography.

    PubMed

    Munoz, Antonio M; Yourik, Paul; Rajagopal, Vaishnavi; Nanda, Jagpreet S; Lorsch, Jon R; Walker, Sarah E

    2017-02-01

    In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

  9. Active yeast ribosome preparation using monolithic anion exchange chromatography

    PubMed Central

    Munoz, Antonio M.; Yourik, Paul; Rajagopal, Vaishnavi; Lorsch, Jon R.

    2017-01-01

    ABSTRACT In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes. PMID:27981882

  10. Antimycotic activity of 4-thioisosteres of flavonoids towards yeast and yeast-like microorganisms.

    PubMed

    Buzzini, Pietro; Menichetti, Stefano; Pagliuca, Chiara; Viglianisi, Caterina; Branda, Eva; Turchetti, Benedetta

    2008-07-01

    Different substituted methoxy- and hydroxy-4-thioisosteres of flavonoids were prepared and their in vitro antimycotic activity towards yeast (Candida spp., Clavispora spp., Cryptococcus spp., Filobasidiella spp., Issatchenkia spp., Pichia spp., Kluyveromyces spp., Saccharomyces spp. and Yarrowia spp.) and yeast-like (Prototheca spp.) microorganisms was tested. Further insights in the biological activities of these antioxidant, oestrogenic and antimicrobial biomimetic derivatives were obtained.

  11. Cell surface engineering of yeast for applications in white biotechnology.

    PubMed

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2011-01-01

    Cell surface engineering is a promising strategy for the molecular breeding of whole-cell biocatalysts. By using this strategy, yeasts can be constructed by the cell surface display of functional proteins; these yeasts are referred to as arming yeasts. Because reactions using arming yeasts as whole-cell biocatalysts occur on the cell surface, materials that cannot enter the cell can be used as reaction substrates. Numerous arming yeasts have therefore been constructed for a wide range of uses such as biofuel production, synthesis of valuable chemicals, adsorption or degradation of environmental pollutants, recovery of rare metal ions, and biosensors. Here, we review the science of yeast cell surface modification as well as current applications and future opportunities.

  12. Dielectric modelling of cell division for budding and fission yeast

    NASA Astrophysics Data System (ADS)

    Asami, Koji; Sekine, Katsuhisa

    2007-02-01

    The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast.

  13. Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator.

    PubMed

    Suchomelová, Petra; Velgová, Denisa; Masek, Tomás; Francis, Dennis; Rogers, Hilary J; Marchbank, Angela M; Lipavská, Helena

    2004-01-01

    During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes. The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M. However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet. Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv. Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division. On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls. Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development. Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots. Instead, they continued to form shoots at low frequencies. Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium. The results indicate that Spcdc25 expression in culture tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins. The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development.

  14. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

    PubMed Central

    Mak, P; Cruz, F D; Chen, S

    1999-01-01

    Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast

  15. Yeast cells can access distinct quiescent states.

    PubMed

    Klosinska, Maja M; Crutchfield, Christopher A; Bradley, Patrick H; Rabinowitz, Joshua D; Broach, James R

    2011-02-15

    We conducted a phenotypic, transcriptional, metabolic, and genetic analysis of quiescence in yeast induced by starvation of prototrophic cells for one of three essential nutrients (glucose, nitrogen, or phosphate) and compared those results with those obtained with cells growing slowly due to nutrient limitation. These studies address two related questions: (1) Is quiescence a state distinct from any attained during mitotic growth, and (2) does the nature of quiescence differ depending on the means by which it is induced? We found that either limitation or starvation for any of the three nutrients elicits all of the physiological properties associated with quiescence, such as enhanced cell wall integrity and resistance to heat shock and oxidative stress. Moreover, the starvations result in a common transcriptional program, which is in large part a direct extrapolation of the changes that occur during slow growth. In contrast, the metabolic changes that occur upon starvation and the genetic requirements for surviving starvation differ significantly depending on the nutrient for which the cell is starved. The genes needed by cells to survive starvation do not overlap the genes that are induced upon starvation. We conclude that cells do not access a unique and discrete G(0) state, but rather are programmed, when nutrients are scarce, to prepare for a range of possible future stressors. Moreover, these survival strategies are not unique to quiescence, but are engaged by the cell in proportion to nutrient scarcity.

  16. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis

    PubMed Central

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

  17. Anti-yeast activity of a food-grade dilution-stable microemulsion.

    PubMed

    Zhang, Hui; Xu, Yaoqi; Wu, Lijiang; Zheng, Xiaodong; Zhu, Songming; Feng, Fengqin; Shen, Lirong

    2010-07-01

    The anti-yeast activities of a food-grade dilution-stable microemulsion against Candida albicans and Saccharomyces cerevisiae have been studied. The weight ratio of the formulated microemulsion is glycerol monolaurate (GML)/propionic acid/Tween 80/sodium benzoate (SB)/water = 3:9:14:14:24. Results of anti-yeast activity on solid medium by agar diffusion method showed that the anti-yeast activity of the microemulsion at 4.8 mg/ml was comparable to that of natamycin at 0.1 mg/ml as positive control. Results of anti-yeast activity in liquid medium by broth dilution method showed that the growth of both C. albicans and S. cerevisiae was completely inhibited when the liquid medium containing 10(6) cfu/ml was treated with 1.2 mg/ml microemulsion, which was determined as minimum fungicidal concentration. The kinetics of killing results showed that the microemulsion killed over 90% yeast cells rapidly within 15 min and caused a complete loss of viability in 120 min. Among the components, SB and GML had a similar anti-yeast activity, followed by propionic acid, while Tween 80 exhibited no activity and could not enhance the anti-yeast activities of these components, and it was revealed that the anti-yeast activity of the microemulsion was attributed to a combination of propionic acid, GML, and SB. The anti-yeast activity of the microemulsion was in good agreement with the leakage of 260-nm absorbing materials and the observation of transmission electron microscopy, indicating that the microemulsion induced the disruption and dysfunction of the cell membrane.

  18. Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

    PubMed Central

    Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

    2016-01-01

    The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

  19. Yeast cell surface display for lipase whole cell catalyst and its applications

    SciTech Connect

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  20. Lipid raft involvement in yeast cell growth and death

    PubMed Central

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na+, K+, and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases. PMID:23087902

  1. The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

    SciTech Connect

    Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer NO is produced from L-arginine in response to elevated temperature in yeast. Black-Right-Pointing-Pointer Tah18 was first identified as the yeast protein involved in NO synthesis. Black-Right-Pointing-Pointer Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.

  2. Uniform Yeast Cell Assembly Based on Microfluidic Microgels

    NASA Astrophysics Data System (ADS)

    Chang, Ya-Wen; He, Peng; Marquez, Manuel; Cheng, Zhengdong; Marquez, Samantha M.

    2011-03-01

    We present a novel microgel templated Yeastosome (Yeast- Celloidosome ) based on self-assembly of yeast cells onto liquid-gel interfaces. To organize living cells onto the surface of the gel particles, strong positive charges were first introduced via LbL (layer by layer) polyelectrolyte decoration on monodisperse agarose microgel templates fabricated with a microfluidic flow focusing device. Native yeasts, bearing negative surface charges can then be driven electrostatically to form a monolayer shell around the gel core. Surface coverage/packing density of the yeast biofilm on varying microgel-to-yeast size ratio assemblies is evaluated with optical microscopy. Mechanical properties of the corresponding shells are characterized with buckling or collapse behavior during drying-hydrating cycle. We demonstrate the capability to fabricate narrow size distribution Yeastosome with a soft hydrogel core. The combination of microfluidic fabrication with cell assembly offers excellent control over inner core properties and could enable further hierarchy bio-structures.

  3. Antioxidant defense parameters as predictive biomarkers for fermentative capacity of active dried wine yeast.

    PubMed

    Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

    2014-08-01

    The production of active dried yeast (ADY) is a common practice in industry for the maintenance of yeast starters and as a means of long term storage. The process, however, causes multiple cell injuries, with oxidative damage being one of the most important stresses. Consequentially, dehydration tolerance is a highly appreciated property in yeast for ADY production. In this study we analyzed the cellular redox environment in three Saccharomyces cerevisiae wine strains, which show markedly different fermentative capacities after dehydration. To measure/quantify the effect of dehydration on the S. cerevisiae strains, we used: (i) fluorescent probes; (ii) antioxidant enzyme activities; (ii) intracellular damage; (iii) antioxidant metabolites; and (iv) gene expression, to select a minimal set of biochemical parameters capable of predicting desiccation tolerance in wine yeasts. Our results show that naturally enhanced antioxidant defenses prevent oxidative damage after wine yeast biomass dehydration and improve fermentative capacity. Based on these results we chose four easily assayable parameters/biomarkers for the selection of industrial yeast strains of interest for ADY production: trehalose and glutathione levels, and glutathione reductase and catalase enzymatic activities. Yeast strains selected in accordance with this process display high levels of trehalose, low levels of oxidized glutathione, a high induction of glutathione reductase activity, as well as a high basal level and sufficient induction of catalase activity, which are properties inherent in superior ADY strains.

  4. The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

    PubMed

    Horowitz, Avital; Lapointe, Jason F; Eid, Rawan; Sheibani, Sara; Gharib, Nada; Jones, Natalie K; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2013-12-01

    The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

  5. A new specific DNA endonuclease activity in yeast mitochondria.

    PubMed

    Sargueil, B; Delahodde, A; Hatat, D; Tian, G L; Lazowska, J; Jacq, C

    1991-02-01

    Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the CO XI gene.

  6. Dynamic changes in brewing yeast cells in culture revealed by statistical analyses of yeast morphological data.

    PubMed

    Ohnuki, Shinsuke; Enomoto, Kenichi; Yoshimoto, Hiroyuki; Ohya, Yoshikazu

    2014-03-01

    The vitality of brewing yeasts has been used to monitor their physiological state during fermentation. To investigate the fermentation process, we used the image processing software, CalMorph, which generates morphological data on yeast mother cells and bud shape, nuclear shape and location, and actin distribution. We found that 248 parameters changed significantly during fermentation. Successive use of principal component analysis (PCA) revealed several important features of yeast, providing insight into the dynamic changes in the yeast population. First, PCA indicated that much of the observed variability in the experiment was summarized in just two components: a change with a peak and a change over time. Second, PCA indicated the independent and important morphological features responsible for dynamic changes: budding ratio, nucleus position, neck position, and actin organization. Thus, the large amount of data provided by imaging analysis can be used to monitor the fermentation processes involved in beer and bioethanol production.

  7. Significant quantities of the glycolytic enzyme phosphoglycerate mutase are present in the cell wall of yeast Saccharomyces cerevisiae.

    PubMed Central

    Motshwene, Precious; Brandt, Wolf; Lindsey, George

    2003-01-01

    NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1 M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present. PMID:12238949

  8. Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast.

    PubMed

    Guaragnella, Nicoletta; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-08-20

    To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H(2)O(2)) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H(2)O(2) and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.

  9. Effects of Selenium Yeast on Oxidative Stress, Growth Inhibition, and Apoptosis in Human Breast Cancer Cells.

    PubMed

    Guo, Chih-Hung; Hsia, Simon; Shih, Min-Yi; Hsieh, Fang-Chin; Chen, Pei-Chung

    2015-01-01

    Recent evidence suggests that selenium (Se) yeast may exhibit potential anti-cancer properties; whereas the precise mechanisms remain unknown. The present study was aimed at evaluating the effects of Se yeast on oxidative stress, growth inhibition, and apoptosis in human breast cancer cells. Treatments of ER-positive MCF-7 and triple-negative MDA-MB-231 cells with Se yeast (100, 750, and 1500 ng Se/mL), methylseleninic acid (MSA, 1500 ng Se/mL), or methylselenocysteine (MSC, 1500 ng Se/mL) at a time course experiment (at 24, 48, 72, and 96 h) were analyzed. Se yeast inhibited the growth of these cancer cells in a dose- and time-dependent manner. Compared with the same level of MSA, cancer cells exposure to Se yeast exhibited a lower growth-inhibitory response. The latter has also lower superoxide production and reduced antioxidant enzyme activities. Furthermore, MSA (1500 ng Se/mL)-exposed non-tumorigenic human mammary epithelial cells (HMEC) have a significant growth inhibitory effect, but not Se yeast and MSC. Compared with MSA, Se yeast resulted in a greater increase in the early apoptosis in MCF-7 cells as well as a lower proportion of early and late apoptosis in MDA-MB-231 cells. In addition, nuclear morphological changes and loss of mitochondrial membrane potential were observed. In conclusion, a dose of 100 to 1500 ng Se/mL of Se yeast can increase oxidative stress, and stimulate growth inhibitory effects and apoptosis induction in breast cancer cell lines, but does not affect non-tumorigenic cells.

  10. A yeast glycolipid biosurfactant, mannosylerythritol lipid, shows potential moisturizing activity toward cultured human skin cells: the recovery effect of MEL-A on the SDS-damaged human skin cells.

    PubMed

    Morita, Tomotake; Kitagawa, Masaru; Suzuki, Michiko; Yamamoto, Shuhei; Sogabe, Atsushi; Yanagidani, Shusaku; Imura, Tomohiro; Fukuoka, Tokuma; Kitamoto, Dai

    2009-01-01

    Mannosylerythritol lipids (MELs) are produced in large amounts from renewable vegetable oils by Pseudozyma antarctica, and are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics and pharmaceuticals, the skin care property of MEL-A, the major component of MELs, was investigated using a three-dimensional cultured human skin model. The skin cells were cultured and treated with sodium dodecyl sulfate (SDS) solution of 1 wt%, and the effects of different lipids on the SDS-damaged cells were then evaluated on the basis of the cell viability. The viability of the damaged cells was markedly recovered by the addition of MEL-A in a dose-dependent manner. Compared to the control, MEL-A solutions of 5 wt% and 10 wt% gave the recovery rate of 73% and 91%, respectively, while ceramide solution of 1 wt% gave the rate of over 100%. This revealed that MEL-A shows a ceramide-like moisturizing activity toward the skin cells. Considering the drawbacks of natural ceramides, namely limited amount and high production cost, the yeast biosurfactants should have a great potential as a novel moisturizer for treating the damaged skin.

  11. Yeast cells-derived hollow core/shell heteroatom-doped carbon microparticles for sustainable electrocatalysis.

    PubMed

    Huang, Xiaoxi; Zou, Xiaoxin; Meng, Yuying; Mikmeková, Eliška; Chen, Hui; Voiry, Damien; Goswami, Anandarup; Chhowalla, Manish; Asefa, Tewodros

    2015-01-28

    The use of renewable resources to make various synthetic materials is increasing in order to meet some of our sustainability challenges. Yeast is one of the most common household ingredients, which is cheap and easy to reproduce. Herein we report that yeast cells can be thermally transformed into hollow, core-shell heteroatom-doped carbon microparticles that can effectively electrocatalyze the oxygen reduction and hydrazine oxidation reactions, reactions that are highly pertinent to fuel cells or renewable energy applications. We also show that yeast cell walls, which can easily be separated from the cells, can produce carbon materials with electrocatalytic activity for both reactions, albeit with lower activity compared with the ones obtained from intact yeast cells. The results reveal that the intracellular components of the yeast cells such as proteins, phospholipids, DNAs and RNAs are indirectly responsible for the latter's higher electrocatalytic activity, by providing it with more heteroatom dopants. The synthetic method we report here can serve as a general route for the synthesis of (electro)catalysts using microorganisms as raw materials.

  12. Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

    PubMed

    Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

    2016-08-01

    The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Hydrolysis of whey lactose using CTAB-permeabilized yeast cells.

    PubMed

    Kaur, Gurpreet; Panesar, Parmjit S; Bera, Manav B; Kumar, Harish

    2009-01-01

    Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.

  14. Hypotonic shocks activate rat TRPV4 in Yeast in the Absence of Polyunsaturated Fatty Acids

    PubMed Central

    Loukin, Stephen H.; Su, Zhenwei; Kung, Ching

    2010-01-01

    Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca2+ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference. PMID:19174160

  15. Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

    PubMed Central

    Tosato, Valentina; Grüning, Nana-Maria; Breitenbach, Michael; Arnak, Remigiusz; Ralser, Markus; Bruschi, Carlo V.

    2013-01-01

    Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (“translocants”), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast. PMID:23346549

  16. Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway.

    PubMed

    Piccirillo, Sarah; Morales, Rita; White, Melissa G; Smith, Keston; Kapros, Tamas; Honigberg, Saul M

    2015-12-01

    Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.

  17. Aroma formation by immobilized yeast cells in fermentation processes.

    PubMed

    Nedović, V; Gibson, B; Mantzouridou, T F; Bugarski, B; Djordjević, V; Kalušević, A; Paraskevopoulou, A; Sandell, M; Šmogrovičová, D; Yilmaztekin, M

    2015-01-01

    Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes.

  18. Biomimetic Yeast Cell Typing-Application of QCMs.

    PubMed

    Seidler, Karin; Polreichová, Miroslava; Lieberzeit, Peter A; Dickert, Franz L

    2009-01-01

    Artificial antibodies represent a key factor in the generation of sensing systems for the selective detection of bioanalytes of variable sizes. With biomimetic surfaces, the important model organism Saccharomyces cerevisiae and several of its growth stages may be detected. Quartz crystal microbalances (QCM) with 10 MHz fundamental frequency and coated with polymers imprinted with synchronized yeast cells are presented, which are able to detect duplex cells with high selectivity. Furthermore, a multichannel quartz crystal microbalance (MQCM) was designed and optimized for the measurement in liquids. This one-chip system based on four-electrode geometry allows the simultaneous detection of four analytes and, thus, provides a monitoring system for biotechnology and process control. For further standardization of the method, synthetic stamps containing plastic yeast cells in different growth stages were produced and utilized for imprinting. Mass-sensitive measurements with such MIPs resulted in the same sensor characteristics as obtained for those imprinted with native yeast cells.

  19. Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells

    PubMed Central

    Weber, Simone Schneider; Parente, Ana Flávia Alves; Borges, Clayton Luiz; Parente, Juliana Alves; Bailão, Alexandre Melo; de Almeida Soares, Célia Maria

    2012-01-01

    Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host. PMID:23272246

  20. Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

    NASA Astrophysics Data System (ADS)

    Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

    2006-02-01

    We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

  1. In vitro activity of voriconazole against Mexican oral yeast isolates.

    PubMed

    Sánchez Vargas, Luis Octavio; Eraso, Elena; Carrillo-Muñoz, Alfonso Javier; Aguirre, José Manuel; Gaitán-Cepeda, Luis Alberto; Quindós, Guillermo

    2010-05-01

    Oral candidiasis is the most prevalent complication in HIV-infected and AIDS patients. Topical antifungal treatment is useful for the initial episodes of oral candidiasis, but most patients suffer more than one episode and fluconazole or itraconazole can help in the management, and voriconazole may represent a useful alternative agent for the treatment of recalcitrant oral and oesophageal candidiasis. The aim of this research was to study the in vitro activity of voriconazole and fluconazole against Mexican oral isolates of clinically relevant yeast. The in vitro susceptibility of 187 oral yeast isolates from HIV-infected and healthy Mexicans was determined for fluconazole and voriconazole by the M44-A disc diffusion method. At 24 h, fluconazole was active against 179 of 187 isolates (95.7 %). Moreover, a 100% susceptibility to voriconazole was observed. Voriconazole and fluconazole are highly active in vitro against oral yeast isolates. This study provides baseline data on susceptibilities to both antifungal agents in Mexico.

  2. Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

    DOEpatents

    Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

    2012-03-20

    Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

  3. The price of independence: cell separation in fission yeast.

    PubMed

    Martín-García, Rebeca; Santos, Beatriz

    2016-04-01

    The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.

  4. Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

    PubMed

    Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

    2015-09-02

    Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

  5. Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

    PubMed

    Petrovic, Uros; Sribar, Jernej; Matis, Maja; Anderluh, Gregor; Peter-Katalinić, Jasna; Krizaj, Igor; Gubensek, Franc

    2005-10-15

    Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

  6. A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

    PubMed Central

    Sacristán-Reviriego, Almudena; Madrid, Marisa; Cansado, José; Martín, Humberto; Molina, María

    2014-01-01

    Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. PMID:24465549

  7. Singularity in polarization: rewiring yeast cells to make two buds.

    PubMed

    Howell, Audrey S; Savage, Natasha S; Johnson, Sam A; Bose, Indrani; Wagner, Allison W; Zyla, Trevin R; Nijhout, H Frederik; Reed, Michael C; Goryachev, Andrew B; Lew, Daniel J

    2009-11-13

    For budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single "winning" cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters.

  8. [Alcohol fermentation: effect of temperature on ethanol accumulation within yeast cells (author's transl)].

    PubMed

    Navarro, J M; Durand, G

    1978-01-01

    During fermentation, yeast growth is rapidly stopped when the concentration of alcohol in the medium increases but fermentive activity is not entirely inhibited until high alcohol concentrations are reached. The rate of alcohol accumulation within the cells and certain kinetic parameters were simultaneously determined in such fermentative processes using Saccharomyces carlsbergensis cells. The growth inhibitory effect of alcohol was related to its retention inside within the cells; i.e. yeast multiplication is stopped when intracellular alcohol concentration reaches a maximum value. Moreover, the higher the temperature, the deeper the inhibitory effect of ethanol and the higher the maximal intracellular alcohol concentration. Activation energy determinations showed that ethanol accumulation within the cells was a consequence of the resistance to its diffusion through the cell wall from within to outside the cell.

  9. Programmed Cell Death Initiation and Execution in Budding Yeast

    PubMed Central

    Strich, Randy

    2015-01-01

    Apoptosis or programmed cell death (PCD) was initially described in metazoans as a genetically controlled process leading to intracellular breakdown and engulfment by a neighboring cell . This process was distinguished from other forms of cell death like necrosis by maintenance of plasma membrane integrity prior to engulfment and the well-defined genetic system controlling this process. Apoptosis was originally described as a mechanism to reshape tissues during development. Given this context, the assumption was made that this process would not be found in simpler eukaryotes such as budding yeast. Although basic components of the apoptotic pathway were identified in yeast, initial observations suggested that it was devoid of prosurvival and prodeath regulatory proteins identified in mammalian cells. However, as apoptosis became extensively linked to the elimination of damaged cells, key PCD regulatory proteins were identified in yeast that play similar roles in mammals. This review highlights recent discoveries that have permitted information regarding PCD regulation in yeast to now inform experiments in animals. PMID:26272996

  10. Non-interferometric quantitative phase imaging of yeast cells

    NASA Astrophysics Data System (ADS)

    Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

  11. A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

    PubMed Central

    Marini, N J; Meldrum, E; Buehrer, B; Hubberstey, A V; Stone, D E; Traynor-Kaplan, A; Reed, S I

    1996-01-01

    In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway. Images PMID:8670805

  12. Involvement of the yeast metacaspase Yca1 in ubp10Delta-programmed cell death.

    PubMed

    Bettiga, Maurizio; Calzari, Luciano; Orlandi, Ivan; Alberghina, Lilia; Vai, Marina

    2004-11-01

    UBP10 encodes a deubiquitinating enzyme of Saccharomyces cerevisiae. Its inactivation results in a complex phenotype characterized by a subpopulation of cells that exhibits the typical cellular markers of apoptosis. Here, we show that additional deletion of YCA1, coding for the yeast metacaspase, suppressed the ubp10 disruptant phenotype. Moreover, YCA1 overexpression, without any external stimulus, had a detrimental effect on growth and viability of ubp10 cells accompanied by an increase of apoptotic cells. This response was completely abrogated by ascorbic acid addition. We also observed that cells lacking UBP10 had an endogenous caspase activity, revealed by incubation in vivo with FITC-labeled VAD-fmk. All these results argue in favour of an involvement of the yeast metacaspase in the active cell death triggered by loss of UBP10 function.

  13. Inhibition of Yap2 activity by MAPKAP kinase Rck1 affects yeast tolerance to cadmium.

    PubMed

    Mazzola, Daiane; Pimentel, Catarina; Caetano, Soraia; Amaral, Catarina; Menezes, Regina; Santos, Claudia N; Eleutherio, Elis; Rodrigues-Pousada, Claudina

    2015-09-14

    Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAPKAP) kinase Rck1. We show that Rck1 deletion confers protection against cadmium toxicity and that the mechanism underlying this observation relies on Yap2. Rck1 removal from the yeast genome potentiates Yap2 activity by increasing protein half-life and delaying its nuclear export. As a consequence, several Yap2 antioxidant targets are over-activated by a mechanism that also requires Yap1. Several genes of the cell wall integrity (CWI) pathway are upregulated under cadmium stress in a Yap2 dependent way. We showed that deletion of CWI genes renders yeast cells more sensitive to cadmium. These findings led us to suggest that in response to cadmium stress Yap2 may serve a dual purpose: oxidative stress attenuation and cell wall maintenance.

  14. Yeast Replicator: A High-Throughput Multiplexed Microfluidics Platform for Automated Measurements of Single-Cell Aging.

    PubMed

    Liu, Ping; Young, Thomas Z; Acar, Murat

    2015-10-20

    The yeast Saccharomyces cerevisiae is a model organism for replicative aging studies; however, conventional lifespan measurement platforms have several limitations. Here, we present a microfluidics platform that facilitates simultaneous lifespan and gene expression measurements of aging yeast cells. Our multiplexed high-throughput platform offers the capability to perform independent lifespan experiments using different yeast strains or growth media. Using this platform in minimal media environments containing glucose, we measured the full lifespan of individual yeast cells in wild-type and canonical gene deletion backgrounds. Compared to glucose, in galactose we observed a 16.8% decrease in replicative lifespan accompanied by an ∼2-fold increase in single-cell oxidative stress levels reported by PSOD1-mCherry. Using PGAL1-YFP to measure the activity of the bistable galactose network, we saw that OFF and ON cells are similar in their lifespan. Our work shows that aging cells are committed to a single phenotypic state throughout their lifespan.

  15. Identification and characterization of antimicrobial activity in two yeast genera.

    PubMed Central

    Bilinski, C A; Innamorato, G; Stewart, G G

    1985-01-01

    A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated. Images PMID:3937494

  16. Cellulosic ethanol production by combination of cellulase-displaying yeast cells.

    PubMed

    Baek, Seung-Ho; Kim, Sujin; Lee, Kyusung; Lee, Jung-Kul; Hahn, Ji-Sook

    2012-12-10

    As an effort to find suitable endoglucanases to generate cellulolytic yeast strains, two fungal endoglucanases, Thermoascus aurantiacus EGI and Trichoderma reesei EGII, and two bacterial endoglucanases, Clostridium thermocellum CelA and CelD, were expressed on the yeast surface, and their surface expression levels, pH- and temperature-dependent enzyme activities, and substrate specificities were analyzed. T. aurantiacus EGI showed similar patterns of pH- and temperature-dependent activities to those of T. reesei EGII which has been widely used due to its high enzyme activity. Although EGII showed higher carboxymethyl cellulose (CMC) degradation activity than EGI, EGI showed better activity toward phosphoric acid swollen cellulose (PASC). For ethanol production from PASC, we combined three types of yeast cells, each displaying T. aurantiacus EGI, T. reesei CBHII (exoglucanase) and Aspergillus aculeatus BGLI (β-glucosidase), instead of co-expressing these enzymes in a single cell. In this system, ethanol production can be easily optimized by adjusting the combination ratio of each cell type. A mixture of cells with the optimized EGI:CBHII:BGLI ratio of 6:2:1 produced 1.3 fold more ethanol (2.1g/l) than cells composed of an equal amount of each cell type, suggesting the usefulness of this system for cellulosic ethanol production.

  17. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients

    PubMed Central

    Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-01-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other. PMID:27077831

  18. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

    PubMed

    Muller, Nicolas; Piel, Matthieu; Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-04-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.

  19. Differential Adsorption of Ochratoxin A and Anthocyanins by Inactivated Yeasts and Yeast Cell Walls during Simulation of Wine Aging

    PubMed Central

    Petruzzi, Leonardo; Baiano, Antonietta; De Gianni, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria; Bevilacqua, Antonio

    2015-01-01

    The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify the intensity of the phenomenon. The aim of this study was to examine OTA adsorption by two strains of Saccharomyces cerevisiae (the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a yeast cell wall preparation. Experiments were conducted using Nero di Troia red wine contaminated with 2 μg/L OTA and supplemented with yeast biomass (20 g/L). The samples were analyzed periodically to assess mycotoxin concentration, chromatic characteristics, and total anthocyanins over 84 days of aging. Yeast cell walls revealed the highest OTA-adsorption in comparison to thermally-inactivated cells (50% vs. 43% toxin reduction), whilst no significant differences were found for the amount of adsorbed anthocyanins in OTA-contaminated and control wines. OTA and anthocyanins adsorption were not competitive phenomena. Unfortunately, the addition of yeast cells to wine could cause color loss; therefore, yeast selection should also focus on this trait to select the best strain. PMID:26516913

  20. Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating.

    PubMed Central

    Nelson, Bryce; Parsons, Ainslie B; Evangelista, Marie; Schaefer, Karen; Kennedy, Kathy; Ritchie, Steven; Petryshen, Tracey L; Boone, Charles

    2004-01-01

    Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion. PMID:15020407

  1. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  2. Origin of irreversibility of cell cycle start in budding yeast.

    PubMed

    Charvin, Gilles; Oikonomou, Catherine; Siggia, Eric D; Cross, Frederick R

    2010-01-19

    Budding yeast cells irreversibly commit to a new division cycle at a regulatory transition called Start. This essential decision-making step involves the activation of the SBF/MBF transcription factors. SBF/MBF promote expression of the G1 cyclins encoded by CLN1 and CLN2. Cln1,2 can activate their own expression by inactivating the Whi5 repressor of SBF/MBF. The resulting transcriptional positive feedback provides an appealing, but as yet unproven, candidate for generating irreversibility of Start. Here, we investigate the logic of the Start regulatory module by quantitative single-cell time-lapse microscopy, using strains in which expression of key regulators is efficiently controlled by changes of inducers in a microfluidic chamber. We show that Start activation is ultrasensitive to G1 cyclin. In the absence of CLN1,2-dependent positive feedback, we observe that Start transit is reversible, due to reactivation of the Whi5 transcriptional repressor. Introduction of the positive feedback loop makes Whi5 inactivation and Start activation irreversible, which therefore guarantees unidirectional entry into S phase. A simple mathematical model to describe G1 cyclin turn on at Start, entirely constrained by empirically measured parameters, shows that the experimentally measured ultrasensitivity and transcriptional positive feedback are necessary and sufficient dynamical characteristics to make the Start transition a bistable and irreversible switch. Our study thus demonstrates that Start irreversibility is a property that arises from the architecture of the system (Whi5/SBF/Cln2 loop), rather than the consequence of the regulation of a single component (e.g., irreversible protein degradation).

  3. Inventions on baker's yeast storage and activation at the bakery plant.

    PubMed

    Gélinas, Pierre

    2010-01-01

    Baker's yeast is the gas-forming ingredient in bakery products. Methods have been invented to properly handle baker's yeast and optimize its activity at the bakery plant. Over the years, incentives for inventions on yeast storage and activation have greatly changed depending on trends in the baking industry. For example, retailer's devices for cutting bulk pressed yeast and techniques for activating dry yeast have now lost their importance. Review of patents for invention indicates that activation of baker's yeast activity has been a very important issue for bakers, for example, with baking ingredients called yeast foods. In the recent years and especially for highly automated bakeries, interest has moved to equipments and processes for optimized storage of liquid cream yeast to thoroughly control dough fermentation and bread quality.

  4. [Changes in the dielectric properties of water in yeast cells during their death].

    PubMed

    Romanov, A N

    2009-01-01

    The dielectric properties of living and dead unicellular microorganisms (yeast) were studied in the microwave range, and the general regularities and differences were revealed. The effect of temperature on the dielectric properties of yeast cells has been studied. The dielectric parameters of water fractions contained in yeast cells were estimated using the refraction model.

  5. Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity.

    PubMed

    Fine, Frédéric; Gervais, Patrick

    2005-01-01

    Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.

  6. Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast.

    PubMed

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1996-08-09

    A hammerhead ribozyme designed to cleave the yeast ADE1 mRNA has been expressed in yeast under the control of a galactose-inducible promoter. RNA prepared from the galactose-induced yeast cultures possesses an activity that cleaves ADE1 mRNA in vitro. However, in spite of high expression levels of the ribozyme, no cleavage activity could be demonstrated in vivo. On the other hand, when the yeast cells expressing hammerhead RNA were treated with the alpha-factor mating pheromone, the level of ADE1 mRNA was reduced by 50%. Similar reductions were observed when this strain was cultured in the presence of lithium acetate or in nitrogen-free medium. Moreover, control experiments in which disabled hammerhead genes were expressed showed no such reductions. Extension of the length of the flanking recognition arms of the ribozyme from a total of 10 to 16 or 24 nucleotides diminished the inhibitory effect of the ribozyme. These data suggest that ribozymes are able to cleave a trans-RNA target in yeast.

  7. Influence of temperature, pH and water activity on "in vitro" inhibition of Penicillium glabrum (Wehmer) Westling by yeasts.

    PubMed

    Sinigaglia, M; Corbo, M R; Ciccarone, C

    1998-08-01

    Four different yeast species (Metschnikowia pulcherrima, Saccharomycopsis vini, Kluyveromyces marxianus, Cryptococcus albidus), isolated from surface of grapes, were evaluated for biocontrol potential against Penicillium glabrum. In order to investigate the influence of temperature, pH, water activity and yeast cell concentration on Penicillium glabrum inhibition, the individual effects and the interaction of these factors were analyzed by means of a Central Composite Design (CCD). All yeast species tested showed antagonistic effects which were more pronounced at high cell concentrations. The other variables affected the antagonistic effect differentially depending on the yeast species. Results of the experimental design showed that the selective success of a competitive microflora is under environmental control; moreover, when microbial cells are subjected to multiple factors, the effects and the reciprocal interactions of the individual variables cannot be independently evaluated.

  8. The puc1 Cyclin Regulates the G1 Phase of the Fission Yeast Cell Cycle in Response to Cell Size

    PubMed Central

    Martín-Castellanos, Cristina; Blanco, Miguel A.; de Prada, José M.; Moreno, Sergio

    2000-01-01

    Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1+ gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1. PMID:10679013

  9. Activation of polyomavirus DNA replication by yeast GAL4 is dependent on its transcriptional activation domains.

    PubMed Central

    Bennett-Cook, E R; Hassell, J A

    1991-01-01

    The polyomavirus replication origin contains transcriptional regulatory sequences. To determine how these elements function in DNA replication, and to learn whether a common mechanism underlies the activation of transcription and DNA replication, we tested whether a well-characterized transcriptional activator, yeast GAL4, was capable of stimulating DNA replication and transcription in the same mammalian cell line. We observed that GAL4 activated polyomavirus DNA replication in mouse cells when its binding site was juxtaposed to the late border of the polyomavirus origin core. Synergistic activation of DNA replication was achieved by multimerization of the GAL4 binding site. Analysis of GAL4 mutant proteins, GAL4 hybrid proteins and mutants of the latter revealed that the activation domains of these transcriptional activators were required to stimulate DNA replication. In agreement with previously published data, the activation domains of GAL4 were also required to enhance transcription in the same mouse cell line. These observations implicate transcriptional activators in Py DNA replication and suggest that similar mechanisms govern the activation of transcription and DNA replication. Images PMID:1849079

  10. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    PubMed

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  11. Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts.

    PubMed

    Schebor, C; Galvagno, M; del Pilar Buera, M; Chirife, J

    2000-01-01

    Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars. The resulting glass transition temperatures were found to decrease with increasing moisture content. The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in baker's yeast. The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO(2)) of dry yeast was studied. First-order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed. Significant losses of fermentative activity were observed in vitrified yeast samples. The dependence of rate constants with temperature was found to follow Arrhenius behavior. The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss.

  12. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

    PubMed Central

    McGrath, J P; Jentsch, S; Varshavsky, A

    1991-01-01

    All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability. Images PMID:1989885

  13. Budding yeast colony growth study based on circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, Devi; Khotimah, S. N.; Viridi, S.

    2016-08-01

    Yeast colony growth can be modelled by using circular granular cells, which can grow and produce buds. The bud growth angle can be set to regulate cell budding pattern. Cohesion force, contact force and Stokes force were adopted to accommodate the behaviour and interactions among cells. Simulation steps are divided into two steps, the explicit step is due to cell growing and implicit step for the cell rearrangement. Only in explicit step that time change was performed. In this study, we examine the influence of cell diameter growth time and reproduction time combination toward the growth of cell number and colony formation. We find a commutative relation between the cell diameter growth time and reproduction time to the specific growth rate. The greater value of the multiplication of the parameters, the smaller specific growth rate is obtained. It also shows a linear correlation between the specific growth rate and colony diameter growth rate.

  14. Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity.

    PubMed

    Sanz, Ramsés; Galceran, Ma Teresa; Puignou, Lluís

    2003-01-01

    Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.

  15. Quantitative proteomic comparison of stationary/G0 phase cells and tetrads in budding yeast

    PubMed Central

    Kumar, Ravinder; Srivastava, Sanjeeva

    2016-01-01

    Most of the microbial cells on earth under natural conditions exist in a dormant condition, commonly known as quiescent state. Quiescent cells exhibit low rates of transcription and translation suggesting that cellular abundance of proteins may be similar in quiescent cells. Therefore, this study aim to compare the proteome of budding yeast cells from two quiescent states viz. stationary phase/G0 and tetrads. Using iTRAQ (isobaric tag for relative and absolute quantitation) based quantitative proteomics we identified 289 proteins, among which around 40 proteins exhibited ±1.5 fold change consistently from the four biological replicates. Proteomics data was validated by western blot and denstiometric analysis of Hsp12 and Spg4. Level of budding yeast 14-3-3 proteins was found to be similar in both the quiescent states, whereas Hsp12 and Spg4 expressed only during stress. FACS (fluorescence-activated cell sorting) analysis showed that budding yeast cells were arrested at G1 stages both in tetrads as well as in stationary phase. We also observed that quiescent states did not express Ime1 (inducer of meiosis). Taken together, our present study demonstrates that the cells in quiescent state may have similar proteome, and accumulation of proteins like Hsp12, Hsp26, and Spg4 may play an important role in retaining viability of the cells during dormancy. PMID:27558777

  16. Calcium modulation of doxorubicin cytotoxicity in yeast and human cells.

    PubMed

    Nguyen, Thi Thuy Trang; Lim, Ying Jun; Fan, Melanie Hui Min; Jackson, Rebecca A; Lim, Kim Kiat; Ang, Wee Han; Ban, Kenneth Hon Kim; Chen, Ee Sin

    2016-03-01

    Doxorubicin is a widely used chemotherapeutic agent, but its utility is limited by cellular resistance and off-target effects. To understand the molecular mechanisms regulating chemotherapeutic responses to doxorubicin, we previously carried out a genomewide search of doxorubicin-resistance genes in Schizosaccharomyces pombe fission yeast and showed that these genes are organized into networks that counteract doxorubicin cytotoxicity. Here, we describe the identification of a subgroup of doxorubicin-resistance genes that, when disrupted, leads to reduced tolerance to exogenous calcium. Unexpectedly, we observed a suppressive effect of calcium on doxorubicin cytotoxicity, where concurrent calcium and doxorubicin treatment resulted in significantly higher cell survival compared with cells treated with doxorubicin alone. Conversely, inhibitors of voltage-gated calcium channels enhanced doxorubicin cytotoxicity in the mutants. Consistent with these observations in fission yeast, calcium also suppressed doxorubicin cytotoxicity in human breast cancer cells. Further epistasis analyses in yeast showed that this suppression of doxorubicin toxicity by calcium was synergistically dependent on Rav1 and Vph2, two regulators of vacuolar-ATPase assembly; this suggests potential modulation of the calcium-doxorubicin interaction by fluctuating proton concentrations within the cellular environment. Thus, the modulatory effects of drugs or diet on calcium concentrations should be considered in doxorubicin treatment regimes.

  17. Human ARF4 expression rescues sec7 mutant yeast cells.

    PubMed Central

    Deitz, S B; Wu, C; Silve, S; Howell, K E; Melançon, P; Kahn, R A; Franzusoff, A

    1996-01-01

    Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. PMID:8668142

  18. Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis.

    PubMed

    Borovikova, Diana; Herynkova, Pavla; Rapoport, Alexander; Sychrova, Hana

    2014-01-01

    Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300 mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S. cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival.

  19. Unidirectional P-body transport during the yeast cell cycle.

    PubMed

    Garmendia-Torres, Cecilia; Skupin, Alexander; Michael, Sean A; Ruusuvuori, Pekka; Kuwada, Nathan J; Falconnet, Didier; Cary, Gregory A; Hansen, Carl; Wiggins, Paul A; Dudley, Aimée M

    2014-01-01

    P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained "hovering" near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions.

  20. Unidirectional P-Body Transport during the Yeast Cell Cycle

    PubMed Central

    Garmendia-Torres, Cecilia; Skupin, Alexander; Michael, Sean A.; Ruusuvuori, Pekka; Kuwada, Nathan J.; Falconnet, Didier; Cary, Gregory A.; Hansen, Carl; Wiggins, Paul A.; Dudley, Aimée M.

    2014-01-01

    P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained “hovering” near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions. PMID:24918601

  1. Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR

    PubMed Central

    Zhang, Yajie; Poirier-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2011-01-01

    Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal 1H2O NMR relaxation time constant (T1) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τi), where τi−1 is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O2/5%CO2 (O2) or 95%N2/5%CO2 (N2). ATP was high during O2, and τi−1 was 3.1 s−1 at 25°C. After changing to N2, ATP decreased and τi−1 was 1.8 s−1. The principal active yeast ion transport protein is the plasma membrane H+-ATPase. Studies using the H+-ATPase inhibitor ebselen or a yeast genetic strain with reduced H+-ATPase found reduced τi−1, notwithstanding high ATP. Steady-state water exchange correlates with H+-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity. PMID:22261073

  2. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    NASA Astrophysics Data System (ADS)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

  3. Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides.

    PubMed

    Giovani, Giovanna; Canuti, Valentina; Rosi, Iolanda

    2010-02-28

    To improve our understanding of the factors involved in polysaccharide release during alcoholic fermentation, we investigated three Saccharomyces cerevisiae strains in fermentation trials conducted at two temperatures (25 degrees C and 32 degrees C) and three sugar concentrations (20%, 23.5%, and 27%), with or without supplementation of grape juice with diammonium phosphate (DAP) or microcrystalline cellulose. In two yeast strains, the release of cell wall polysaccharides increased significantly with an increase in fermentation temperature and sugar concentration of the grape juice; the polysaccharide release was greater in stressed conditions, in which the cells were less viable and less metabolically active. In the third strain, the average amount of polysaccharides released into the medium decreased significantly at 32 degrees C with 27% sugar, and increased in grape juice supplemented with DAP. Thus, this strain released more polysaccharides when conditions were nearer to optimal and the yeast cells were more viable and metabolically active. Our results suggest that the yeast strains released cell wall polysaccharides via different mechanisms, and that the cell wall integrity pathway may account for some of the differences in polysaccharide release among the strains.

  4. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

    PubMed Central

    Ball, David A.

    2016-01-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947

  5. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

    PubMed

    Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

    2016-12-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.

  6. Systematic identification of cell size regulators in budding yeast

    PubMed Central

    Soifer, Ilya; Barkai, Naama

    2014-01-01

    Cell size is determined by a complex interplay between growth and division, involving multiple cellular pathways. To identify systematically processes affecting size control in G1 in budding yeast, we imaged and analyzed the cell cycle of millions of individual cells representing 591 mutants implicated in size control. Quantitative metric distinguished mutants affecting the mechanism of size control from the majority of mutants that have a perturbed size due to indirect effects modulating cell growth. Overall, we identified 17 negative and dozens positive size control regulators, with the negative regulators forming a small network centered on elements of mitotic exit network. Some elements of the translation machinery affected size control with a notable distinction between the deletions of parts of small and large ribosomal subunit: parts of small ribosomal subunit tended to regulate size control, while parts of the large subunit affected cell growth. Analysis of small cells revealed additional size control mechanism that functions in G2/M, complementing the primary size control in G1. Our study provides new insights about size control mechanisms in budding yeast. PMID:25411401

  7. [Export of an invertase by yeast cells (Candida utilis)].

    PubMed

    Alekseeva, O V; Sabirzianova, T A; Celiakh, I O; Kalebina, T S; Kulaev, I S

    2014-01-01

    Export and accumulation of various forms of invertase (EC 3.2.1.26) in the cell wall and culture liquid of the yeast Candida utilis was investigated. It was found that the high-molecular-weight CW-form of invertase is present in the cell wall. This form is not exported into the culture liquid, and it is by a third more glycosylated than the previously described exported S-form. It was shown that one of the two liquid forms of invertase exported into the culture-the glycosylated S-form--is retained in the cell wall, while the other one--the nonglycosylated F-form--was not detected in the cell wall. Based on these results, as well as data on the distribution dynamics of the enzyme in the culture liquid and in the cell wall during different growth stages of a yeast culture, we suggested that the nonglycosylated form was exported into the culture liquid via the zone of abnormal cell wall permeability and the glycosylated forms of this enzyme (both exported and nonexported) did not use this pathway (the degree of N-glycosylation is an important factor determining the final localization of the enzyme).

  8. Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

    PubMed Central

    Rodal, Avital A.; Kozubowski, Lukasz; Goode, Bruce L.; Drubin, David G.; Hartwig, John H.

    2005-01-01

    Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. PMID:15525671

  9. Preparation of corncob grits as a carrier for immobilizing yeast cells for ethanol production.

    PubMed

    Lee, Sang-Eun; Lee, Choon Geun; Kang, Do Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

    2012-12-01

    In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

  10. The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

    PubMed

    Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M

    2016-05-19

    Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.

  11. Diploid yeast cells yield homozygous spontaneous mutations

    NASA Technical Reports Server (NTRS)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  12. Global Dynamical Properties of the Yeast Cell Cycle Network

    NASA Astrophysics Data System (ADS)

    Tang, Chao

    2004-03-01

    The interactions between proteins, DNA, and RNA in living cells constitute molecular networks that govern various cellular functions. To investigate the global dynamical properties and stabilities of such networks, we studied the network regulating the cell division (cell cycle) of the budding yeast. With the use of both discrete (Boolean) and continuous (ODEs) dynamical models, it was demonstrated that the cell-cycle network is extremely stable and robust for its function. The biological stationary state--the G1 state--is a global attractor of the dynamics. The biological pathway--the cell-cycle sequence of protein states--is a globally attracting trajectory of the dynamics. These properties are largely preserved with respect to small perturbations to the network. These results suggest that cellular regulatory networks are robustly designed for their functions.

  13. Modified arabinoxylan rice bran (MGN-3/Biobran) enhances yeast-induced apoptosis in human breast cancer cells in vitro.

    PubMed

    Ghoneum, Mamdooh; Gollapudi, Sastry

    2005-01-01

    We have recently reported that phagocytosis of killed Saccharomyces cerevisiae, baker's yeast, induced apoptosis in human breast cancer cell lines MCF-7, ZR-75-1 and HCC70. In this study we have evaluated the effect of treatment with MGN-3, a modified arabinoxylan from rice bran, on phagocytosis and yeast-induced apoptosis in breast cancer cells. Cancer cells were cultured with yeast at a ratio of 1:10 in the absence or presence of MGN-3, and the percentages of phagocytic and apoptotic cancer cells were examined by flow cytometry and by cytospin preparations. Cancer cells treated with MGN-3 exhibited increased percentages of attachment (200%) and uptake of yeast (313%) by MCF-7 cells at 0.5 hr, as compared with cells without MGN-3. In addition, treatment with MGN-3 resulted in a 2 fold increase in the percentage of apoptotic MCF-7 cells, 2.5 fold for ZR-75 cells and 1.8 fold for HCC70 cells. MGN-3 effect was dose-dependent and associated with increased activation of caspases 8 and 9 in MCF-7 cells, and caspases 8, 9 and 3 in HCC70 cells. This data demonstrates that MGN-3 accelerates phagocytosis-induced apoptosis of cancer cells, which may represent a novel therapeutic strategy for the treatment of breast cancer.

  14. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    SciTech Connect

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  15. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    PubMed

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  16. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-05

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  17. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA

    PubMed Central

    Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

    2014-01-01

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ0). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent. PMID:28357227

  18. Facile and quantitative electrochemical detection of yeast cell apoptosis

    NASA Astrophysics Data System (ADS)

    Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

    2014-03-01

    An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.

  19. The yeast cell-cycle network is robustly designed

    NASA Astrophysics Data System (ADS)

    Li, Fangting; Long, Tao; Lu, Ying; Ouyang, Qi; Tang, Chao

    2004-04-01

    The interactions between proteins, DNA, and RNA in living cells constitute molecular networks that govern various cellular functions. To investigate the global dynamical properties and stabilities of such networks, we studied the cell-cycle regulatory network of the budding yeast. With the use of a simple dynamical model, it was demonstrated that the cell-cycle network is extremely stable and robust for its function. The biological stationary state, the G1 state, is a global attractor of the dynamics. The biological pathway, the cell-cycle sequence of protein states, is a globally attracting trajectory of the dynamics. These properties are largely preserved with respect to small perturbations to the network. These results suggest that cellular regulatory networks are robustly designed for their functions.

  20. [Methods of increasing the activity of extracellular esterase, beta-fructofuranosidase and proteases of wine yeast].

    PubMed

    Abdurazakova, S Kh; Salomov, Kh T

    1975-01-01

    Upon regular fermentation changes in the activity of the enzymes esterase, beta-fructofuranosidase and protease of the yeast Saccharomyces mini of the Parkent I race were examined. The maximum activity of the enzymes occurred in the stationary phase of the yeast growth. An increase in the activity of the above enzymes was shown possible during a prolonged stabilization of the stationary conditions in the process of a continuous chemostat cultivation of wine yeast.

  1. Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1.

    PubMed

    Guaragnella, Nicoletta; Bobba, Antonella; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-01-04

    To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

  2. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    SciTech Connect

    Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  3. Mechanisms of electron transfer between a styrylquinolinium dye and yeast in biofuel cell.

    PubMed

    Hubenova, Yolina; Bakalska, Rumyana; Hubenova, Eleonora; Mitov, Mario

    2016-12-01

    In the present study, the influence of the recently synthesized styrylquinolinium dye 4-{(E)-2-[4-(dimethylamino)naphthalen-1-yl]ethenyl}-1-methylquinolinium iodide (DANSQI) on the intracellular processes as well as the electrical outputs of Candida melibiosica 2491 yeast-based biofuel cell was investigated. The addition of nanomolar quantities of DANSQI to the yeast suspension results in an increase of the current outputs right after the startup of the biofuel cells, associated with an electrooxidation of the dye on the anode. After that, the formed cation radical of the dye penetrates the yeast cells, provoking a set of intracellular changes. Studies of the subcellular anolyte fractions show that 1μM dye increased the peroxisomal catalase activity 30-times (1.15±0.06Unit/mg protein) and over twice the mitochondrial cytochrome c oxidase activity (92±5Unit/mg protein). The results obtained by electrochemical and spectrophotometric analyses let to the supposition that the dye acts as subcellular shuttle, on account of its specific intramolecular charge transfer properties. The transition between its benzoid, quinolyl radical and ion forms and their putative role for the extracellular and intracellular charge transfer mechanisms are discussed.

  4. A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

    PubMed Central

    Robinow, C. F.; Marak, J.

    1966-01-01

    The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope. PMID:5331666

  5. Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines

    PubMed Central

    Chandel, Avinash; Das, Krishna K.; Bachhawat, Anand K.

    2016-01-01

    Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward-facing channels: the plasma membrane, Cch1p, and the vacuolar calcium channel, Yvc1p. Deletion of these channels partially rescues cells from glutathione depletion–induced cell death. Subsequent investigations on the Yvc1p channel, a homologue of the mammalian TRP channels, revealed that the channel is activated by glutathionylation. Yvc1p has nine cysteine residues, of which eight are located in the cytoplasmic regions and one on the transmembrane domain. We show that three of these cysteines, Cys-17, Cys-79, and Cys-191, are specifically glutathionylated. Mutation of these cysteines to alanine leads to a loss in glutathionylation and a concomitant loss in calcium channel activity. We further investigated the mechanism of glutathionylation and demonstrate a role for the yeast glutathione S-transferase Gtt1p in glutathionylation. Yvc1p is also deglutathionylated, and this was found to be mediated by the yeast thioredoxin, Trx2p. A model for redox activation and deactivation of the yeast Yvc1p channel is presented. PMID:27708136

  6. Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.

    PubMed

    Lim, Sungwon; Glasgow, Jeff E; Filsinger Interrante, Maria; Storm, Erica M; Cochran, Jennifer R

    2017-03-16

    Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.

  7. Feasibility of measuring ferricyanide reduction by yeasts to estimate their activity during alcoholic fermentation in wine-making conditions.

    PubMed

    Roustan, Jean-Louis; Sablayrolles, Jean-Marie

    2003-01-01

    We assessed the feasibility of measuring the extracellular reduction of ferricyanide in the presence of an intermediate carrier (menadione) as a means of estimating the activity of yeasts during alcoholic fermentation. A spectrophotometric and a potentiometric approach were used. Comparison of specific reductase activity and gas production rate during the stationary phase indicated that measuring the menadione-catalyzed reduction of ferricyanide provides a good estimate of the total activity of the yeast cells in a fermenting must. The response observed following the addition of an electron acceptor (acetaldehyde) confirmed that the reductase activity of menadione is dependent on the availability of NADH. The stability of menadione in the fermentation medium, as assessed by the potentiometric method, suggested that electrochemical reoxidation of the ferrocyanide can act as a substitute for the addition of an electron acceptor when studying the redox regulation of fermenting yeasts.

  8. Cell-wall composition and structure of yeast cells and conjugation tubes of Tremella mesenterica.

    PubMed

    Reid, I D; Bartnicki-Garcia, S

    1976-09-01

    Cell walls prepared from vegetative yeast cells and from hormone-induced conjugation tubes of the basidiomycete Tremella mesenterica had similar compositions. Evidence was found for 1,3-alpha-glucan (yeast 38%, tube 25%), 1,3-beta-1,6-beta-glucan (yeast 33%, tube 48%) and chitin (both less than 3%) in the walls. The walls also contained xylose (5 to 7%), mannose (6%), glucuronic acid (approx. 2%), and traces of galactose. Protein amounted to less than 2% of the wall weight. The cell capsule was very insoluble and could not be removed from the cell wall. The conjugation hormone did not appear to exert its effect on cell shape by causing gross changes in wall composition.

  9. Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells

    PubMed Central

    Armstrong, Christine A.

    2017-01-01

    Aberrant activation of telomerase occurs in 85–90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments. PMID:28330934

  10. Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

    PubMed

    Armstrong, Christine A; Tomita, Kazunori

    2017-03-01

    Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.

  11. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  12. Cell-cycle analyses using thymidine analogues in fission yeast.

    PubMed

    Anda, Silje; Boye, Erik; Grallert, Beata

    2014-01-01

    Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU) and 5-Chloro-2'-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2'-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.

  13. Process engineering for bioflavour production with metabolically active yeasts - a mini-review.

    PubMed

    Carlquist, Magnus; Gibson, Brian; Karagul Yuceer, Yonca; Paraskevopoulou, Adamantini; Sandell, Mari; Angelov, Angel I; Gotcheva, Velitchka; Angelov, Angel D; Etschmann, Marlene; de Billerbeck, Gustavo M; Lidén, Gunnar

    2015-01-01

    Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industries. The production of flavours can take place by either extraction from plant materials, chemical synthesis, biological conversion of precursor molecules or de novo biosynthesis. The latter alternatives are gaining importance through the rapidly growing fields of systems biology and metabolic engineering, giving efficient production hosts for the so-called 'bioflavours', which are natural flavour and/or fragrance compounds obtained with cell factories or enzymatic systems. Yeasts are potential production hosts for bioflavours. In this mini-review, we give an overview of bioflavour production in yeasts from the process-engineering perspective. Two specific examples, production of 2-phenylethanol and vanillin, are used to illustrate the process challenges and strategies used.

  14. Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

    PubMed Central

    Smith, Ida M.; Christensen, Jeffrey E.; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  15. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    PubMed

    Smith, Ida M; Christensen, Jeffrey E; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  16. Raman scattering evidence of hydrohalite formation on frozen yeast cells.

    PubMed

    Okotrub, K A; Surovtsev, N V

    2013-02-01

    We studied yeast cells in physiological solution during freezing by Raman microspectroscopy technique. The purpose was to find out the origin of a sharp peak near ∼3430cm(-1) in Raman spectrum of frozen mammalian cells, observed earlier (J. Dong et al., Biophys. J. 99 (2010) 2453), which presumably could be used as an indicator of intracellar ice appearance. We have shown that this line (actually doublet of 3408 and 3425cm(-1)) corresponds to Raman spectrum of hydrohalite (NaCl⋅2H(2)O), which is formed as the result of the eutectic crystallization of the liquid solution around the cells. We also show that the spatial distribution of hydrohalite in the sample significantly depends on the cooling rate. At lower cooling rate (1°C/min), products of eutectic crystallization form layer on the cell surface which thickness varies for different cells and can reach ∼1μm in thickness. At higher cooling rate (20°C/min), the hydrohalite distribution appears more homogeneous, in the sample, and the eutectic crystallization layer around the cells was estimated to be less than ∼20nm. These experimental results are consistent with scenarios predicted by the two-factor hypothesis for freezing induced cell injury. This work demonstrates a potential of Raman microspectroscopy to study peculiarities of the eutectic crystallization around single cells in vivo with the high spatial resolution.

  17. Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Tsai, C S; Shi, J L; Beehler, B W; Beck, B

    1992-12-01

    The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored. The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture. On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum. Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate. The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized. By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs. Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase. Acetate greatly enhances the acetyl CoA synthetase activity. Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities. Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented.

  18. TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

    PubMed

    Oro, Lucia; Zara, Severino; Fancellu, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Ciani, Maurizio; Comitini, Francesca

    2014-05-01

    Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a β-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding β-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

  19. Hydrothermal decomposition of yeast cells for production of proteins and amino acids.

    PubMed

    Lamoolphak, Wiwat; Goto, Motonobu; Sasaki, Mitsuru; Suphantharika, Manop; Muangnapoh, Chirakarn; Prommuag, Chattip; Shotipruk, Artiwan

    2006-10-11

    This study examines hydrothermal decomposition of Baker's yeast cells, used as a model for spent Brewer's yeast waste, into protein and amino acids. The reaction was carried out in a closed batch reactor at various temperatures between 100 and 250 degrees C. The reaction products were separated into water-soluble and solid residue. The results demonstrated that the amount of yeast residue decreased with increasing hydrolysis temperature. After 20 min reaction in water at 250 degrees C, 78% of yeast was decomposed. The highest amount of protein produced was also obtained at this condition and was found to be 0.16 mg/mg dry yeast. The highest amount of amino acids (0.063 mg/mg dry yeast) was found at the lowest temperature tested after 15 min. The hydrolysis product obtained at 200 degrees C was tested as a nutrient source for yeast growth. The growth of yeast cells in the culture medium containing 2 w/v% of this product was comparable to that of the cells grown in the medium containing commercial yeast extract at the same concentration. These results demonstrated the feasibility of using subcritical water to potentially decompose proteinaceous waste such as spent Brewer's yeast while recovering more useful products.

  20. Stochastic model of yeast cell-cycle network

    NASA Astrophysics Data System (ADS)

    Zhang, Yuping; Qian, Minping; Ouyang, Qi; Deng, Minghua; Li, Fangting; Tang, Chao

    2006-07-01

    Biological functions in living cells are controlled by protein interaction and genetic networks. These molecular networks should be dynamically stable against various fluctuations which are inevitable in the living world. In this paper, we propose and study a stochastic model for the network regulating the cell cycle of the budding yeast. The stochasticity in the model is controlled by a temperature-like parameter β. Our simulation results show that both the biological stationary state and the biological pathway are stable for a wide range of “temperature”. There is, however, a sharp transition-like behavior at βc, below which the dynamics are dominated by noise. We also define a pseudo energy landscape for the system in which the biological pathway can be seen as a deep valley.

  1. How cells recognize damaged DNA: Clues from xeroderma pigmentosum and yeast

    SciTech Connect

    Chu, G.; Chang, E.; Patterson, M. )

    1990-01-01

    Xeroderma pigmentosum (XP) is characterized by the defective excision repair of DNA damaged by many agents, including ultraviolet radiation (UV) and cisplatin. We have identified a factor in human cells that recognizes multiple forms of DNA damage and is absent in XP complementation group E. Denoted XPE binding factor, it is expressed at five-fold higher levels in tumor cell lines resistant to the antitumor drug cisplatin. Finally, although it does not have photoreactivating activity, XPE binding factor shares multiple binding characteristics with yeast photolyase, suggesting that it is the human homolog of photolyase.

  2. Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

    PubMed

    Makushok, Tatyana; Alves, Paulo; Huisman, Stephen Michiel; Kijowski, Adam Rafal; Brunner, Damian

    2016-05-19

    Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.

  3. Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

    PubMed

    Takagi, Toshiyuki; Yokoi, Takahiro; Shibata, Toshiyuki; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae.

  4. eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

    PubMed Central

    Menacho-Márquez, Mauricio; Rodríguez-Hernández, Carlos J; Villaronga, M Ángeles; Pérez-Valle, Jorge; Gadea, José; Belandia, Borja; Murguía, José R

    2015-01-01

    β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses. PMID:25590579

  5. Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

    PubMed Central

    Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

    2013-01-01

    SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

  6. Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

    SciTech Connect

    Sifontes, Ángela B.; González, Gema; Tovar, Leidy M.; Méndez, Franklin J.; Gomes, Maria E.; Cañizales, Edgar; Niño-Vega, Gustavo; Villalobos, Hector; Brito, Joaquin L.

    2013-02-15

    Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m{sup 2} g{sup −1}. -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N{sub 2} adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity, respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m{sup 2} g{sup −1}.

  7. A mitochondria-targeting artemisinin derivative with sharply increased antitumor but depressed anti-yeast and anti-malaria activities

    PubMed Central

    Sun, Chen; Cao, Yu; Zhu, Pan; Zhou, Bing

    2017-01-01

    The potent anti-malarial drug artemisinins are additionally anti-tumorigenic and inhibitory to yeast growth. The action mechanism of artemisinins, however, is not well understood. Heme and mitochondrial membrane are both suggested to be involved in the action of artemisinins. Because heme is also synthesized in the mitochondrion, mitochondria appear to be a critical organelle for artemisinins’ activities. In this study, we synthesized a mitochondria-targeting artemisinin derivative by conjugating triphenylphosphonium (TPP) to artelinic acid (ARTa). ARTa-TPP displays far more potent anti-tumorigenic activity than its parent compound. In contrast, ARTa-TPP is much less active against yeast respiration growth and malarial parasites. Notably, ARTa-TPP is also associated with increased toxicity to other kinds of control mammalian cells. These results suggest divergent action modes for artemisinins against cancer cells and malaria or yeast cells. We conclude that mitochondrial targeting could substantially elevate the anticancer potency of artemisinins, but the accompanied increased toxicity to normal cells raises an alert. The mechanism regarding the opposing effects of TPP conjugation to ARTa on its anticancer and anti-malarial/anti-yeast potencies is discussed based on our current understandings of artemisinins’ action. PMID:28368011

  8. A mitochondria-targeting artemisinin derivative with sharply increased antitumor but depressed anti-yeast and anti-malaria activities.

    PubMed

    Sun, Chen; Cao, Yu; Zhu, Pan; Zhou, Bing

    2017-04-03

    The potent anti-malarial drug artemisinins are additionally anti-tumorigenic and inhibitory to yeast growth. The action mechanism of artemisinins, however, is not well understood. Heme and mitochondrial membrane are both suggested to be involved in the action of artemisinins. Because heme is also synthesized in the mitochondrion, mitochondria appear to be a critical organelle for artemisinins' activities. In this study, we synthesized a mitochondria-targeting artemisinin derivative by conjugating triphenylphosphonium (TPP) to artelinic acid (ARTa). ARTa-TPP displays far more potent anti-tumorigenic activity than its parent compound. In contrast, ARTa-TPP is much less active against yeast respiration growth and malarial parasites. Notably, ARTa-TPP is also associated with increased toxicity to other kinds of control mammalian cells. These results suggest divergent action modes for artemisinins against cancer cells and malaria or yeast cells. We conclude that mitochondrial targeting could substantially elevate the anticancer potency of artemisinins, but the accompanied increased toxicity to normal cells raises an alert. The mechanism regarding the opposing effects of TPP conjugation to ARTa on its anticancer and anti-malarial/anti-yeast potencies is discussed based on our current understandings of artemisinins' action.

  9. Enhanced leavening properties of baker's yeast by reducing sucrase activity in sweet dough.

    PubMed

    Zhang, Cui-Ying; Lin, Xue; Feng, Bing; Liu, Xiao-Er; Bai, Xiao-Wen; Xu, Jia; Pi, Li; Xiao, Dong-Guang

    2016-07-01

    Leavening ability in sweet dough is required for the commercial applications of baker's yeast. This property depends on many factors, such as glycolytic activity, sucrase activity, and osmotolerance. This study explored the importance of sucrase level on the leavening ability of baker's yeast in sweet dough. Furthermore, the baker's yeast strains with varying sucrase activities were constructed by deleting SUC2, which encodes sucrase or replacing the SUC2 promoter with the VPS8/TEF1 promoter. The results verify that the sucrase activity negatively affects the leavening ability of baker's yeast strains under high-sucrose conditions. Based on a certain level of osmotolerance, sucrase level plays a significant role in the fermentation performance of baker's yeast, and appropriate sucrase activity is an important determinant for the leavening property of baker's yeast in sweet dough. Therefore, modification on sucrase activity is an effective method for improving the leavening properties of baker's yeast in sweet dough. This finding provides guidance for the breeding of industrial baker's yeast strains for sweet dough leavening. The transformants BS1 with deleted SUC2 genetic background provided decreased sucrase activity (a decrease of 39.3 %) and exhibited enhanced leavening property (an increase of 12.4 %). Such a strain could be useful for industrial applications.

  10. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared towards the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically-expressed enzymes. Here we show that the addit...

  11. [Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae].

    PubMed

    Komakhin, R A; Komakhina, V V

    2008-01-01

    Double-stranded DNA breaks are currently thought to initiate homologous DNA recombination during meiosis. These breaks are mediated by several proteins, the key protein is Spol1p. Spo11 proteins being encoded by the highly conserved orthologs of SPO11 are present in most eukaryotes ranging from plants to man and are structurally similar to the subunit A of the archaea topoisomerase VI. The SPO11 of S. cerevisiae is currently known to be expressed during prophase I. It encodes a topoisomerase II that is apparently active as a dimer. Neither its localization in the native cells nor its nuclear localisation signals have been described in the literature. We report the expression of the coding region of SPO11 and its truncated variants C-terminally tagged by the egfp reporter in yeast. As judged by the EGFP fluorescence, the Spo11 p-EGFP fusion was localized in vegetative yeast nuclei whereas Spo11pdelta-EGFP lacking 25 N-terminal amino acids of Spollp was localized in cytoplasm. Nineteen N-terminal amino acids of Spo11p fused to EGFP made some reporter to be localized in the nucleus. Thus, we conclude that N-terminal part of Spo11p is a nuclear localization signal that is not specific for prophase I and is used to import proteins in vegetative yeast cells.

  12. Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum.

    PubMed

    Abe, M; Nishida, I; Minemura, M; Qadota, H; Seyama, Y; Watanabe, T; Ohya, Y

    2001-07-20

    1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

  13. Methods of Synchronization of Yeast Cells for the Analysis of Cell Cycle Progression.

    PubMed

    Juanes, M Angeles

    2017-01-01

    Cell division is a fascinating and fundamental process that sustains life. By this process, unicellular organisms reproduce and multicellular organisms sustain development, growth, and tissue repair. Division of a mother cell gives rise to two daughter cells according to an ordered set of events within four successive phases called G1 (gap1), S (DNA Synthesis), G2 (gap2), and M (Mitosis) phase. How these different phases are orchestrated to ensure the physical separation of the two daughter cells is a tightly regulated process. Indeed, inappropriate cell division could lead to uncontrolled cell proliferation and ultimately to cancer. Saccharomyces cerevisiae is an excellent model system for unraveling the secrets of cell division. A large community of researchers has chosen budding yeast as a model because of its advantages: rapid growth in simple and economical media, tractable genetics, powerful biochemistry, cell biology, and proteomics approaches. Furthermore, the cell cycle mechanisms, as elucidated in yeast, are conserved in higher eukaryotes. The ability to synchronize and get large numbers of cells in a particular stage of the cell cycle is crucial to properly explore the mechanisms of the cell cycle. An overview of the most common yeast synchronization techniques has been compiled in this chapter.

  14. Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells.

    PubMed

    Spearman, Maureen; Chan, Sarah; Jung, Vince; Kowbel, Vanessa; Mendoza, Meg; Miranda, Vivian; Butler, Michael

    2016-09-10

    Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

  15. Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell.

    PubMed

    Christwardana, Marcelinus; Kwon, Yongchai

    2017-02-01

    Membraneless microbial fuel cell (MFC) employing new microbial catalyst formed as yeast cultivated from Saccharomyces cerevisiae and carbon nanotube (yeast/CNT) is suggested. To analyze its catalytic activity and performance and stability of MFC, several characterizations are performed. According to the characterizations, the catalyst shows excellent catalytic activities by facile transfer of electrons via reactions of NAD, FAD, cytochrome c and cytochrome a3, while it induces high maximum power density (MPD) (344mW·m(-2)). It implies that adoption of yeast induces increases in catalytic activity and MFC performance. Furthermore, MPD is maintained to 86% of initial value even after eight days, showing excellent MFC stability.

  16. Apple Can Act as Anti-Aging on Yeast Cells

    PubMed Central

    Palermo, Vanessa; Mattivi, Fulvio; Silvestri, Romano; La Regina, Giuseppe; Falcone, Claudio; Mazzoni, Cristina

    2012-01-01

    In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrence of cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such as ascorbic acid (vitamin C) and polyphenols. The substances that hinder the presence of free radicals are also able to protect cells from aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples and their components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate that all the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all apple components. PMID:22970337

  17. Effect of source-separated urine storage on estrogenic activity detected using bioluminescent yeast Saccharomyces cerevisiae.

    PubMed

    Jaatinen, Sanna; Kivistö, Anniina; Palmroth, Marja R T; Karp, Matti

    2016-09-01

    The objective was to demonstrate that a microbial whole cell biosensor, bioluminescent yeast, Saccharomyces cerevisiae (BMAEREluc/ERα) can be applied to detect overall estrogenic activity from fresh and stored human urine. The use of source-separated urine in agriculture removes a human originated estrogen source from wastewater influents, subsequently enabling nutrient recycling. Estrogenic activity in urine should be diminished prior to urine usage in agriculture in order to prevent its migration to soil. A storage period of 6 months is required for hygienic reasons; therefore, estrogenic activity monitoring is of interest. The method measured cumulative female hormone-like activity. Calibration curves were prepared for estrone, 17β-estradiol, 17α- ethinylestradiol and estriol. Estrogen concentrations of 0.29-29,640 μg L(-1) were detectable while limit of detection corresponded to 0.28-35 μg L(-1) of estrogens. The yeast sensor responded well to fresh and stored urine and gave high signals corresponding to 0.38-3,804 μg L(-1) of estrogens in different urine samples. Estrogenic activity decreased during storage, but was still higher than in fresh urine implying insufficient storage length. The biosensor was suitable for monitoring hormonal activity in urine and can be used in screening anthropogenic estrogen-like compounds interacting with the receptor.

  18. The occurrence of killer activity in yeasts isolated from natural habitats.

    PubMed

    Wójcik, Monika; Kordowska-Wiater, Monika

    2015-01-01

    Yeast's ability to restrict the growth and kill other yeasts, fungi and bacteria has been known for over 50 years. Killer activity was detected in yeasts deposited in the world collections or isolated from natural habitats. In this study, isolates from the forest environment, leaves of fruit trees, flower petals, cereals and frozen fruit have been screened in terms of their killer activities. Killer activity was tested on strains belonging to six yeast species: Candida, Rhodotorula, Pichia, Pachysolen, Yarrowia, Trichosporon. The reference strains were Kluyveromyces lactis Y-6682 and Kluyveromyces marxinanus Y-8281, well-known to be sensitive to yeast killer toxins. Among one hundred and two tested strains, 24 (23.5% of isolates) showed positive killer action, and 10 (9.8% of the isolates) a weak killer action against at least one sensitive reference strain. The highest killer activity was observed among isolates from forest soil and flowers.

  19. Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.

    PubMed

    Leshin, Jonathan A; Rakauskaitė, Rasa; Dinman, Jonathan D; Meskauskas, Arturas

    2010-01-01

    One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

  20. Continuous beer fermentation using immobilized yeast cell bioreactor systems.

    PubMed

    Brányik, Tomás; Vicente, António A; Dostálek, Pavel; Teixeira, José A

    2005-01-01

    Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO(2) removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.

  1. Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences.

    PubMed Central

    Tan, S; Ammerer, G; Richmond, T J

    1988-01-01

    Pheromone receptor transcription factor (PRTF) and MAT alpha 1 are protein transcription factors that are involved in the regulation of the alpha-specific genes in Saccharomyces cerevisiae. We have expressed MAT alpha 1 as a fusion protein in Escherichia coli and purified it from inclusion bodies in milligram quantities. The MAT alpha 1 protein was obtained after specific cleavage of the fusion protein. Quantitative band shift electrophoresis was used to determine the equilibrium dissociation constants that describe the multicomponent binding equilibrium between the PRTF and MAT alpha 1 proteins, and alpha-specific STE3 upstream activating sequence (UAS) DNA. The dissociation constant for the complex of PRTF and the a-specific UAS of STE2 was also measured and found to be 5.9 X 10(-11) M, only three times less than that for the PRTF-STE3 UAS complex. Analyses of these complexes by DNase I footprinting demonstrate that the PRTF binding site is confined to the palindromic P-box sequence in the case of the STE3 UAS, but extends symmetrically from this central region to cover 28 bp for the STE2 UAS. When MAT alpha 1 is bound to the PRTF-STE3 complex, the region of DNA protected is enlarged to that seen for the PRTF-STE2 complex. Our results using these two purified factors in vitro suggest that PRTF has nearly the same affinity for a- and alpha-specific UAS elements and that transcriptional activation requires a particular conformational state for the PRTF-DNA complex which occurs in the PRTF-STE2 and MAT alpha 1-PRTF-STE3 complexes, but not in the PRTF-STE3 complex. Images PMID:2854061

  2. Fission Yeast Receptor of Activated C Kinase (RACK1) Ortholog Cpc2 Regulates Mitotic Commitment through Wee1 Kinase*

    PubMed Central

    Núñez, Andrés; Franco, Alejandro; Soto, Teresa; Vicente, Jero; Gacto, Mariano; Cansado, José

    2010-01-01

    In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G1/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G2/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G1/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes. PMID:20974849

  3. TORC1 activity is partially reduced under nitrogen starvation conditions in sake yeast Kyokai no. 7, Saccharomyces cerevisiae.

    PubMed

    Nakazawa, Nobushige; Sato, Aya; Hosaka, Masahiro

    2016-03-01

    Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells.

  4. Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

    PubMed

    Pericolini, Eva; Gabrielli, Elena; Ballet, Nathalie; Sabbatini, Samuele; Roselletti, Elena; Cayzeele Decherf, Amélie; Pélerin, Fanny; Luciano, Eugenio; Perito, Stefano; Jüsten, Peter; Vecchiarelli, Anna

    2017-01-02

    Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

  5. Live fast, die soon: cell cycle progression and lifespan in yeast cells

    PubMed Central

    Jiménez, Javier; Bru, Samuel; Ribeiro, Mariana; Clotet, Josep

    2015-01-01

    Our understanding of lifespan has benefited enormously from the study of a simple model, the yeast Saccharomyces cerevisiae. Although a unicellular organism, yeasts undergo many of the processes directly related with aging that to some extent are conserved in mammalian cells. Nutrient-limiting conditions have been involved in lifespan extension, especially in the case of caloric restriction, which also has a direct impact on cell cycle progression. In fact, other environmental stresses (osmotic, oxidative) that interfere with normal cell cycle progression also influence the lifespan of cells, indicating a relationship between lifespan and cell cycle control. In the present review we compile and discuss new findings related to how cell cycle progression is regulated by other nutrients. We centred this review on the analysis of phosphate, also give some attention to nitrogen, and the impact of these nutrients on lifespan. PMID:28357278

  6. Response to oxidative stress in Paracoccidioides yeast cells as determined by proteomic analysis.

    PubMed

    de Arruda Grossklaus, Daciene; Bailão, Alexandre Melo; Vieira Rezende, Tereza Cristina; Borges, Clayton Luiz; de Oliveira, Milton Adriano Pelli; Parente, Juliana Alves; de Almeida Soares, Célia Maria

    2013-05-01

    An efficient oxidative stress response is important to the fungal pathogen Paracoccidioides to survive within the human host. In this study, oxidative stress was mimicked by exposure of yeast cells to hydrogen peroxide (2 mM H2O2). To investigate the effect of H2O2 on the proteome of Paracoccidioides, we used a large scale 2-DE protein gel electrophoresis approach to analyze differentially expressed proteins/isoforms that were detected in early (2 h) and in late (6 h) oxidative stress treatments. All proteins/isoforms were grouped based on their functional categories that revealed a global activation of antioxidant enzymes, such as catalase, superoxide dismutase, cytochrome C peroxidase and thioredoxin. A view of the metabolic cell profile, as determined by proteomics, depicted a shift in the yeast cells metabolism as suggested by the activation of the pentose phosphate pathway, a great source of cellular reducing power in the form of NADPH. Additionally, in silico analyzes depicted 34 oxidoreductases proteins/isoforms putatively involved with defense against oxidative stress. Confirmatory assays of enzymatic activity, flow cytometry, transcript levels and NADPH measurements, produced data in agreement with proteomic analysis.

  7. A Highly Conserved Kinase Is an Essential Component for Stress Tolerance in Yeast and Plant Cells

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Hee; van Montagu, Marc; Verbruggen, Nathalie

    1999-05-01

    Osmotic stress (drought, salt stress) is a major limiting factor for crop productivity in the world. Because cellular responses to osmotic stress are thought to be conserved in eukaryotes and because yeast is much more amenable than plants to genetic research, a functional strategy has been performed to identify limiting steps in osmotolerance of plants based on the complementation of yeast with a plant library. A new plant cDNA that encodes a functional homologue of the yeast Dbf2 kinase enhances salt, drought, cold, and heat tolerance upon overexpression in yeast as well as in transgenic plant cells.

  8. Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability.

    PubMed

    Patterson, Melissa N; Scannapieco, Alison E; Au, Pak Ho; Dorsey, Savanna; Royer, Catherine A; Maxwell, Patrick H

    2015-10-01

    Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.

  9. Beta-glucan-depleted, glycopeptide-rich extracts from Brewer's and Baker's yeast (Saccharomyces cerevisiae) lower interferon-gamma production by stimulated human blood cells in vitro.

    PubMed

    Williams, Roderick; Dias, Daniel A; Jayasinghe, Nirupama; Roessner, Ute; Bennett, Louise E

    2016-04-15

    Regulation of the human immune system requires controlled pro- and anti-inflammatory responses for host defence against infection and disease states. Yeasts (Saccharomyces cerevisiae), as used in brewing and baking, are mostly known for ability to stimulate the human immune-system predominantly reflecting the pro-inflammatory cell wall β-glucans. However, in this study, using food-compatible processing methods, glycopeptide-enriched and β-glucan-depleted products were each prepared from Brewer's and Baker's yeasts, which suppressed production of interferon-γ (IFN-γ) in human whole blood cell assay, signifying that anti-inflammatory factors are also present in yeast. Anti-inflammatory bioactivities of products prepared from Brewer's and Baker's yeast were compared with the commercial yeast product, Epicor®. While unfractionated Epicor was inactive, the C18 resin-binding fractions of Brewer's and Baker's yeast products and Epicor dose-dependently lowered IFN-γ, demonstrating that Epicor also contained both pro-inflammatory (β-glucans) and anti-inflammatory components. Anti-inflammatory activity was attributed to C18 resin-binding species glyco-peptides in Epicor and experimental yeast products. This study demonstrated that pro- and anti-inflammatory factors could be resolved and enriched in yeasts by suitable processing, with potential to improve specific activities.

  10. Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast

    PubMed Central

    Madrid, Marisa; Vázquez-Marín, Beatriz; Franco, Alejandro; Soto, Teresa; Vicente-Soler, Jero; Gacto, Mariano; Cansado, José

    2016-01-01

    In eukaryotic cells, the highly conserved Target of Rapamycin (TOR) and the Mitogen Activated Protein Kinase (MAPK) signaling pathways elicit adaptive responses to extra- and intracellular conditions by regulating essential cellular functions. However, the nature of the functional relationships between both pathways is not fully understood. In the fission yeast Schizosaccharomyces pombe the cell integrity MAPK pathway (CIP) regulates morphogenesis, cell wall structure and ionic homeostasis. We show that the Rab GTPase Ryh1, a TORC2 complex activator, cross-activates the CIP and its core member, the MAPK Pmk1, by two distinct mechanisms. The first one involves TORC2 and its downstream effector, Akt ortholog Gad8, which together with TORC1 target Psk1 increase protein levels of the PKC ortholog Pck2 during cell wall stress or glucose starvation. Also, Ryh1 activates Pmk1 in a TORC2-independent fashion by prompting plasma membrane trafficking and stabilization of upstream activators of the MAPK cascade, including PDK ortholog Ksg1 or Rho1 GEF Rgf1. Besides, stress-activated Pmk1 cross-inhibits Ryh1 signaling by decreasing the GTPase activation cycle, and this ensures cell growth during alterations in phosphoinositide metabolism. Our results reveal a highly intricate cross-regulatory relationship between both pathways that warrants adequate cell adaptation and survival in response to environmental changes. PMID:27876895

  11. Modelling of Yeast Mating Reveals Robustness Strategies for Cell-Cell Interactions

    PubMed Central

    Chen, Weitao; Nie, Qing; Yi, Tau-Mu; Chou, Ching-Shan

    2016-01-01

    Mating of budding yeast cells is a model system for studying cell-cell interactions. Haploid yeast cells secrete mating pheromones that are sensed by the partner which responds by growing a mating projection toward the source. The two projections meet and fuse to form the diploid. Successful mating relies on precise coordination of dynamic extracellular signals, signaling pathways, and cell shape changes in a noisy background. It remains elusive how cells mate accurately and efficiently in a natural multi-cell environment. Here we present the first stochastic model of multiple mating cells whose morphologies are driven by pheromone gradients and intracellular signals. Our novel computational framework encompassed a moving boundary method for modeling both a-cells and α-cells and their cell shape changes, the extracellular diffusion of mating pheromones dynamically coupled with cell polarization, and both external and internal noise. Quantification of mating efficiency was developed and tested for different model parameters. Computer simulations revealed important robustness strategies for mating in the presence of noise. These strategies included the polarized secretion of pheromone, the presence of the α-factor protease Bar1, and the regulation of sensing sensitivity; all were consistent with data in the literature. In addition, we investigated mating discrimination, the ability of an a-cell to distinguish between α-cells either making or not making α-factor, and mating competition, in which multiple a-cells compete to mate with one α-cell. Our simulations were consistent with previous experimental results. Moreover, we performed a combination of simulations and experiments to estimate the diffusion rate of the pheromone a-factor. In summary, we constructed a framework for simulating yeast mating with multiple cells in a noisy environment, and used this framework to reproduce mating behaviors and to identify strategies for robust cell-cell interactions. PMID

  12. Biological significance of nuclear localization of mitogen-activated protein kinase Pmk1 in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-07-27

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level.

  13. Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast*

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M. Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-01-01

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level. PMID:22685296

  14. Microchannel-free collection and single-cell isolation of yeast cells in a suspension using liquid standing wave

    NASA Astrophysics Data System (ADS)

    Matsutani, Akihiro; Takada, Ayako

    2016-11-01

    We demonstrate a microchannel-free collection method at nodes of liquid standing waves by the vertical vibration of a suspension including yeast cells. The pattern formation of the collection of cells using standing waves in a suspension was investigated by varying the frequency and waveform of vibrations. The single-cell isolation of yeast cells was achieved using a microenclosure array set at the nodes. In addition, we succeeded in the microchannel-free collection of yeast cells in a suspension, where patterns were formed by tapping vibration. The proposed technique is very simple and we believe that it will be useful for single-cell analysis and investigation.

  15. Zygosaccharomyces rouxii Trk1 is an efficient potassium transporter providing yeast cells with high lithium tolerance.

    PubMed

    Zimmermannova, Olga; Salazar, Ana; Sychrova, Hana; Ramos, Jose

    2015-06-01

    Zygosaccharomyces rouxii is an osmotolerant yeast growing in the presence of high concentrations of salts and/or sugars. The maintenance of intracellular potassium homeostasis is essential for osmostress adaptation. Zygosaccharomyces rouxii is endowed with only one typical potassium transporter (ZrTrk1). We characterized ZrTrk1 activity and its contribution to various physiological parameters in detail. Our results show that ZrTrk1 is a high-affinity K(+) transporting system efficiently discriminating between K(+) and Li(+) and indicate the presence of another, currently unknown K(+) importing system with a low affinity in Z. rouxii cells. Upon ZrTrk1 heterologous expression in Saccharomyces cerevisiae, it confers cells with a remarkably high lithium tolerance (even to wild-type strains) due to preventing Li(+) influx into cells, and is able to complement a plasma-membrane hyperpolarization and cell sensitivity to cationic compounds caused by the lack of endogenous K(+) transporters. Intracellular pH measurements with pHluorin, whose coding sequence was integrated into the genome, showed that the expression of ZrTrk1 also complements a decrease in intracellular pH in S. cerevisiae trk1Δ trk2Δ cells. Our data corroborate a tight connection between potassium and proton transporters in yeasts and provide new insights into Z. rouxii cation homeostasis and the basis of its high osmotolerance.

  16. Identification of yeast proteins necessary for cell-surface function of a potassium channel.

    PubMed

    Haass, Friederike A; Jonikas, Martin; Walter, Peter; Weissman, Jonathan S; Jan, Yuh-Nung; Jan, Lily Y; Schuldiner, Maya

    2007-11-13

    Inwardly rectifying potassium (Kir) channels form gates in the cell membrane that regulate the flow of K(+) ions into and out of the cell, thereby influencing the membrane potential and electrical signaling of many cell types, including neurons and cardiomyocytes. Kir-channel function depends on other cellular proteins that aid in the folding of channel subunits, assembly into tetrameric complexes, trafficking of quality-controlled channels to the plasma membrane, and regulation of channel activity at the cell surface. We used the yeast Saccharomyces cerevisiae as a model system to identify proteins necessary for the functional expression of a mammalian Kir channel at the cell surface. A screen of 376 yeast strains, each lacking one nonessential protein localized to the early secretory pathway, identified seven deletion strains in which functional expression of the Kir channel at the plasma membrane was impaired. Six deletions were of genes with known functions in trafficking and lipid biosynthesis (sur4Delta, csg2Delta, erv14Delta, emp24Delta, erv25Delta, and bst1Delta), and one deletion was of an uncharacterized gene (yil039wDelta). We provide genetic and functional evidence that Yil039wp, a conserved, phosphoesterase domain-containing protein, which we named "trafficking of Emp24p/Erv25p-dependent cargo disrupted 1" (Ted1p), acts together with Emp24p/Erv25p in cargo exit from the endoplasmic reticulum (ER). The seven yeast proteins identified in our screen likely impact Kir-channel functional expression at the level of vesicle budding from the ER and/or the local lipid environment at the plasma membrane.

  17. Aptamer-guided gene targeting in yeast and human cells

    PubMed Central

    Ruff, Patrick; Koh, Kyung Duk; Keskin, Havva; Pai, Rekha B.; Storici, Francesca

    2014-01-01

    Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting. PMID:24500205

  18. Performance of a Yeast-mediated Biological Fuel Cell

    PubMed Central

    Gunawardena, Anuradh; Fernando, Sandun; To, Filip

    2008-01-01

    Saccharomyces cerevisiae present in common Baker’s yeast was used in a microbial fuel cell in which glucose was the carbon source. Methylene blue was used as the electronophore in the anode compartment, while potassium ferricyanide and methylene blue were tested as electron acceptors in the cathode compartment. Microbes in a mediator-free environment were used as the control. The experiment was performed in both open and closed circuit configurations under different loads ranging from 100 kΩ to 400Ω. The eukaryotic S. cerevisiae-based fuel cell showed improved performance when methylene blue and ferricyanide were used as electron mediators, rendering a maximum power generation of 146.71±7.7 mW/m3. The fuel cell generated a maximum open circuit voltage of 383.6±1.5 mV and recorded a maximum efficiency of 28±1.8 % under 100 kΩ of external load. PMID:19325724

  19. MECHANISM OF GLUCOSE TRANSPORT ACROSS THE YEAST CELL MEMBRANE

    PubMed Central

    Cirillo, Vincent P.

    1962-01-01

    Cirillo, Vincent P. (Seton Hall College of Medicine and Dentistry, Jersey City, N.J.). Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84:485–491. 1962.—The kinetics of d-glucose and l-sorbose transport was studied in Saccharomyces cerevisiae inhibited with iodoacetic acid under nitrogen to prevent glucose metabolism. d-Glucose was found to compete with l-sorbose for a common membrane transport system with an apparent affinity greater than 25 times that of sorbose. A comparison of the net rate of glucose and sorbose transport at 50 and 500 mm external concentration showed that glucose transport is greater than that of sorbose from the lower concentration, but sorbose transport is greater than glucose at the higher concentration. This reversal of transport rate of two sugars with markedly different affinities is predicted by the membrane carrier theory. A further prediction of carrier theory was confirmed by the demonstration that the rate of glucose transport into fructose-loaded cells is greater than into unloaded cells. PMID:14021412

  20. Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells

    PubMed Central

    Hur, Jae H.; Bahadorani, Sepehr; Graniel, Jacqueline; Koehler, Christopher L.; Ulgherait, Matthew; Rera, Michael; Jones, D. Leanne; Walker, David W.

    2013-01-01

    A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging. PMID:24038661

  1. Killer activity of yeasts isolated from natural environments against some medically important Candida species.

    PubMed

    Vadkertiová, Renata; Sláviková, Elena

    2007-01-01

    Twenty-five yeast cultures, mainly of human origin, belonging to four pathogenic yeast species--Candida albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis were tested for their sensitivity to ten basidiomycetous and eleven ascomycetous yeast species isolated from the water and soil environments and from tree leaves. The best killer activity among basidiomycetous species was exhibited by Rhodotorula glutinis, and R. mucilaginosa. The other carotenoid producing species Cystofilobasidium capitatum, Sporobolomyces salmonicolor, and S. roseus were active only against about 40% of the tested strains and exhibited weak activity. The broadest killer activity among ascomycetous yeasts was shown by the strains Pichia anomala and Metschnikowia pulcherrima. The species Debaryomyces castellii, Debaryomyces hansenii, Hanseniaspora guilliermondii, Pichia membranifaciens, and Williopsis californica did not show any killer activity. The best killer activity exhibited the strains isolated from leafy material. The lowest activity pattern was found among strains originating from soil environment.

  2. Aminopyrrolic synthetic receptors for monosaccharides: a class of carbohydrate-binding agents endowed with antibiotic activity versus pathogenic yeasts.

    PubMed

    Nativi, Cristina; Francesconi, Oscar; Gabrielli, Gabriele; De Simone, Irene; Turchetti, Benedetta; Mello, Tommaso; Di Cesare Mannelli, Lorenzo; Ghelardini, Carla; Buzzini, Pietro; Roelens, Stefano

    2012-04-16

    The biological activity of a set of structurally related aminopyrrolic synthetic receptors for monosaccharides has been tested versus yeast and yeast-like microorganisms and compared to their binding affinity toward mannosides. Antibiotic activity comparable to that of well-known polyene (amphotericin B) or azole (ketoconazole) drugs has been found for some members of the family, along with a general correlation with binding abilities. A systematic structure-activity-affinity investigation shed light on the structural and functional requirements necessary for antibiotic activity and identified the tripodal compound 1 as the most potent compound of the set. Together with toxicity tests and inhibitor localization experiments performed through fluorescence microscopy, these studies led to the characterization of a new class of carbohydrate binding agents possessing antibiotic activity, in which pyrrolic groups precisely structured on a tripodal architecture appear to be responsible for permeability through the cell wall of pathogens, as well as for antibiotic activity inside the cytoplasm.

  3. Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis.

    PubMed

    Schoborg, Jennifer A; Hodgman, C Eric; Anderson, Mark J; Jewett, Michael C

    2014-05-01

    Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

  4. A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival*

    PubMed Central

    Beckley, Janel R.; Chen, Jun-Song; Yang, Yanling; Peng, Junmin; Gould, Kathleen L.

    2015-01-01

    Deubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival. PMID:26412298

  5. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species.

  6. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  7. Imaging of the Actin Cytoskeleton and Mitochondria in Fixed Budding Yeast Cells.

    PubMed

    Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is widely used as a model system to study the organization and function of the cytoskeleton. In the past, its small size, rounded shape, and rigid cell wall created obstacles to explore the cell biology of this model eukaryote. It is now possible to acquire and analyze high-resolution and super-resolution multidimensional images of the yeast cell. As a result, imaging of yeast has emerged as an important tool in eukaryotic cell biology. This chapter describes labeling methods and optical approaches for visualizing the cytoskeleton and interactions of the actin cytoskeleton with mitochondria in fixed yeast cells using wide-field and super-resolution fluorescence microscopy.

  8. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  9. Cell dualism: presence of cells with alternative membrane potentials in growing populations of bacteria and yeasts.

    PubMed

    Ivanov, Volodymyr; Rezaeinejad, Saeid; Chu, Jian

    2013-10-01

    It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ⁻-cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ⁺-cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC⁻) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ⁻-cells was 93.6 ± 1.8 % for bacteria and 90.4 ± 4.0 % for yeasts and the content of live Δψ⁺-cells was 0.9 ± 0.3 % for bacteria and 2.4 ± 0.7 % for yeasts. Hypothetically, existence of Δψ⁺-cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K⁺, Na⁺, and Ca²⁺ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ(⁻-cells to Δψ⁺-cells can be explained by slow influx of K⁺ ions into Δψ⁻-cell to the trigger level of K⁺ concentration ("compression of potassium spring"), which is forming "alternative" Δψ⁺-cell for a short period, following with fast efflux of K⁺ ions out of Δψ⁺-cell ("release of potassium spring") returning cell to normal Δψ⁻ state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ⁻- and Δψ⁺- cells.

  10. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  11. SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription.

    PubMed

    Sterner, David E; Belotserkovskaya, Rimma; Berger, Shelley L

    2002-09-03

    Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (HIS3 and TRP3), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated SPT7 resulted in derepressed HIS3 transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation.

  12. SUMO functions in constitutive transcription and during activation of inducible genes in yeast.

    PubMed

    Rosonina, Emanuel; Duncan, Sarah M; Manley, James L

    2010-06-15

    Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

  13. Using dielectrophoresis to study the dynamic response of single budding yeast cells to Lyticase.

    PubMed

    Tang, Shi-Yang; Yi, Pyshar; Soffe, Rebecca; Nahavandi, Sofia; Shukla, Ravi; Khoshmanesh, Khashayar

    2015-05-01

    Budding yeast cells are quick and easy to grow and represent a versatile model of eukaryotic cells for a variety of cellular studies, largely because their genome has been widely studied and links can be drawn with higher eukaryotes. Therefore, the efficient separation, immobilization, and conversion of budding yeasts into spheroplast or protoplast can provide valuable insight for many fundamentals investigations in cell biology at a single cell level. Dielectrophoresis, the induced motion of particles in non-uniform electric fields, possesses a great versatility for manipulation of cells in microfluidic platforms. Despite this, dielectrophoresis has been largely utilized for studying of non-budding yeast cells and has rarely been used for manipulation of budding cells. Here, we utilize dielectrophoresis for studying the dynamic response of budding cells to different concentrations of Lyticase. This involves separation of the budding yeasts from a background of non-budding cells and their subsequent immobilization onto the microelectrodes at desired densities down to single cell level. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts, in a highly dynamic process that depends on the concentration of Lyticase. We also introduce a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes.

  14. Extracellular electron transfer in yeast-based biofuel cells: A review.

    PubMed

    Hubenova, Yolina; Mitov, Mario

    2015-12-01

    This paper reviews the state-of-the art of the yeast-based biofuel cell research and development. The established extracellular electron transfer (EET) mechanisms in the presence and absence of exogenous mediators are summarized and discussed. The approaches applied for improvement of mediator-less yeast-based biofuel cells performance are also presented. The overview of the literature shows that biofuel cells utilizing yeasts as biocatalysts generate power density in the range of 20 to 2440 mW/m(2), which values are comparable with the power achieved when bacteria are used instead. The electrons' origin and the contribution of the glycolysis, fermentation, aerobic respiration, and phosphorylation to the EET are commented. The reported enhanced current generation in aerobic conditions presumes reconsideration of some basic MFC principles. The challenges towards the practical application of the yeast-based biofuel cells are outlined.

  15. Deposition of Coatings from Live Yeast Cells and Large Particles by “Convective-Sedimentation” Assembly

    PubMed Central

    Jerrim, Lindsey B.; Velev, Orlin D.

    2009-01-01

    Convective assembly at high volume fraction was used for the rapid deposition of uniform, close-packed coatings of Saccharomyces cerevisiae yeast cells onto glass slides. A computational model was developed to calculate the thickness profiles of such coatings for different set of conditions. Both the experiments and the numerical simulations demonstrated that the deposition process is strongly affected by the presence of sedimentation. The deposition device was inclined to increase the uniformity of the coatings by causing the cells to sediment toward the three-phase contact line. In accordance with the simulation, the experiments showed that both increasing the angle of the device and decreasing the angle between the slides increased the uniformity of the deposited coatings. Finally, the “convective-sedimentation” assembly method was used to deposit mixed layers of live cells and large latex particles as an example of immobilized biologically active composite coatings. PMID:19366200

  16. Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii.

    PubMed

    Ludovico, Paula; Sansonetty, Filipe; Silva, Manuel T; Côrte-Real, Manuela

    2003-03-01

    Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.

  17. How do yeast cells become tolerant to high ethanol concentrations?

    PubMed

    Snoek, Tim; Verstrepen, Kevin J; Voordeckers, Karin

    2016-08-01

    The brewer's yeast Saccharomyces cerevisiae displays a much higher ethanol tolerance compared to most other organisms, and it is therefore commonly used for the industrial production of bioethanol and alcoholic beverages. However, the genetic determinants underlying this yeast's exceptional ethanol tolerance have proven difficult to elucidate. In this perspective, we discuss how different types of experiments have contributed to our understanding of the toxic effects of ethanol and the mechanisms and complex genetics underlying ethanol tolerance. In a second part, we summarize the different routes and challenges involved in obtaining superior industrial yeasts with improved ethanol tolerance.

  18. Fast automated yeast cell counting algorithm using bright-field and fluorescence microscopic images

    PubMed Central

    2013-01-01

    Background The faithful determination of the concentration and viability of yeast cells is important for biological research as well as industry. To this end, it is important to develop an automated cell counting algorithm that can provide not only fast but also accurate and precise measurement of yeast cells. Results With the proposed method, we measured the precision of yeast cell measurements by using 0%, 25%, 50%, 75% and 100% viability samples. As a result, the actual viability measured with the proposed yeast cell counting algorithm is significantly correlated to the theoretical viability (R2 = 0.9991). Furthermore, we evaluated the performance of our algorithm in various computing platforms. The results showed that the proposed algorithm could be feasible to use with low-end computing platforms without loss of its performance. Conclusions Our yeast cell counting algorithm can rapidly provide the total number and the viability of yeast cells with exceptional accuracy and precision. Therefore, we believe that our method can become beneficial for a wide variety of academic field and industries such as biotechnology, pharmaceutical and alcohol production. PMID:24215650

  19. Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

    PubMed

    Chen, Ke-Quan; Li, Jian; Ma, Jiang-Feng; Jiang, Min; Wei, Ping; Liu, Zhong-Min; Ying, Han-Jie

    2011-01-01

    The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.

  20. Optical spectral analysis of ultra-weak photon emission from tissue culture and yeast cells

    NASA Astrophysics Data System (ADS)

    Nerudová, Michaela; Červinková, Kateřina; Hašek, Jiří; Cifra, Michal

    2015-01-01

    Optical spectral analysis of the ultra-weak photon emission (UPE) could be utilized for non-invasive diagnostic of state of biological systems and for elucidation of underlying mechanisms of UPE generation. Optical spectra of UPE from differentiated HL-60 cells and yeast cells (Saccharomyces cerevisiae) were investigated. Induced photon emission of neutrophil-like cells and spontaneous photon emission of yeast cells were measured using highly sensitive photomultiplier module Hamamatsu H7360-01 in a thermally regulated light-tight chamber. The respiratory burst of neutrophil-like HL-60 cells was induced with the PMA (phorbol 12-myristate, 13-acetate). PMA activates an assembly of NADPH oxidase, which induces a rapid formation of reactive oxygen species (ROS). Long-pass edge filters (wavelength 350, from 400 to 600 with 25 nm resolution and 650 nm) were used for optical spectral analysis. Propagation of error of indirect measurements and standard deviation were used to assess reliability of the measured spectra. Results indicate that the photon emission from both cell cultures is detectable in the six from eight examined wavelength ranges with different percentage distribution of cell suspensions, particularly 450-475, 475-500, 500-525, 525-550, 550-575 and 575-600 nm. The wavelength range of spectra from 450 to 550 nm coincides with the range of photon emission from triplet excited carbonyls (350-550 nm). The both cells cultures emitted photons in wavelength range from 550 to 600 nm but this range does not correspond with any known emitter. To summarize, we have demonstrated a clear difference in the UPE spectra between two organisms using rigorous methodology and error analysis.

  1. Peroxide Sensors for the Fission Yeast Stress-activated Mitogen-activated Protein Kinase Pathway

    PubMed Central

    Buck, Vicky; Quinn, Janet; Pino, Teresa Soto; Martin, Humberto; Saldanha, Jose; Makino, Kozo; Morgan, Brian A.; Millar, Jonathan B.A.

    2001-01-01

    The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast. PMID:11179424

  2. Yeast-like cell formation and glutathione metabolism in autolysing cultures of Penicillium chrysogenum.

    PubMed

    Pócsi, I; Molnár, Z; Pusztahelyi, T; Varecza, Z; Emri, T

    2007-12-01

    The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi.

  3. sup 31 P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

    SciTech Connect

    Brindle, K.; Braddock, P.; Fulton, S. )

    1990-04-03

    Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creating kinase activities similar to those found in mammalian heart muscle. {sup 31}P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts.

  4. Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

    PubMed Central

    Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2006-01-01

    During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994

  5. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform

    PubMed Central

    Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt

    2017-01-01

    With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar, a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. PMID:28179544

  7. Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform.

    PubMed

    Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt; Lhoussaine, Cédric; Hersen, Pascal; Batt, Gregory

    2017-02-01

    With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar, a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others.

  8. Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Petelinc, Tanja; Polak, Tomaž; Demšar, Lea; Jamnik, Polona

    2013-01-01

    We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds. PMID:23409133

  9. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    PubMed

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

  10. Cell size and budding during starvation of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Johnston, G C

    1977-01-01

    When starved for nitrogen, cells of the yeast Saccharomyces cerevisiae produced abnormally small cells. Nonetheless, during starvation, only cells of a size characteristic of growing cells were capable of initiating a bud. Even when growth was severely limited, some event(s) in G1 required growth to a critical size for completion. PMID:334753

  11. Gis1 and Rph1 regulate glycerol and acetate metabolism in glucose depleted yeast cells.

    PubMed

    Orzechowski Westholm, Jakub; Tronnersjö, Susanna; Nordberg, Niklas; Olsson, Ida; Komorowski, Jan; Ronne, Hans

    2012-01-01

    Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1.

  12. Gis1 and Rph1 Regulate Glycerol and Acetate Metabolism in Glucose Depleted Yeast Cells

    PubMed Central

    Nordberg, Niklas; Olsson, Ida; Komorowski, Jan; Ronne, Hans

    2012-01-01

    Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1. PMID:22363679

  13. Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

    PubMed

    Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

    2013-05-17

    Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

  14. Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur.

    PubMed

    Juntachai, Weerapong; Kajiwara, Susumu

    2015-10-01

    Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity.

  15. Cell-Cell Communication in Yeast Using Auxin Biosynthesis and Auxin Responsive CRISPR Transcription Factors.

    PubMed

    Khakhar, Arjun; Bolten, Nicholas J; Nemhauser, Jennifer; Klavins, Eric

    2016-04-15

    An engineering framework for synthetic multicellular systems requires a programmable means of cell-cell communication. Such a communication system would enable complex behaviors, such as pattern formation, division of labor in synthetic microbial communities, and improved modularity in synthetic circuits. However, it remains challenging to build synthetic cellular communication systems in eukaryotes due to a lack of molecular modules that are orthogonal to the host machinery, easy to reconfigure, and scalable. Here, we present a novel cell-to-cell communication system in Saccharomyces cerevisiae (yeast) based on CRISPR transcription factors and the plant hormone auxin that exhibits several of these features. Specifically, we engineered a sender strain of yeast that converts indole-3-acetamide (IAM) into auxin via the enzyme iaaH from Agrobacterium tumefaciens. To sense auxin and regulate transcription in a receiver strain, we engineered a reconfigurable library of auxin-degradable CRISPR transcription factors (ADCTFs). Auxin-induced degradation is achieved through fusion of an auxin-sensitive degron (from IAA corepressors) to the CRISPR TF and coexpression with an auxin F-box protein. Mirroring the tunability of auxin perception in plants, our family of ADCTFs exhibits a broad range of auxin sensitivities. We characterized the kinetics and steady-state behavior of the sender and receiver independently as well as in cocultures where both cell types were exposed to IAM. In the presence of IAM, auxin is produced by the sender cell and triggers deactivation of reporter expression in the receiver cell. The result is an orthogonal, rewireable, tunable, and, arguably, scalable cell-cell communication system for yeast and other eukaryotic cells.

  16. Saccharomyces cerevisiae Produces a Yeast Substance that Exhibits Estrogenic Activity in Mammalian Systems

    NASA Astrophysics Data System (ADS)

    Feldman, David; Stathis, Peter A.; Hirst, Margaret A.; Price Stover, E.; Do, Yung S.; Kurz, Walter

    1984-06-01

    Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

  17. Evaluation of the efficiency of different disruption methods on yeast cell wall preparation for β-glucan isolation.

    PubMed

    Bzducha-Wróbel, Anna; Błażejak, Stanisław; Kawarska, Anna; Stasiak-Różańska, Lidia; Gientka, Iwona; Majewska, Ewa

    2014-12-15

    Selected methods for yeast cell disruption were evaluated to establish their suitability for cell wall preparation in the process of β-glucan isolation. The effect of different disruption methods on contents of total saccharides, β-glucans and proteins in the produced cell walls preparations was analyzed. The degree of cell wall purification from intracellular components was established on the basis of the ratio of solubilised material. The investigated methods included: cell exposure to hot water (autoclaving), thermally-induced autolysis, homogenization in a bead mill, sonication and their combinations. Experimental systems were prepared in water (pH 5.0 and pH 7.0) and Tris-HCl buffer (pH 8.0). The Saccharomyces cerevisiae yeast cell wall preparations with the highest degree of cytosol component release and purification of β-glucans were produced by 30 min of cell homogenization with zirconium-glass beads (0.5 mm in diameter). This was confirmed by the highest ratio of solubilised material (approx. 64%-67%). The thus-produced preparations contained ca. 60% of total saccharides, 13%-14% of β(1,3)/(1,6)-glucans, and approx. 35% of crude proteins. Similar results were obtained after autolysis coupled with bead milling as well as with sonication, but the time required for these processes was more than 24 h. Homogenization in a bead mill could be valuable for general isolation procedures because allows one to eliminate the different autolytic activity of various yeast strains.

  18. Quantitative description of ion transport via plasma membrane of yeast and small cells

    PubMed Central

    Volkov, Vadim

    2015-01-01

    Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions. PMID:26113853

  19. Quantitative description of ion transport via plasma membrane of yeast and small cells.

    PubMed

    Volkov, Vadim

    2015-01-01

    Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions.

  20. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    PubMed

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  1. Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

    PubMed Central

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm

    2015-01-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity. PMID:25691663

  2. Metabolic regulation and maximal reaction optimization in the central metabolism of a yeast cell

    NASA Astrophysics Data System (ADS)

    Kasbawati, Gunawan, A. Y.; Hertadi, R.; Sidarto, K. A.

    2015-03-01

    Regulation of fluxes in a metabolic system aims to enhance the production rates of biotechnologically important compounds. Regulation is held via modification the cellular activities of a metabolic system. In this study, we present a metabolic analysis of ethanol fermentation process of a yeast cell in terms of continuous culture scheme. The metabolic regulation is based on the kinetic formulation in combination with metabolic control analysis to indicate the key enzymes which can be modified to enhance ethanol production. The model is used to calculate the intracellular fluxes in the central metabolism of the yeast cell. Optimal control is then applied to the kinetic model to find the optimal regulation for the fermentation system. The sensitivity results show that there are external and internal control parameters which are adjusted in enhancing ethanol production. As an external control parameter, glucose supply should be chosen in appropriate way such that the optimal ethanol production can be achieved. For the internal control parameter, we find three enzymes as regulation targets namely acetaldehyde dehydrogenase, pyruvate decarboxylase, and alcohol dehydrogenase which reside in the acetaldehyde branch. Among the three enzymes, however, only acetaldehyde dehydrogenase has a significant effect to obtain optimal ethanol production efficiently.

  3. Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.

    PubMed Central

    Menees, T M; Sandmeyer, S B

    1994-01-01

    Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription. Images PMID:7969160

  4. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

    2008-09-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

  5. Distribution of the trehalase activation response and the regulatory trehalase gene among yeast species.

    PubMed

    Soto, T; Fernández, J; Cansado, J; Vicente, J; Gacto, M

    1997-12-01

    In Saccharomyces cerevisiae and other yeasts the activity of regulatory trehalases increases in response to the addition of glucose and to thermal changes in the extracellular medium. We have performed an screening on the extent of this response among different representative yeast species and the results show that this ability is displayed only by a few members of the Saccharomycetaceae family. However, all yeasts examined contain a gene related to that coding for regulatory trehalase in S. cerevisiae. This finding reveals that the operational distinction between regulatory and nonregulatory trehalase in yeasts is not a property of the enzyme by itself but relays on the expression of accompanying mechanisms able to modulate trehalase activity.

  6. Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.

    PubMed

    Striebeck, Alexander; Robinson, David A; Schüttelkopf, Alexander W; van Aalten, Daan M F

    2013-09-11

    The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of ScMnn9 in complex with GDP and Mn(2+), defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, ScMnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for ScMnn9 'priming' α-1,6-mannosyltransferase activity. Strikingly, both the presence of the ScMnn9 protein and its product, but not ScMnn9 catalytic activity, are required to activate subsequent ScVan1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

  7. Yeast cell metabolism investigated by CO{_2} production and soft X-ray irradiation

    NASA Astrophysics Data System (ADS)

    Masini, A.; Batani, D.; Previdi, F.; Milani, M.; Pozzi, A.; Turcu, E.; Huntington, S.; Takeyasu, H.

    1999-01-01

    Results obtained using a new technique for studying cell metabolism are presented. The technique, consisting in CO2 production monitoring, has been applied to Saccharomyces cerevisiae yeast cells. Also the cells were irradiated using the soft X-ray laser-plasma source at Rutherford Appleton Laboratory with the aim of producing a damage of metabolic processes at the wall level, responsible for fermentation, without great interference with respiration, taking place in mitochondria, and DNA activity. The source was calibrated with PIN diodes and X-ray spectrometers and used Teflon stripes as target, emitting X-rays at about 0.9 keV, with a very low penetration in biological material. X-ray doses delivered to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. Immediately after irradiation, the damage to metabolic activity was measured again by monitoring CO2 production. Results showed a general reduction in gas production by irradiated samples, together with non-linear and non-monotone response to dose. There was also evidence of oscillations in cell metabolic activity and of X-ray induced changes in oscillation frequency.

  8. Yeast AMP-activated Protein Kinase Monitors Glucose Concentration Changes and Absolute Glucose Levels*

    PubMed Central

    Bendrioua, Loubna; Smedh, Maria; Almquist, Joachim; Cvijovic, Marija; Jirstrand, Mats; Goksör, Mattias; Adiels, Caroline B.; Hohmann, Stefan

    2014-01-01

    Analysis of the time-dependent behavior of a signaling system can provide insight into its dynamic properties. We employed the nucleocytoplasmic shuttling of the transcriptional repressor Mig1 as readout to characterize Snf1-Mig1 dynamics in single yeast cells. Mig1 binds to promoters of target genes and mediates glucose repression. Mig1 is predominantly located in the nucleus when glucose is abundant. Upon glucose depletion, Mig1 is phosphorylated by the yeast AMP-activated kinase Snf1 and exported into the cytoplasm. We used a three-channel microfluidic device to establish a high degree of control over the glucose concentration exposed to cells. Following regimes of glucose up- and downshifts, we observed a very rapid response reaching a new steady state within less than 1 min, different glucose threshold concentrations depending on glucose up- or downshifts, a graded profile with increased cell-to-cell variation at threshold glucose concentrations, and biphasic behavior with a transient translocation of Mig1 upon the shift from high to intermediate glucose concentrations. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching data demonstrate that Mig1 shuttles constantly between the nucleus and cytoplasm, although with different rates, depending on the presence of glucose. Taken together, our data suggest that the Snf1-Mig1 system has the ability to monitor glucose concentration changes as well as absolute glucose levels. The sensitivity over a wide range of glucose levels and different glucose concentration-dependent response profiles are likely determined by the close integration of signaling with the metabolism and may provide for a highly flexible and fast adaptation to an altered nutritional status. PMID:24627493

  9. Nitric Oxide-Mediated Antioxidative Mechanism in Yeast through the Activation of the Transcription Factor Mac1

    PubMed Central

    Nasuno, Ryo; Aitoku, Miho; Manago, Yuki; Nishimura, Akira; Sasano, Yu; Takagi, Hiroshi

    2014-01-01

    The budding yeast Saccharomyces cerevisiae possesses various defense mechanisms against environmental stresses that generate reactive oxygen species, leading to growth inhibition or cell death. Our recent study showed a novel antioxidative mechanism mediated by nitric oxide (NO) in yeast cells, but the mechanism underlying the oxidative stress tolerance remained unclear. We report here one of the downstream pathways of NO involved in stress-tolerance mechanism in yeast. Our microarray and real-time quantitative PCR analyses revealed that exogenous NO treatment induced the expression of genes responsible for copper metabolism under the control of the transcription factor Mac1, including the CTR1 gene encoding high-affinity copper transporter. Our ChIP analysis also demonstrated that exogenous NO enhances the binding of Mac1 to the promoter region of target genes. Interestingly, we found that NO produced under high-temperature stress conditions increased the transcription level of the CTR1 gene. Furthermore, NO produced during exposure to high temperature also increased intracellular copper content, the activity of Cu,Zn-superoxide dismutase Sod1, and cell viability after exposure to high-temperature in a manner dependent on Mac1. NO did not affect the expression of the MAC1 gene, indicating that NO activates Mac1 through its post-translational modification. Based on the results shown here, we propose a novel NO-mediated antioxidative mechanism that Mac1 activated by NO induces the CTR1 gene, leading to an increase in cellular copper level, and then Cu(I) activates Sod1. This is the first report to unveil the mechanism of NO-dependent antioxidative system in yeast. PMID:25423296

  10. Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells.

    PubMed

    Cuesta, Alberto; Rodríguez, Alejandro; Salinas, Irene; Meseguer, José; Esteban, M Angeles

    2007-03-01

    The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h post-injection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens.

  11. Effect of Evolutionary Adaption on Xylosidase Activity in Thermotolerant Yeast Isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3.

    PubMed

    Behera, Shuvashish; Sharma, Nilesh K; Arora, Richa; Kumar, Sachin

    2016-08-01

    Efficient use of xylose along with glucose is necessary for the economic production of lignocellulosic based biofuels. Xylose transporters play an important role in the microorganisms for efficient utilization of xylose. In the present study, a novel method has been developed for a rapid assay of xylose transport activity in the xylose-utilizing isolates and other known yeasts. An assay was conducted to compare the activity of β-xylosidase using p-nitrophenyl-β-D-xylopyranoside (pNPX) in the intact, intracellular, and extracellular yeasts cells showing xylose transporter. Saccharomyces cerevisiae (MTCC 170) showed no xylosidase activity, while little growth was observed in the xylose-containing medium. Although other yeasts, i.e., Kluyveromyces marxianus NIRE-K1 (MTCC 5933), K. marxianus NIRE-K3 (MTCC 5934), and Candida tropicalis (MTCC 230), showed xylosidase activity in intact, intracellular, and extracellular culture. The xylosidase activity in intact cell was higher than that of extracellular and intracellular activity in all the yeast cells. The enzyme activity was higher in case of K. marxianus NIRE-K1 and K. marxianus NIRE-K3 rather than the C. tropicalis. Further, better xylosidase activity was observed in adapted K. marxianus cells which were 2.79-28.46 % higher than that of native (non-adapted) strains, which indicates the significant improvement in xylose transportation.

  12. Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal.

    PubMed

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

    2012-04-01

    Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from "Mezcal de Oaxaca" were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave tequilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry.

  13. The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

    SciTech Connect

    Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P.; Nath, Utpal

    2011-07-01

    Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.

  14. Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

    PubMed Central

    Merín, María Gabriela; Martín, María Carolina; Rantsiou, Kalliopi; Cocolin, Luca; de Ambrosini, Vilma Inés Morata

    2015-01-01

    Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking. PMID:26413065

  15. Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape.

    PubMed

    Merín, María Gabriela; Martín, María Carolina; Rantsiou, Kalliopi; Cocolin, Luca; de Ambrosini, Vilma Inés Morata

    2015-01-01

    Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

  16. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells.

    PubMed

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti

  17. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    PubMed Central

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti

  18. Time-dependent profiling of metabolites from Snf1 mutant and wild type yeast cells.

    PubMed

    Humston, Elizabeth M; Dombek, Kenneth M; Hoggard, Jamin C; Young, Elton T; Synovec, Robert E

    2008-11-01

    The effect of sampling time in the context of growth conditions on a dynamic metabolic system was investigated in order to assess to what extent a single sampling time may be sufficient for general application, as well as to determine if useful kinetic information could be obtained. A wild type yeast strain (W) was compared to a snf1Delta mutant yeast strain (S) grown in high-glucose medium (R) and in low-glucose medium containing ethanol (DR). Under these growth conditions, different metabolic pathways for utilizing the different carbon sources are expected to be active. Thus, changes in metabolite levels relating to the carbon source in the growth medium were anticipated. Furthermore, the Snf1 protein kinase complex is required to adapt cellular metabolism from fermentative R conditions to oxidative DR conditions. So, differences in intracellular metabolite levels between the W and S yeast strains were also anticipated. Cell extracts were collected at four time points (0.5, 2, 4, 6 h) after shifting half of the cells from R to DR conditions, resulting in 16 sample classes (WR, WDR, SR, SDR) x (0.5, 2, 4, 6 h). The experimental design provided time course data, so temporal dependencies could be monitored in addition to carbon source and strain dependencies. Comprehensive two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOFMS) was used with discovery-based data mining algorithms ( Anal. Chem. 2006, 78, 5068-5075 (ref 1); J. Chromatogr., A 2008, 1186, 401-411 (ref 2)) to locate regions within the 2D chromatograms (i.e., metabolites) that provided chemical selectivity between the 16 sample classes. These regions were mathematically resolved using parallel factor analysis to positively identify the metabolites and to acquire quantitative results. With these tools, 51 unique metabolites were identified and quantified. Various time course patterns emerged from these data, and principal component analysis (PCA) was utilized as

  19. Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase.

    PubMed

    Helmstaedt, K; Krappmann, S; Braus, G H

    2001-09-01

    Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

  20. Ammonium Is Toxic for Aging Yeast Cells, Inducing Death and Shortening of the Chronological Lifespan

    PubMed Central

    Santos, Júlia

    2012-01-01

    Here we show that in aging Saccharomyces cerevisiae (budding yeast) cells, NH4+ induces cell death associated with shortening of chronological life span. This effect is positively correlated with the concentration of NH4+ added to the culture medium and is particularly evident when cells are starved for auxotrophy-complementing amino acids. NH4+-induced cell death is accompanied by an initial small increase of apoptotic cells followed by extensive necrosis. Autophagy is inhibited by NH4+, but this does not cause a decrease in cell viability. We propose that the toxic effects of NH4+ are mediated by activation of PKA and TOR and inhibition of Sch9p. Our data show that NH4+ induces cell death in aging cultures through the regulation of evolutionary conserved pathways. They may also provide new insights into longevity regulation in multicellular organisms and increase our understanding of human disorders such as hyperammonemia as well as effects of amino acid deprivation employed as a therapeutic strategy. PMID:22615903

  1. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    PubMed Central

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%. PMID:27213160

  2. Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

    PubMed

    Todorova, Atanaska; Pesheva, Margarita; Iliev, Ivan; Bardarov, Krum; Todorova, Teodora

    2017-02-01

    Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 μg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 μg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

  3. Anhydrobiosis in yeast: influence of calcium and magnesium ions on yeast resistance to dehydration-rehydration.

    PubMed

    Trofimova, Yuliya; Walker, Graeme; Rapoport, Alexander

    2010-07-01

    The influence of calcium and magnesium ions on resistance to dehydration in the yeast, Saccharomyces cerevisiae, was investigated. Magnesium ion availability directly influenced yeast cells' resistance to dehydration and, when additionally supplemented with calcium ions, this provided further significant increase of yeast resistance to dehydration. Gradual rehydration of dry yeast cells in water vapour indicated that both magnesium and calcium may be important for the stabilization of yeast cell membranes. In particular, calcium ions were shown for the first time to increase the resistance of yeast cells to dehydration in stress-sensitive cultures from exponential growth phases. It is concluded that magnesium and calcium ion supplementations in nutrient media may increase the dehydration stress tolerance of S. cerevisiae cells significantly, and this finding is important for the production of active dry yeast preparations for food and fermentation industries.

  4. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors

    PubMed Central

    Sasvari, Zsuzsanna; Alatriste Gonzalez, Paulina; Nagy, Peter D.

    2014-01-01

    To combat viral infections, plants possess innate and adaptive immune pathways, such as RNA silencing, R gene and recessive gene-mediated resistance mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF) are also involved in limiting plant virus replication. This review discusses novel CIRFs with antiviral functions, many of them RNA-binding proteins or affecting the RNA binding activities of viral replication proteins. The CIRFs against tombusviruses have been identified in yeast (Saccharomyces cerevisiae), which is developed as an advanced model organism. Grouping of the identified CIRFs based on their known cellular functions and subcellular localization in yeast reveals that TBSV replication is limited by a wide variety of host gene functions. Yeast proteins with the highest connectivity in the network map include the well-characterized Xrn1p 5′–3′ exoribonuclease, Act1p actin protein and Cse4p centromere protein. The protein network map also reveals an important interplay between the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. These restriction factors frequently target the RNA-binding region in the viral replication proteins, thus interfering with the recruitment of the viral RNA for replication and the assembly of the membrane-bound viral replicase. Although many of the characterized CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function as “guardians” of the cellular Hsp70 chaperone system, which is subverted efficiently by TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones. PMID:25157258

  5. Measurement of mass, density, and volume during the cell cycle of yeast

    PubMed Central

    Bryan, Andrea K.; Goranov, Alexi; Amon, Angelika; Manalis, Scott R.

    2010-01-01

    Cell growth comprises changes in both mass and volume—two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell’s three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications. PMID:20080562

  6. Yeast Recombination Enhancer Is Stimulated by Transcription Activation

    PubMed Central

    Ercan, Sevinc; Reese, Joseph C.; Workman, Jerry L.; Simpson, Robert T.

    2005-01-01

    Saccharomyces cerevisiae mating type switching is a gene conversion event that exhibits donor preference. MATa cells choose HMLα for recombination, and MATα cells choose HMRa. Donor preference is controlled by the recombination enhancer (RE), located between HMLα and MATa on the left arm of chromosome III. A number of a-cell specific noncoding RNAs are transcribed from the RE locus. Mcm1 and Fkh1 regulate RE activity in a cells. Here we show that Mcm1 binding is required for both the transcription of the noncoding RNAs and Fkh1 binding. This requirement can be bypassed by inserting another promoter into the RE. Moreover, the insertion of this promoter increases donor preference and opens the chromatin structure around the conserved domains of RE. Additionally, we determined that the level of Fkh1 binding positively correlates with the level of donor preference. We conclude that the role of Mcm1 in RE is to open chromatin around the conserved domains and activate transcription; this facilitates Fkh1 binding and the level of this binding determines the level of donor preference. PMID:16135790

  7. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis

    PubMed Central

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas

    2016-01-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

  8. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

    PubMed

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas; Klipp, Edda

    2016-09-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells.

  9. Monitoring the osmotic response of single yeast cells through force measurement in the environmental scanning electron microscope

    NASA Astrophysics Data System (ADS)

    Jansson, Anna; Nafari, Alexandra; Hedfalk, Kristina; Olsson, Eva; Svensson, Krister; Sanz-Velasco, Anke

    2014-02-01

    We present a measurement system that combines an environmental scanning electron microscope (ESEM) and an atomic force microscope (AFM). This combination enables studies of static and dynamic mechanical properties of hydrated specimens, such as individual living cells. The integrated AFM sensor provides direct and continuous force measurement based on piezoresistive force transduction, allowing the recording of events in the millisecond range. The in situ ESEM-AFM setup was used to study Pichia pastoris wild-type yeast cells. For the first time, a quantified measure of the osmotic response of an individual yeast cell inside an ESEM is presented. With this technique, cell size changes due to humidity variations can be monitored with nanometre accuracy. In addition, mechanical properties were extracted from load-displacement curves. A Young's modulus of 13-15 MPa was obtained for the P. pastoris yeast cells. The developed method is highly interesting as a complementary tool for the screening of drugs directed towards cellular water transport activity and provides new possibilities of studying mechanosensitive regulation of aquaporins.

  10. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

    PubMed Central

    Suauam, Pitipreya; Yingyongnarongkul, Boon-ek; Palaga, Tanapat; Miyakawa, Tokichi; Yompakdee, Chulee

    2015-01-01

    Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant. PMID:26313553

  11. Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast.

    PubMed Central

    Strich, Randy; Mallory, Michael J; Jarnik, Michal; Cooper, Katrina F

    2004-01-01

    Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute. PMID:15342503

  12. Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors.

    PubMed Central

    Gaudon, C; Chambon, P; Losson, R

    1999-01-01

    Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution. We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors. Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain. When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own. By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha. Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs. Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity. PMID:10205176

  13. Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.

    PubMed

    Sasano, Yu; Watanabe, Daisuke; Ukibe, Ken; Inai, Tomomi; Ohtsu, Iwao; Shimoi, Hitoshi; Takagi, Hiroshi

    2012-04-01

    Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.

  14. Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts.

    PubMed

    Fierro-Risco, Jesús; Rincón, Ana María; Benítez, Tahía; Codón, Antonio C

    2013-08-01

    Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.

  15. One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

    PubMed Central

    Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; Li, Song; Smith, Jennifer J.; Ratushny, Alexander V.; Aitchison, John D.

    2016-01-01

    Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAY extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations. PMID:27856698

  16. Permeabilization of yeast Saccharomyces cerevisiae cell walls using nanosecond high power electrical pulses

    NASA Astrophysics Data System (ADS)

    Stirke, A.; Zimkus, A.; Balevicius, S.; Stankevic, V.; Ramanaviciene, A.; Ramanavicius, A.; Zurauskiene, N.

    2014-12-01

    The electrical field-induced changes of the yeast Saccharomyces cerevisiae cells permeabilization to tetraphenylphosphonium (TPP+) ions were studied using square-shaped, nanosecond duration high power electrical pulses. It was obtained that pulses having durations ranging from 10 ns to 60 ns, and generating electric field strengths up to 190 kV/cm significantly (up to 65 times) increase the absorption rate of TPP+ ions without any detectible influence on the yeast cell viability. The modelling of the TPP+ absorption process using a second order rate equation demonstrates that depending on the duration of the pulses, yeast cell clusters of different sizes are homogeniously permeabilized. It was concluded, that nanosecond pulse-induced permeabilization can be applied to increase the operational speed of whole cell biosensors.

  17. Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae.

    PubMed

    Wloch-Salamon, D M; Bem, A E

    2013-02-01

    The occurrence of programmed cell death in unicellular organisms is a subject that arouses great interest of theoreticians and experimental scientists. Already found evolutionarily conserved genes and metabolic pathways confirmed its existence in yeast, protozoa and even bacteria. In the yeast Saccharomyces cerevisiae, at least three main types of death are distinguished: apoptosis, necrosis and autophagy. Their classification suggested by the Nomenclature Committee on Cell Death initially based on the morphological characteristics has now been extended to include the measurable biochemical characteristics. Several laboratory methods previously used to detect the types of cell death of higher eucaryotes and later developed and successfully used for the analysis of yeast cells are here critically reviewed. Their advantages and limitations are described.

  18. Functional genomics in the study of yeast cell polarity: moving in the right direction.

    PubMed

    Styles, Erin; Youn, Ji-Young; Mattiazzi Usaj, Mojca; Andrews, Brenda

    2013-01-01

    The budding yeast Saccharomyces cerevisiae has been used extensively for the study of cell polarity, owing to both its experimental tractability and the high conservation of cell polarity and other basic biological processes among eukaryotes. The budding yeast has also served as a pioneer model organism for virtually all genome-scale approaches, including functional genomics, which aims to define gene function and biological pathways systematically through the analysis of high-throughput experimental data. Here, we outline the contributions of functional genomics and high-throughput methodologies to the study of cell polarity in the budding yeast. We integrate data from published genetic screens that use a variety of functional genomics approaches to query different aspects of polarity. Our integrated dataset is enriched for polarity processes, as well as some processes that are not intrinsically linked to cell polarity, and may provide new areas for future study.

  19. Psychrotrophic yeast Yarrowia lipolytica NCYC 789 mediates the synthesis of antimicrobial silver nanoparticles via cell-associated melanin

    PubMed Central

    2013-01-01

    A psychrotrophic marine strain of the ascomycetous yeast Yarrowia lipolytica (NCYC 789) synthesized silver nanoparticles (AgNPs) in a cell-associated manner. These nanostructures were characterized by UV-Visible spectroscopy and scanning electron microscope-energy dispersive spectrometer (SEM-EDS) analysis. The brown pigment (melanin) involved in metal-interactions was obtained from the cells. This extracted pigment also mediated the synthesis of silver nanoparticles that were characterized by a variety of analytical techniques. The melanin-derived nanoparticles displayed antibiofilm activity. This paper thus reports the synthesis of AgNPs by the biotechnologically important yeast Y. lipolytica; proposes a possible mechanism involved in the synthetic process and describes the use of the bio-inspired nanoparticles as antibiofilm agents. PMID:23758863

  20. The SPA2 protein of yeast localizes to sites of cell growth

    PubMed Central

    1989-01-01

    A yeast gene, SPA2, was isolated with human anti-spindle pole autoantibodies. The SPA2 gene was fused to the Escherichia coli trpE gene, and polyclonal antibodies were prepared to the fusion protein. Immunofluorescence experiments indicate that the SPA2 gene product has a sharply polarized distribution in yeast cells. In budded cells the SPA2 protein is present at the tip of the bud; in unbudded cells, it is localized to one edge of the cell. When a-cells are induced to form schmoos with alpha-factor, the SPA2 protein is found at the tip of the schmoo. These areas of SPA2 localization correspond to cellular sites expected to be involved in bud formation and/or cell growth. The SPA2 antigen is present in a-cells, alpha-cells, and a/alpha-diploid cells, but is absent in mutant cells in which the SPA2 gene has been disrupted. spa2 mutant cells are viable, but display defects in the direction and control of cell growth. Compared to wild-type cells, spa2 mutant cells have slightly altered budding patterns. Entry into stationary phase is impaired for spa2 mutants, and mutants with one particular allele, spa2-7, form multiple buds under nutrient-limiting conditions. Thus, SPA2 is a newly identified yeast gene that is involved in the direction and control of cell division, and whose gene product localizes to the site of cell growth. PMID:2647769

  1. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

    PubMed Central

    Lazzarino, D A; Boldogh, I; Smith, M G; Rosand, J; Pon, L A

    1994-01-01

    Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis. Images PMID:7812049

  2. Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway.

    PubMed

    Salomon, Dor; Bosis, Eran; Dar, Daniel; Nachman, Iftach; Sessa, Guido

    2012-11-01

    The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) is the causal agent of speck disease in tomato. Pst pathogenicity depends on a type III secretion system that delivers effector proteins into host cells, where they promote disease by manipulating processes to the advantage of the pathogen. Previous studies identified seven Pst effectors that inhibit growth when expressed in yeast under normal growth conditions, suggesting that they interfere with cellular processes conserved in yeast and plants. We hypothesized that effectors also target conserved cellular processes that are required for yeast growth only under stress conditions. We therefore examined phenotypes induced by expression of Pst effectors in yeast grown in the presence of various stressors. Out of 29 effectors tested, five (HopX1, HopG1, HopT1-1, HopH1 and AvrPtoB) displayed growth inhibition phenotypes only in combination with stress conditions. Viability assays revealed that the HopX1 effector caused loss of cell viability under prolonged osmotic stress. Using transcription reporters, we found that HopX1 attenuated the activation of the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway, which is responsible for yeast survival under osmotic stress, while other MAPK pathways were mildly affected by HopX1. Interestingly, HopX1-mediated phenotypes in yeast were dependent on the putative transglutaminase catalytic triad of the effector. This study enlarges the pool of phenotypes available for the functional analysis of Pst type III effectors in yeast, and exemplifies how analysis of phenotypes detected in yeast under stress conditions can lead to the identification of eukaryotic cellular processes affected by bacterial effectors.

  3. Yeast clathrin has a distinctive light chain that is important for cell growth

    PubMed Central

    1990-01-01

    The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino- terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue- specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells. PMID:2211819

  4. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    PubMed Central

    Chaillot, Julien; Cook, Michael A.; Corbeil, Jacques; Sellam, Adnane

    2016-01-01

    One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. PMID:28040776

  5. Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering.

    PubMed

    Hasunuma, Tomohisa; Kondo, Akihiko

    2012-01-01

    To build an energy and material secure future, a next generation of renewable fuels produced from lignocellulosic biomass is required. Although lignocellulosic biomass, which represents an abundant, inexpensive and renewable source for bioethanol production, is of great interest as a feedstock, the complicated ethanol production processes involved make the cost of producing bioethanol from it higher compared to corn starch and cane juice. Therefore, consolidated bioprocessing (CBP), which combines enzyme production, saccharification and fermentation in a single step, has gained increased recognition as a potential bioethanol production system. CBP requires a highly engineered microorganism developed for several different process-specific characteristics. The dominant strategy for engineering a CBP biocatalyst is to express multiple components of a cellulolytic system from either fungi or bacteria in the yeast Saccharomyces cerevisiae. The development of recombinant yeast strains displaying cellulases and hemicellulases on the cell surface represents significant progress toward realization of CBP. Regardless of the process used for biomass hydrolysis, CBP-enabling microorganisms encounter a variety of toxic compounds produced during biomass pretreatment that inhibit microbial growth and ethanol yield. Systems biology approaches including disruptome screening, transcriptomics, and metabolomics have been recently exploited to gain insight into the molecular and genetic traits involved in tolerance and adaptation to the fermentation inhibitors. In this review, we focus on recent advances in development of yeast strains with both the ability to directly convert lignocellulosic material to ethanol and tolerance in the harsh environments containing toxic compounds in the presence of ethanol.

  6. Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase.

    PubMed

    Buchhaupt, Markus; Guder, Jan Christopher; Etschmann, Maria Magdalena Walburga; Schrader, Jens

    2012-01-01

    Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.

  7. Growth model and metabolic activity of brewing yeast biofilm on the surface of spent grains: a biocatalyst for continuous beer fermentation.

    PubMed

    Brányik, Tomás; Vicente, António A; Kuncová, Gabriela; Podrazký, Ondrej; Dostálek, Pavel; Teixeira, José A

    2004-01-01

    In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate (q(im)) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble-column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (X(im)act) of the total immobilized biomass (X(im)) was subsequently estimated at approximately X(im)act = 0.12 g(IB) g(C)(-1) (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell-to-carrier adhesion and cell-to-cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (X(im)act) and the maximum attainable biomass load (X(im)max) was included into the model. Since the average biofilm thickness was estimated at ca. 10 microm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble-column reactor. Good agreement between model simulations and experimental data was achieved.

  8. Pulse-transmission Oscillators: Autonomous Boolean Models and the Yeast Cell Cycle

    NASA Astrophysics Data System (ADS)

    Sevim, Volkan; Gong, Xinwei; Socolar, Joshua

    2010-03-01

    Models of oscillatory gene expression typically involve a constitutively expressed or positively autoregulated gene which is repressed by a negative feedback loop. In Boolean representations of such systems, which include the repressilator and relaxation oscillators, dynamical stability stems from the impossibility of satisfying all of the Boolean rules at once. We consider a different class of networks, in which oscillations are due to the transmission of a pulse of gene activation around a ring. Using autonomous Boolean modeling methods, we show how the circulating pulse can be stabilized by decoration of the ring with certain feedback and feed-forward motifs. We then discuss the relation of these models to ODE models of transcriptional networks, emphasizing the role of explicit time delays. Finally, we show that a network recently proposed as a generator of cell cycle oscillations in yeast contains the motifs required to support stable transmission oscillations.

  9. Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

    SciTech Connect

    Singh, Bhag; Gupta, Radhey S. . E-mail: gupta@mcmaster.ca

    2006-08-04

    Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.

  10. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  11. Immobilised cells of Pachysolen tannophilus yeast for ethanol production from crude glycerol.

    PubMed

    Stepanov, Nikolay; Efremenko, Elena

    2017-01-25

    Screening among naturally occurring yeast strains of Pachysolen spp. that are capable of producing ethanol from glycerol under aerobic conditions identified the most active culture, P. tannophilus Y -475. Conversion of glycerol by this producer immobilised in poly(vinyl alcohol) cryogel resulted in a 90% yield of ethanol relative to the theoretical limit. The maximum rate of alcohol accumulation was 0.64±0.01gL(-1)h(-1) at a 25gL(-1) concentration of glycerol in the culture medium. We demonstrated the efficacy of reusing immobilised cells (for a minimum of 16 working cycles for batch mode of crude glycerol conversion to ethanol) and the possibility of long-term (for a minimum of 140h) use of the cells in continuous mode with a maximum process productivity of 0.63±0.02gL(-1)h(-1), while the medium dilution rate in the reactor was 0.062±0.001h(-1). Reduction of metabolic activity did not exceed 5-7% relative to baseline. Immobilised cells were demonstrated to withstand long-term storage in frozen form for at least 2 years while retaining high metabolic activity.

  12. Nitrogen-dependent calcineurin activation in the yeast Hansenula polymorpha.

    PubMed

    Rodríguez, Celia; Galindo, Luis R; Siverio, José M

    2013-04-01

    Non-preferred nitrogen sources, unlike preferred ones, raised total cell Ca(2+) content and expression of ENA1, a very well-known calcineurin-regulated gene. This indicates calcineurin activation is regulated by nitrogen source. Nitrogen catabolite repression (NCR) and nitrate induction mechanisms, both regulating nitrate assimilation in Hansenula polymorpha, are controlled by calcineurin. Concerning NCR, lack of calcineurin (cnb1 mutant) decreased nitrate-assimilation gene expression, levels of the transcription factor Gat1 and growth in several nitrogen sources. We found that the role of calcineurin in NCR was mediated by Crz1 via Gat1. Regarding nitrate induction, calcineurin also affects the levels of transcription factors Gat2 and Yna2 involved in this process. We conclude that Ca(2+) and calcineurin play a central role in nitrogen signalling and assimilation. Thus, the nitrogen source modulates Ca(2+) content and calcineurin activation. Calcineurin in turn regulates nitrogen assimilation genes.

  13. Comparison of various techniques for determining viability of Paracoccidioides brasiliensis yeast-form cells.

    PubMed Central

    Restrepo, A; Cano, L E; de Bedout, C; Brummer, E; Stevens, D A

    1982-01-01

    The viability of Paracoccidioides brasiliensis yeast-form cells was determined by colony-forming units, direct fluorescent staining, and production of germ tubes in slide culture. The first procedure was unreliable and time consuming; the latter two showed better correlation with hemacytometer total cell counts and required significantly less time. PMID:7107858

  14. Proliferation enhancement of budding yeast and mammalian cells with periodic oxygen radical treatment

    NASA Astrophysics Data System (ADS)

    Mori, Yosuke; Kobayashi, Jun; Murata, Tomiyasu; Hahizume, Hiroshi; Hori, Masaru; Ito, Masafumi

    2015-09-01

    Recently, nonequilibrium atmospheric-pressure plasmas have been intensively studied for biological applications. However, the each effect of species in plasmas to biological tissue has not been clarified yet because various factors exist in the plasmas. Accordingly, we have studied effects of atomic oxygen dose on cell growth such as budding yeast and mouse NIH3T3 fibroblasts of mammalian cells. Both of cells were suspended with PBS, and treated using oxygen radical source. In order to prevent the radicals from reacting with the ambient air, the treatment region was surrounded by a plastic cover and purged with Ar. The proliferative effect of 15 % was observed at the O3Pj dose of around 1 . 0 ×1017 cm-3 in NIH3T3 cells as well as in yeast cells. Moreover, periodic oxygen treatment enhanced the effect in budding yeast cells. The best interval of periodic oxygen radical treatment was around 2 hours, which is almost the same period as that of their cell cycle. With the optimum interval time, we have investigated the effect of the number of the treatments. As the number of treatments increases, the growth rate of budding yeast cells was gradually enhanced and saturated at thrice treatments. This work was partly supported by JSPS KAKENHI Grant Numbers 26286072 and project for promoting Research Center in Meijo University.

  15. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  16. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    PubMed Central

    Vaz, Aline B. M.; Rosa, Luiz H.; Vieira, Mariana L. A.; de Garcia, Virginia; Brandão, Luciana R.; Teixeira, Lia C. R. S.; Moliné, Martin; Libkind, Diego; van Broock, Maria; Rosa, Carlos A.

    2011-01-01

    The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates. PMID:24031709

  17. Chromatin Assembly in a Yeast Whole-Cell Extract

    NASA Astrophysics Data System (ADS)

    Schultz, Michael C.; Hockman, Darren J.; Harkness, Troy A. A.; Garinther, Wendy I.; Altheim, Brent A.

    1997-08-01

    A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

  18. Aeromonas hydrophila typing scheme based on patterns of agglutination with erythrocytes and yeast cells.

    PubMed Central

    Adams, D; Atkinson, H M; Woods, W H

    1983-01-01

    An agglutination typing scheme has been developed for strains of Aeromonas hydrophila. Primary agglutination typing is based on testing agar-grown A. hydrophila cells with human, horse, rat, and guinea pig erythrocytes and Saccharomyces cerevisiae cells. Further subdivision of primary groups is based firstly on whether yeast cell agglutination is inhibited by a D-mannose polymer, yeast mannan, and secondly on patterns of inhibition of hemagglutination by yeast mannan and the monomeric sugars L-fucose, D-galactose, and D-mannose. A total of 320 isolates were tested, and these were divisible into 39 distinct types on the basis of this scheme. Application of this typing scheme in the future to isolates of A. hydrophila known to be associated with human infection may enable correlations to be made between particular agglutination types and human pathogenicity. PMID:6841579

  19. Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells.

    PubMed

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2013-01-01

    G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

  20. CDC37 is required for p60v-src activity in yeast.

    PubMed Central

    Dey, B; Lightbody, J J; Boschelli, F

    1996-01-01

    Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form. Images PMID:8885235

  1. Oxidative stress activates FUS1 and RLM1 transcription in the yeast Saccharomyces cerevisiae in an oxidant-dependent Manner.

    PubMed

    Staleva, Liliana; Hall, Andrea; Orlow, Seth J

    2004-12-01

    Mating in haploid Saccharomyces cerevisiae occurs after activation of the pheromone response pathway. Biochemical components of this pathway are involved in other yeast signal transduction networks. To understand more about the coordination between signaling pathways, we used a "chemical genetic" approach, searching for compounds that would activate the pheromone-responsive gene FUS1 and RLM1, a reporter for the cell integrity pathway. We found that catecholamines (l-3,4-hydroxyphenylalanine [l-dopa], dopamine, adrenaline, and noradrenaline) elevate FUS1 and RLM1 transcription. N-Acetyl-cysteine, a powerful antioxidant in yeast, completely reversed this effect, suggesting that FUS1 and RLM1 activation in response to catecholamines is a result of oxidative stress. The oxidant hydrogen peroxide also was found to activate transcription of an RLM1 reporter. Further genetic analysis combined with immunoblotting revealed that Kss1, one of the mating mitogen-activated protein kinases (MAPKs), and Mpk1, an MAPK of the cell integrity pathway, participated in l-dopa-induced stimulation of FUS1 and RLM1 transcription. We also report that Mpk1 and Hog1, the high osmolarity MAPK, were phosphorylated upon induction by hydrogen peroxide. Together, our results demonstrate that cells respond to oxidative stress via different signal transduction machinery dependent upon the nature of the oxidant.

  2. Mold-inhibitory activity of different yeast species during airtight storage of wheat grain.

    PubMed

    Adel Druvefors, Ulrika; Schnürer, Johan

    2005-02-01

    The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (<10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C. fennica and C. silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.

  3. Ethanol production potential from fermented rice noodle wastewater treatment using entrapped yeast cell sequencing batch reactor

    NASA Astrophysics Data System (ADS)

    Siripattanakul-Ratpukdi, Sumana

    2012-03-01

    Fermented rice noodle production generates a large volume of starch-based wastewater. This study investigated the treatment of the fermented rice noodle wastewater using entrapped cell sequencing batch reactor (ECSBR) compared to traditional sequencing batch reactor (SBR). The yeast cells were applied because of their potential to convert reducing sugar in the wastewater to ethanol. In present study, preliminary treatment by acid hydrolysis was performed. A yeast culture, Saccharomyces cerevisiae, with calcium alginate cell entrapment was used. Optimum yeast cell loading in batch experiment and fermented rice noodle treatment performances using ECSBR and SBR systems were examined. In the first part, it was found that the cell loadings (0.6-2.7 × 108 cells/mL) did not play an important role in this study. Treatment reactions followed the second-order kinetics with the treatment efficiencies of 92-95%. In the second part, the result showed that ECSBR performed better than SBR in both treatment efficiency and system stability perspectives. ECSBR maintained glucose removal of 82.5 ± 10% for 5-cycle treatment while glucose removal by SBR declined from 96 to 40% within the 5-cycle treatment. Scanning electron microscopic images supported the treatment results. A number of yeast cells entrapped and attached onto the matrix grew in the entrapment matrix.

  4. Effects of aging and heat treatment on whole yeast cells and yeast cell walls and on adsorption of ochratoxin A in a wine model system.

    PubMed

    Nunez, Y P; Pueyo, E; Carrascosa, A V; Martínez-Rodríguez, A J

    2008-07-01

    A wine model was evaluated to determine the influence of aging on the ability of whole yeast cells (WY) and yeast cell walls (YCW) to remove ochratoxin A (OTA). Aging and autolysis were monitored for 214 h in the model wine. The original concentration of OTA in the model wine was 10 microg/liter, and WY and YCW were added at a final concentration of 1 g/liter. YCW mannoproteins were involved in the removal of OTA from the model wine through adsorption mechanisms. Aging affected the capacity of WY to remove OTA, but YCW removal capacity remained constant during aging. A previous heat treatment (85 degrees C for 10 min) of WY and YCW increased their removal capacity and increased the efficiency of the decontamination process.

  5. The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

    PubMed

    Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

    2011-03-01

    Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

  6. Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk.

    PubMed

    Melville, Priscilla A; Benites, Nilson R; Ruz-Peres, Monica; Yokoya, Eugenio

    2011-11-01

    The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

  7. Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display

    PubMed Central

    Wen, Fei; Sethi, Dhruv K.; Wucherpfennig, Kai W.; Zhao, Huimin

    2011-01-01

    Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. PMID:21752831

  8. Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain.

    PubMed

    van Nierop, Sandra N E; Axcell, Barry C; Cantrell, Ian C; Rautenbach, Marina

    2008-10-01

    The brewing of beer involves two major biological systems, namely malted barley (malt) and yeast. Both malt and yeast show natural variation and assessing the impact of differing malts on yeast performance is important in the optimisation of the brewing process. Currently, the brewing industry uses well-established tests to assess malt quality, but these frequently fail to predict malt-associated problem fermentations, such as incomplete fermentations, premature yeast flocculation (PYF) and gushing of the final beer product. Antimicrobial compounds, and in particular antiyeast compounds in malt, may be one of the unknown and unmeasured malt factors leading to problem fermentations. In this study, the adaptation of antimicrobial assays for the determination of antiyeast activity in malt is described. Our adapted assay was able to detect differing antiyeast activities in nine malt samples. For this sample set, malts associated with PYF during fermentation and gushing activity in beer showed high antiyeast activity. Both PYF and gushing are malt quality issues associated with fungal infection of barley in the field which may result in elevated antimicrobial activity in the barley grain. Also, two more malts that passed the normal quality control tests were also observed to have high antiyeast activity and such malts must be considered as suspect. Based on our results, this assay is a useful measure of malt quality as it quantifies the antiyeast activity in malt which may adversely impact on brewery fermentation.

  9. The effect of cell density on the production of xylitol from D-xylose by yeast.

    PubMed

    Cao, N J; Tang, R; Gong, C S; Chen, L F

    1994-01-01

    The rate of xylitol production from D-xylose increased with increasing yeast cell density. The optimal temperature for xylitol production is 36 degrees C, and the optimal pH range is from 4.0 to 6.0. At high initial yeast cell concentration of 26 mg/mL, 210 g/L of xylitol was produced from 260 g/L of D-xylose after 96 h of incubation with an indicated yield of 81% of the theoretical value.

  10. [Production of plant-derived natural products in yeast cells - A review].

    PubMed

    Wang, Dong; Dai, Zhubo; Zhang, Xueli

    2016-03-04

    Plant-derived natural products (PNPs) have been widely used in pharmaceutical and nutritional fields. So far, the main method to produce PNPs is extracting them from their original plants, however, there remains lots of problems. With the concept of synthetic biology, construction of yeast cell factories for production of PNPs provides an alternative way. In this review, we will focus on PNPs' market and application, research progress for production of artemisinin, research progress for production of terpenes, alkaloids and polyunsaturated fatty acid (PUFAs) and recent technology development to give a brief introduction of construction of yeast cells for production of PNPs.

  11. Soft x-ray-controlled dose deposition in yeast cells: techniques, model, and biological assessment

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Batani, Dimitri; Conti, Aldo; Masini, Alessandra; Costato, Michele; Pozzi, Achille; Turcu, I. C. Edmond

    1996-12-01

    A procedure is presented to release soft x-rays onto yeast cell membrane allegedly damaging the resident enzymatic processes connected with fermentation. The damage is expected to be restricted to regulating fermentation processes without interference with respiration. By this technique fermentation is followed leading to CO2 production, and respiration resulting in global pressure measurements. A solid state pressure sensor system has been developed linked to a data acquisition system. Yeast cells cultures have been investigated at different concentrations and with different nutrients. A non-monotone response in CO2 production as a function of the delivered x-ray dose is observed.

  12. Nur1 Dephosphorylation Confers Positive Feedback to Mitotic Exit Phosphatase Activation in Budding Yeast

    PubMed Central

    Godfrey, Molly; Kuilman, Thomas; Uhlmann, Frank

    2015-01-01

    Substrate dephosphorylation by the cyclin-dependent kinase (Cdk)-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later on, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle. PMID:25569132

  13. Airlift-driven fibrous-bed bioreactor for continuous production of glucoamylase using immobilized recombinant yeast cells.

    PubMed

    Kilonzo, Peter; Margaritis, Argyrios; Bergougnou, Maurice

    2009-08-10

    Continuous production of a fungal glucoamylase by immobilized recombinant Saccharomyces cerevisiae strain C468 containing plasmid pGAC9. Yeast cells were immobilized on hydrophilic cotton cloth in an inverse internal loop airlift-driven bioreactor. Free-cell culture in the airlift and stirred tank bioreactors confirmed the plasmid instability of the recombinant yeast. Enhanced glucoamylase productivity and plasmid stability were observed both in the free and immobilized cell cultures in the airlift bioreactor system. The glucoamylase level of the free-cell culture in the airlift bioreactor was approximately 20% higher than that in the in stirred tank bioreactor due to high cell density (cell dry weight/volume of bioreactor) and fraction of the plasmid-carrying cells. A potentially high glucoamylase activity of 161U/L and a corresponding volumetric productivity of 3.5U/Lh were achieved when a cell density of approximately 85g/L (or 12.3g/g fiber) was attained in the fibrous-bed immobilized cell bioreactor system. The stable glucoamylase production was achieved after five generations, at which time a fraction of approximately 62% of the plasmid-carrying cells was realized in the immobilized cell system. Plasmid stability was increased for the immobilized cells during continuous culture at the operating dilution rate. The volumetric and specific productivities and fraction of plasmid-carrying cells in the immobilized cell system were higher than in the free-cell counterpart, however. This was in part due to the high viability (approximately 80%) in the immobilized cell system and the selective immobilization of the plasmid-carrying cells in the fibrous bed, and perhaps increased plasmid copy number.

  14. Production of Formaldehyde by Detergent-Treated Cells of a Methanol Yeast, Candida boidinii S2 Mutant Strain AOU-1

    PubMed Central

    Sakai, Yasuyoshi; Tani, Yoshiki

    1988-01-01

    Treatment of cells of a methanol yeast, Candida boidinii, with the cationic detergent cetyldimethylbenzyl-ammonium chloride (Cation M2) improved the production of formaldehyde. Formaldehyde production was improved twofold with respect to the initial amount of formaldehyde and 1.61-fold with respect to the final amount of formaldehyde after a 12-h reaction under optimized detergent treatment conditions. The treatment caused formaldehyde and formate dehydrogenases to leak out of the cells more rapidly than catalase, but there was no leakage of alcohol oxidase. The improvement in formaldehyde production was considered to be due to the increased permeability of yeast cell membranes and to lower activities of formaldehyde and formate dehydrogenases in Cation M2-treated cells than in intact cells. Changes in the ultrastructure of the cells were observed upon Cation M2 treatment. Several developed peroxisomes were observed in intact cells. After Cation M2 treatment, the cells were obviously damaged, and several peroxisomes seemed to have fused with each other. Images PMID:16347563

  15. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

    PubMed Central

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-01-01

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  16. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  17. Enhanced Direct Ethanol Production by Cofactor Optimization of Cell Surface-Displayed Xylose Isomerase in Yeast.

    PubMed

    Sasaki, Yusuke; Takagi, Toshiyuki; Motone, Keisuke; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2017-04-10

    Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d-xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC-displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co(2+) ), especially enhanced the activity by 46-fold. Co(2+) also contributed to d-xylose fermentation, and ethanol yields and xylose consumption rates were improved by 6.0- and 2.7-fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC-displaying yeast showed the faster d-xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co-culture system. A xylan-degrading yeast strain was established by displaying two kinds of xylanases; endo-1,4-β-xylanase (Xyn11B) from Saccharophagus degradans, and β-xylosidase (XlnD) from Aspergillus niger. The yeast co-culture system enabled fine-tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)-displaying yeast and XylC-displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10(13) : 1.39 × 10(13) : 2.78 × 10(13) molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co-culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. This article is protected by copyright. All rights reserved.

  18. Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

    PubMed

    Ashe, Mark P; Bill, Roslyn M

    2011-06-01

    Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

  19. Attachment of MAL32-encoded maltase on the outside of yeast cells improves maltotriose utilization.

    PubMed

    Dietvorst, J; Blieck, L; Brandt, R; Van Dijck, P; Steensma, H Y

    2007-01-01

    The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an alpha-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of alpha-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.

  20. Duplication of the Yeast Spindle Pole Body Once per Cell Cycle

    PubMed Central

    Rüthnick, Diana

    2016-01-01

    The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. Centrosomes and SPBs duplicate exactly once per cell cycle by mechanisms that use the mother structure as a platform for the assembly of the daughter. The conserved Sfi1 and centrin proteins are essential components of the SPB duplication process. Sfi1 is an elongated molecule that has, in its center, 20 to 23 binding sites for the Ca2+-binding protein centrin. In the yeast Saccharomyces cerevisiae, all Sfi1 N termini are in contact with the mother SPB whereas the free C termini are distal to it. During S phase and early mitosis, cyclin-dependent kinase 1 (Cdk1) phosphorylation of mainly serine residues in the Sfi1 C termini blocks the initiation of SPB duplication (“off” state). Upon anaphase onset, the phosphatase Cdc14 dephosphorylates Sfi1 (“on” state) to promote antiparallel and shifted incorporation of cytoplasmic Sfi1 molecules into the half-bridge layer, which thereby elongates into the bridge. The Sfi1 C termini of the two Sfi1 layers localize in the bridge center, whereas the N termini of the newly assembled Sfi1 molecules are distal to the mother SPB. These free Sfi1 N termini then assemble the new SPB in G1 phase. Recruitment of Sfi1 molecules into the anaphase SPB and bridge formation were also observed in Schizosaccharomyces pombe, suggesting that the Sfi1 bridge cycle is conserved between the two organisms. Thus, restricting SPB duplication to one event per cell cycle requires only an oscillation between Cdk1 kinase and Cdc14 phosphatase activities. This clockwork regulates the “on”/“off” state of the Sfi1-centrin receiver. PMID:26951196

  1. Dectin-1 mediates in vitro phagocytosis of Candida albicans yeast cells by retinal microglia.

    PubMed

    Maneu, Victoria; Yáñez, Alberto; Murciano, Celia; Molina, Andrés; Gil, María Luisa; Gozalbo, Daniel

    2011-10-01

    We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b(+) retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b(+) cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.

  2. Cell-to-cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community.

    PubMed

    Campbell, Kate; Vowinckel, Jakob; Ralser, Markus

    2016-09-01

    Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self-establishing metabolically cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in single-cell heterogeneity. Cells within SeMeCo communities showed to be highly heterogeneous in their stress tolerance, while the survival of each cell under heat or oxidative stress, was strongly determined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidant (H2 O2 , diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild-type populations. Moreover, stress heterogeneity did not establish as sole consequence of metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell to cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.

  3. Reliable cell cycle commitment in budding yeast is ensured by signal integration

    NASA Astrophysics Data System (ADS)

    Tang, Chao

    2014-03-01

    Cells have to make reliable decisions in response to external and/or internal signals that can be noisy and varying. For budding yeast Saccharomyces cerevisiae, cells decide whether and when to commit to cell division at the Start checkpoint. The decision is irreversible and has the physiological significance for coordinating cell growth with cell division. The trigger of the Start, the G1 cyclin Cln3 is a dynamic sensor of the nutrient and cellular conditions with low copy number and rapid turnover time. Here we quantitatively investigate how cells process the information from Cln3 to make the Start decision. By using an inducible Cln3 and monitoring the time cell waits before Start transition (G1 length), we find that G1 length is inversely proportional to Cln3 concentration, which implies that Start is triggered when the integration of Cln3 concentration over time exceeds certain threshold. We identify the Start repressor, Whi5 as the integrator. The instantaneous kinase activity of Cln3-Cdk1 is recorded over time on the phosphorylated Whi5, and the decision is made only when the phosphorylation level of Whi5 reaches a threshold. Furthermore, we find that Whi5 plays an important role for coordinating growth and division - cells modulate Whi5 level in different nutrient conditions to adjust the Start threshold. The strategy of signal integration, which reduces noise and minimizes error and uncertainty, has been found in decision-making behaviors of animals. Our work shows that it is adopted at the cellular level, suggesting a general design principle that may be widely implemented in decision-making and signaling systems.

  4. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    PubMed

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.

  5. A systems-level approach for metabolic engineering of yeast cell factories.

    PubMed

    Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

    2012-03-01

    The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.

  6. Isolation of two cell populations from yeast during high-level alcoholic fermentation that resemble quiescent and nonquiescent cells from the stationary phase on glucose.

    PubMed

    Benbadis, Laurent; Cot, Marlène; Rigoulet, Michel; Francois, Jean

    2009-12-01

    High-level production of bioethanol (140 g L(-1) in 45 h) in aerated fed-batch cultures of Saccharomyces cerevisiae was shown to be linked to the length of a production phase uncoupled to the growth. The induction of this phase was characterized by metabolic and morphologic changes reminiscent of those occurring in the stationary phase of growth on glucose. Global transcriptomic analysis of ethanol-stressed yeast cells in the uncoupling phase harboured features similar to those from stationary-phase cells on glucose. Two distinct cellular populations were isolated by Percoll density-gradient centrifugation in this uncoupling phase. The lower fraction was enriched by yeast cells that were mostly uniform in size and opalescent, containing a large amount of glycogen and trehalose, and exhibiting high respiratory activity. In contrast, the upper fraction was characterized by cells heterogeneous in size, with one to several small buds, which did not contain storage carbohydrates and which exhibited a poor respiratory competence while retaining a high relative glycolytic activity. These results are discussed in terms of a possible induction of a state similar to the quiescence state previously observed from yeast stationary-phase cultures, in response to ethanol toxicity, whose acquisition may be critical for performing high-level alcoholic fermentation.

  7. Central Roles of Small GTPases in the Development of Cell Polarity in Yeast and Beyond

    PubMed Central

    Park, Hay-Oak; Bi, Erfei

    2007-01-01

    Summary: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating. PMID:17347519

  8. Oscillatory Dynamics of Cell Cycle Proteins in Single Yeast Cells Analyzed by Imaging Cytometry

    PubMed Central

    Ball, David A.; Marchand, Julie; Poulet, Magaly; Baumann, William T.; Chen, Katherine C.; Tyson, John J.; Peccoud, Jean

    2011-01-01

    Progression through the cell division cycle is orchestrated by a complex network of interacting genes and proteins. Some of these proteins are known to fluctuate periodically during the cell cycle, but a systematic study of the fluctuations of a broad sample of cell-cycle proteins has not been made until now. Using time-lapse fluorescence microscopy, we profiled 16 strains of budding yeast, each containing GFP fused to a single gene involved in cell cycle regulation. The dynamics of protein abundance and localization were characterized by extracting the amplitude, period, and other indicators from a series of images. Oscillations of protein abundance could clearly be identified for Cdc15, Clb2, Cln1, Cln2, Mcm1, Net1, Sic1, and Whi5. The period of oscillation of the fluorescently tagged proteins is generally in good agreement with the inter-bud time. The very strong oscillations of Net1 and Mcm1 expression are remarkable since little is known about the temporal expression of these genes. By collecting data from large samples of single cells, we quantified some aspects of cell-to-cell variability due presumably to intrinsic and extrinsic noise affecting the cell cycle. PMID:22046265

  9. Chromosome or chromatin condensation leads to meiosis or apoptosis in stationary yeast (Saccharomyces cerevisiae) cells.

    PubMed

    Yang, Hui; Ren, Qun; Zhang, Zhaojie

    2006-12-01

    When starved of essential nutrients, yeast cells cease mitotic division and enter an alternative state called the 'stationary phase'. In this paper, we report that stationary cells enter two major pathways: meiosis and apoptosis. Using transmission electron microscopy, five types of cell were identified in the stationary phase: (1) cells with chromosome condensed nuclei; (2) cells with normal, homogeneously stained nuclei; (3) sporulated cells; (4) apoptotic cells, in which chromatin, but not individual chromosomes, was condensed; and (5) dead cells, in which nuclei and cytoplasm were degraded. Further evidence using live cell imaging and mutation analysis suggested that cells with condensed chromosomes underwent meiosis, whereas chromatin condensed cells underwent apoptotic cell death. Cells with homogeneous nuclei are believed to be in the true resting state and undergo cell death when starvation continues. Chromosome or chromatin condensation may serve as a hallmark of life or death for stationary cells.

  10. Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation

    PubMed Central

    Lin, Tien-chen; Neuner, Annett; Schlosser, Yvonne T; Scharf, Annette ND; Weber, Lisa; Schiebel, Elmar

    2014-01-01

    Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains. DOI: http://dx.doi.org/10.7554/eLife.02208.001 PMID:24842996

  11. Metabolism of benzoquinone by yeast cells and oxidative characteristics of corresponding hydroquinone: application to highly sensitive measurement of yeast cell density by using benzoquinone and a chemiluminescent probe.

    PubMed

    Tsukatani, Tadayuki; Ide, Seiji; Ukeda, Hiroyuki; Matsumoto, Kiyoshi

    2004-07-01

    The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2-*) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2 x 10(3) - 4.8 x 10(4) cells/ml. The detection limit was 4.8 x 10(2) cells/ml.

  12. Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

    PubMed

    Sharkey, Michael A; Chebbi, Ahmed; McDonnell, Kevin A; Staunton, Claire; Dowling, Denis P

    2015-06-07

    The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

  13. Antimitochondrial effects of thioacetamide and ethylenethiourea in human and yeast cell cultures.

    PubMed

    Diala, E; Mittwoch, U; Wilkie, D

    1980-07-01

    Cytological studies in the light microscope showed that thioacetamide (TAA) depressed the mitotic index in cultures of skin fibroblasts at the lowest concentrations used (100 μg/ml). At high concentration (1 mg/ml), TAA tended to cause aberration in nuclear morphology. Ethylenethiourea (ETU) had no effect on either mitotic index or nuclear morphology at 1 mg/ml. Fibroblast cultures treated with 1 mg/ml TAA and cultures grown in the presence of 2 mg/ml ETU were studied by electron microscopy. In some TAA-treated cells there was unfolding of the nuclear membrane and enlargement and granulation of the nucleolus, but these effects were not correlated. In all cells, TAA caused severe and characteristic damage to the majority of mitochondria, whether or not there were nuclear aberrations. The organelle showed extensive swelling of the cristae of the inner membrane and an increase in matrix density. Ultrastructure of other cell components appeared to be unaffected by this treatment. In ETU-treated cells some less severe swelling of inner mitochondrial membranes was seen and only in a minority of cells, whilst all other cell structures appeared normal. Similar membrane swelling and increase in matrix density was seen in isolated rat liver mitochondria after incubation with TAA, indicating a direct antimitochondrial effect of the carcinogen.When yeast cells were treated with TAA and ETU, primary antimitochondrial activity of these compounds was apparent from (1) inhibition of growth in non-fermentable medium, (2) selective blockage of mitochondrial protein synthesis and (3) induction of mitochondrial mutations. TAA was much more effective than ETU in all these respects.

  14. “In vitro” antifungal activity of ozonized sunflower oil on yeasts from onychomycosis

    PubMed Central

    Guerrer, L.V.; Cunha, K. C.; Nogueira, M. C. L.; Cardoso, C. C.; Soares, M. M. C. N.; Almeida, M. T. G.

    2012-01-01

    The “in vitro” antifungal activity of ozonized sunflower oil (Bioperoxoil®) was tested on 101 samples of yeasts originating from onychomycosis using the disk diffusion method. The oil was efficacious against several clinical fungal strains: Candida parapsilosis, Candida albicans, Trichosporon asahii, Candida tropicalis and Candida guilliermondii. PMID:24031958

  15. Catalase activity is stimulated by H(2)O(2) in rich culture medium and is required for H(2)O(2) resistance and adaptation in yeast.

    PubMed

    Martins, Dorival; English, Ann M

    2014-01-01

    Catalases are efficient scavengers of H2O2 and protect cells against H2O2 stress. Examination of the H2O2 stimulon in Saccharomyces cerevisiae revealed that the cytosolic catalase T (Ctt1) protein level increases 15-fold on H2O2 challenge in synthetic complete media although previous work revealed that deletion of the CCT1 or CTA1 genes (encoding peroxisomal/mitochondrial catalase A) does not increase the H2O2 sensitivity of yeast challenged in phosphate buffer (pH 7.4). This we attributed to our observation that catalase activity is depressed when yeast are challenged with H2O2 in nutrient-poor media. Hence, we performed a systematic comparison of catalase activity and cell viability of wild-type yeast and of the single catalase knockouts, ctt1∆ and cta1∆, following H2O2 challenge in nutrient-rich medium (YPD) and in phosphate buffer (pH 7.4). Ctt1 but not Cta1 activity is strongly induced by H2O2 when cells are challenged in YPD but suppressed when cells are challenged in buffer. Consistent with the activity results, exponentially growing ctt1∆ cells in YPD are more sensitive to H2O2 than wild-type or cta1∆ cells, whereas in buffer all three strains exhibit comparable H2O2 hypersensitivity. Furthermore, catalase activity is increased during adaptation to sublethal H2O2 concentrations in YPD but not in buffer. We conclude that induction of cytosolic Ctt1 activity is vital in protecting yeast against exogenous H2O2 but this activity is inhibited by H2O2 when cells are challenged in nutrient-free media.

  16. An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells.

    PubMed

    Patterson, James O; Swaffer, Matthew; Filby, Andrew

    2015-07-01

    Fission yeast (Schizosaccharomyces pombe) is an excellent model organism for studying eukaryotic cell division because many of the underlying principles and key regulators of cell cycle biology are conserved from yeast to humans. As such it can be employed as tool for understanding complex human diseases that arise from dis-regulation in cell cycle controls, including cancers. Conventional Flow Cytometry (CFC) is a high-throughput, multi-parameter, fluorescence-based single cell analysis technology. It is widely used for studying the mammalian cell cycle both in the context of the normal and disease states by measuring changes in DNA content during the transition through G1, S and G2/M using fluorescent DNA-binding dyes. Unfortunately analysis of the fission yeast cell cycle by CFC is not straightforward because, unlike mammalian cells, cytokinesis occurs after S-phase meaning that bi-nucleated G1 cells have the same DNA content as mono-nucleated G2 cells and cannot be distinguished using total integrated fluorescence (pulse area). It has been elegantly shown that the width of the DNA pulse can be used to distinguish G2 cells with a single 2C foci versus G1 cells with two 1C foci, however the accuracy of this measurement is dependent on the orientation of the cell as it traverses the laser beam. To this end we sought to improve the accuracy of the fission yeast cell cycle analysis and have developed an Imaging Flow Cytometry (IFC)-based method that is able to preserve the high throughput, objective analysis afforded by CFC in combination with the spatial and morphometric information provide by microscopy. We have been able to derive an analysis framework for subdividing the yeast cell cycle that is based on intensiometric and morphometric measurements and is thus robust against orientation-based miss-classification. In addition we can employ image-based metrics to define populations of septated/bi-nucleated cells and measure cellular dimensions. To our knowledge

  17. Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

    PubMed

    Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

    2012-01-01

    In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

  18. Metabolically engineered methylotrophic yeast cells and enzymes as sensor biorecognition elements.

    PubMed

    Gonchar, Mykhailo; Maidan, Mykola; Korpan, Yaroslav; Sibirny, Volodymyr; Kotylak, Zbigniew; Sibirny, Andrei

    2002-08-01

    An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated. It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors.

  19. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  20. Intracellular yeasts in endothelial cells of a great blue heron (Ardea herodias).

    PubMed

    Millins, Caroline; Hill, Janet E; Wobeser, Gary

    2010-01-01

    Intracellular organisms in the endothelial cells of several organs of an adult great blue heron (Ardea herodias) were identified as a yeast in the family Saccharomycetales based on ultrastructural morphology and sequence data from the ribosomal RNA operon. Morphologically similar organisms of unknown identity have been described previously in Muscovy (Cairina moschata) and domestic (Anas platyrhynchos domestica) ducks.

  1. Yeast cell wall supplementation alters the performance and health of beef heifers during the receiving period

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was designed to determine the effect of feeding yeast cell wall (YCW) on performance of newly received crossbred heifers (n = 140; 225 ± 9.4 kg) Heifers were sorted by source (n = 2) and arranged in a completely randomized block design (35 pens; 7 pens/treatment; 4 heifers/pen). Heifers were...

  2. Making artificial honey using yeast cells from salivary glands of honey bees.

    PubMed

    Kathiresan, K; Srinivasan, K

    2005-07-01

    The salivary glands of a honey bee, Apis cerana and the yeast cells isolated from these glands were studied for their effects on sucrose solution. This solution exhibited lowered pH and increased levels of fructose and total amino acids as the time of incubation proceeded. The solution thus made was similar to the natural honey.

  3. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    PubMed

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.

  4. Yeast cell wall supplementation alters the metabolic responses of crossbred heifers to an endotoxin challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study examined the effect of feeding yeast cell wall (YCW) products on the metabolic responses of newly-received heifers to endotoxin challenge. Heifers (n = 24; 219 ± 2.4 kg) were separated into treatment groups receiving a Control diet (n = 8), YCW-A (2.5 grams/heifer/d; n = 8) or YCW-C (2.5 ...

  5. Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase.

    PubMed

    Kucerova, Lucia; Poturnajova, Martina; Tyciakova, Silvia; Matuskova, Miroslava

    2012-03-01

    Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.

  6. The role of oxygen in yeast metabolism during high cell density brewery fermentations.

    PubMed

    Verbelen, P J; Saerens, S M G; Van Mulders, S E; Delvaux, F; Delvaux, F R

    2009-04-01

    The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.

  7. Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

    PubMed Central

    Chasman, Deborah; Ho, Yi-Hsuan; Berry, David B; Nemec, Corey M; MacGilvray, Matthew E; Hose, James; Merrill, Anna E; Lee, M Violet; Will, Jessica L; Coon, Joshua J; Ansari, Aseem Z; Craven, Mark; Gasch, Audrey P

    2014-01-01

    Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology. PMID:25411400

  8. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    DOE PAGES

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

    2016-03-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

  9. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    PubMed Central

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

    2016-01-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

  10. Current progress in high cell density yeast bioprocesses for bioethanol production.

    PubMed

    Westman, Johan O; Franzén, Carl Johan

    2015-08-01

    High capital costs and low reaction rates are major challenges for establishment of fermentation-based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.

  11. A model of yeast cell-cycle regulation based on multisite phosphorylation

    PubMed Central

    Barik, Debashis; Baumann, William T; Paul, Mark R; Novak, Bela; Tyson, John J

    2010-01-01

    In order for the cell's genome to be passed intact from one generation to the next, the events of the cell cycle (DNA replication, mitosis, cell division) must be executed in the correct order, despite the considerable molecular noise inherent in any protein-based regulatory system residing in the small confines of a eukaryotic cell. To assess the effects of molecular fluctuations on cell-cycle progression in budding yeast cells, we have constructed a new model of the regulation of Cln- and Clb-dependent kinases, based on multisite phosphorylation of their target proteins and on positive and negative feedback loops involving the kinases themselves. To account for the significant role of noise in the transcription and translation steps of gene expression, the model includes mRNAs as well as proteins. The model equations are simulated deterministically and stochastically to reveal the bistable switching behavior on which proper cell-cycle progression depends and to show that this behavior is robust to the level of molecular noise expected in yeast-sized cells (∼50 fL volume). The model gives a quantitatively accurate account of the variability observed in the G1-S transition in budding yeast, which is governed by an underlying sizer+timer control system. PMID:20739927

  12. Apoptosis induced by ultraviolet radiation is enhanced by amplitude modulated radiofrequency radiation in mutant yeast cells.

    PubMed

    Markkanen, Ari; Penttinen, Piia; Naarala, Jonne; Pelkonen, Jukka; Sihvonen, Ari-Pekka; Juutilainen, Jukka

    2004-02-01

    The aim of this study was to investigate whether radiofrequency (RF) electromagnetic field (EMF) exposure affects cell death processes of yeast cells. Saccharomyces cerevisiae yeast cells of the strains KFy417 (wild-type) and KFy437 (cdc48-mutant) were exposed to 900 or 872 MHz RF fields, with or without exposure to ultraviolet (UV) radiation, and incubated simultaneously with elevated temperature (+37 degrees C) to induce apoptosis in the cdc48-mutated strain. The RF exposure was carried out in a special waveguide exposure chamber where the temperature of the cell cultures can be precisely controlled. Apoptosis was analyzed using the annexin V-FITC method utilizing flow cytometry. Amplitude modulated (217 pulses per second) RF exposure significantly enhanced UV induced apoptosis in cdc48-mutated cells, but no effect was observed in cells exposed to unmodulated fields at identical time-average specfic absorption rates (SAR, 0.4 or 3.0 W/kg). The findings suggest that amplitude modulated RF fields, together with known damaging agents, can affect the cell death process in mutated yeast cells. Bioelectromagnetics 25:127-133, 2004.

  13. Study of budding yeast colony formation and its characterizations by using circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  14. Efficient yeast cell-surface display of exo- and endo-cellulase using the SED1 anchoring region and its original promoter

    PubMed Central

    2014-01-01

    Background The recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for direct conversion of lignocellulosic materials to ethanol. However, the cellulolytic activities of the conventional cellulase-displaying yeast strains are insufficient for the hydrolysis of cellulose. In this study, we constructed novel gene cassettes for the efficient cellulose utilization by cellulase-displaying yeast strains. Results The novel gene cassettes for the cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reeseii endoglucanase II (EGII) were constructed using the promoter and the GPI anchoring region derived from Saccharomyces cerevisiae SED1. The gene cassettes were integrated into the S. cerevisiae genome, then the β-glucosidase activity of these recombinant strains was evaluated. We revealed that simultaneous utilization of the SED1 promoter and Sed1 anchoring domain in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The β-glucosidase activity of recombinant yeast cells transduced with the novel gene cassette was 8.4-fold higher than that of a conventional strain. The novel EGII-displaying strain also achieved 106-fold higher hydrolysis activity against the water-insoluble cellulose than a conventional strain. Furthermore, direct ethanol production from hydrothermally processed rice straw was improved by the display of T. reeseii EGII using the novel gene cassette. Conclusions We have developed novel gene cassettes for the efficient cell-surface display of exo- and endo-type cellulolytic enzymes. The results suggest that this gene cassette has the wide applicability for cell-surface display and that cellulase-displaying yeasts have significant potential for cost-effective bioethanol production from lignocellulosic biomass. PMID:24423072

  15. Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

    PubMed Central

    Narayan, Monisha; Chou, Ching-Shan; Park, Hay-Oak

    2013-01-01

    Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model. PMID:23437206

  16. Systematic exploration of ubiquitin sequence, E1 activation efficiency, and experimental fitness in yeast

    PubMed Central

    Roscoe, Benjamin P.; Bolon, Daniel N. A.

    2014-01-01

    The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) as well as overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by two fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation. PMID:24862281

  17. [Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

    PubMed

    Ma, Dong-Mei; Bai, Jun-Jie; Jian, Qing; Lao, Hai-Hua; Ye, Xing; Luo, Jian-Ren

    2003-09-01

    Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher

  18. Overexpression of the yeast transcriptional activator ADR1 induces mutation of the mitochondrial genome.

    PubMed

    Cherry, J R; Denis, C L

    1989-05-01

    It was previously observed that increased dosages of the ADR1 gene, which encodes a yeast transcriptional activator required for alcohol dehydrogenase II (ADH II) expression, cause a decreased rate of growth in medium containing ethanol as the carbon source. Here we show that observed reduction in growth rate is mediated by the ADR1 protein which, when overexpressed, increases the frequency of cytoplasmic petites. Unlike previously characterized mutations known to potentiate petite formation, the ADR1 effect is dominant, with the petite frequency rising concomitantly with increasing ADR1 dosage. The ability of ADR1 to increase the frequency of mitochondrial mutation is correlated with its ability to activate ADH II transcription but is independent of the level of ADH II being expressed. Based on restoration tests using characterized mit- strains, ADR1 appears to cause non-specific deletions within the mitochondrial genome to produce rho- petites. Pedigree analysis of ADR1-overproducing strains indicates that only daughter cells become petite. This pattern is analogous to that observed for petite induction by growth at elevated temperature and by treatment with the acridine dye euflavine. One strain resistant to ADR1-induced petite formation displayed cross-resistance to petite mutation by growth at elevated temperature and euflavine treatment, yet was susceptible to petite induction by ethidium bromide. These results suggest that ADR1 overexpression disrupts the fidelity of mitochondrial DNA replication or repair.

  19. Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

    PubMed

    Farbo, Maria Grazia; Urgeghe, Pietro Paolo; Fiori, Stefano; Marceddu, Salvatore; Jaoua, Samir; Migheli, Quirico

    2016-01-18

    Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20 μg/kg OTA and after 48 h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72-120 h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed.

  20. Monitoring the cell cycle by multi-kinase-dependent regulation of Swe1/Wee1 in budding yeast.

    PubMed

    Lee, Kyung S; Asano, Satoshi; Park, Jung-Eun; Sakchaisri, Krisada; Erikson, Raymond L

    2005-10-01

    In eukaryotes, G(2)/M transition is induced by the activation of cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. Recent advances in our understanding of mitotic entry in budding yeast has revealed that these cells utilize the level of Swe1 (Wee1 ortholog) phosphorylation as a means of monitoring cell cycle progression and of coordinating morphogenetic events with mitotic entry. Swe1 is phosphorylated by at least three distinct kinases at different stages of the cell cycle. This cumulative phosphorylation leads to the hyperphosphorylation and degradation of Swe1 through ubiquitin-mediated proteolysis. Thus, Swe1 functions as an important cell cycle modulator that integrates multiple upstream signals from prior cell cycle events before its ultimate degradation permits passage into mitosis.

  1. Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

    PubMed

    Baranwal, Shivani; Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S

    2014-09-01

    Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

  2. Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability.

    PubMed

    Iinoya, Kaoru; Kotani, Tetsuya; Sasano, Yu; Takagi, Hiroshi

    2009-06-01

    The budding yeast Saccharomyces cerevisiae Sigma1278b has the MPR1 gene, which confers resistance to the proline analogue azetidine-2-carboxylate (AZC). This gene encodes an N-acetyltransferase Mpr1 that detoxifies AZC, and the homologous genes have been found in many yeasts. Recently, we found that Mpr1 protects yeast cells by reducing the intracellular reactive oxygen species (ROS) levels under oxidative stresses, such as heat-shock, freezing, or ethanol treatment. Unlike the known antioxidant enzymes, Mpr1 is thought to acetylate toxic metabolite(s) involved in ROS generation via oxidative events. To improve the enzymatic functions of Mpr1, we applied PCR random mutagenesis to MPR1. The mutagenized plasmid library was introduced into the S. cerevisiae S288C strain lacking MPR1, and we successfully isolated two Mpr1 variants with higher AZC resistance (K63R and F65L/L117V). Interestingly, overexpression of the K63R variant was found to increase cell viability or decrease intracellular ROS levels after exposure to H(2)O(2) or ethanol compared with the wild-type Mpr1. In vitro studies with the recombinant enzymes showed that the catalytic efficiency of the K63R variant for AZC and acetyl-CoA was higher than that of the wild-type Mpr1 and that the F65L mutation greatly enhanced the thermal stability. The mutational analysis and molecular modeling suggest that an alpha-helix containing Lys63 and Phe65 has important roles in the function of Mpr1. In addition, the wild-type and K63R variant Mpr1 reduced intracellular ROS levels under ethanol stress conditions on haploid sake yeast cells. These results suggest that engineering Mpr1 might be useful in breeding oxidative stress-tolerant yeast strains.

  3. A role for calcium in the regulation of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Franco, Alejandro; Soto, Teresa; Vicente-Soler, Jero; Paredes, Vanessa; Madrid, Marisa; Gacto, Mariano; Cansado, José

    2003-01-01

    Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca2+-binding proteins. In this study we provide direct evidence for physical binding of Ca2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe, and show that aspartic residues at positions 97 and 108 in the conserved putative Ca2+-binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca2+ partially resemble those devoid of the ntp1+ gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca2+-binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca2+ binding through the Ca2+-binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi. PMID:12943532

  4. SNAP-, CLIP- and Halo-tag labelling of budding yeast cells.

    PubMed

    Stagge, Franziska; Mitronova, Gyuzel Y; Belov, Vladimir N; Wurm, Christian A; Jakobs, Stefan

    2013-01-01

    Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6'-carboxy isomers and not the 5'-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6'- and 5'-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.

  5. High-throughput tracking of single yeast cells in a microfluidic imaging matrix.

    PubMed

    Falconnet, D; Niemistö, A; Taylor, R J; Ricicova, M; Galitski, T; Shmulevich, I; Hansen, C L

    2011-02-07

    Time-lapse live cell imaging is a powerful tool for studying signaling network dynamics and complexity and is uniquely suited to single cell studies of response dynamics, noise, and heritable differences. Although conventional imaging formats have the temporal and spatial resolution needed for such studies, they do not provide the simultaneous advantages of cell tracking, experimental throughput, and precise chemical control. This is particularly problematic for system-level studies using non-adherent model organisms such as yeast, where the motion of cells complicates tracking and where large-scale analysis under a variety of genetic and chemical perturbations is desired. We present here a high-throughput microfluidic imaging system capable of tracking single cells over multiple generations in 128 simultaneous experiments with programmable and precise chemical control. High-resolution imaging and robust cell tracking are achieved through immobilization of yeast cells using a combination of mechanical clamping and polymerization in an agarose gel. The channel and valve architecture of our device allows for the formation of a matrix of 128 integrated agarose gel pads, each allowing for an independent imaging experiment with fully programmable medium exchange via diffusion. We demonstrate our system in the combinatorial and quantitative analysis of the yeast pheromone signaling response across 8 genotypes and 16 conditions, and show that lineage-dependent effects contribute to observed variability at stimulation conditions near the critical threshold for cellular decision making.

  6. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  7. Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.

    PubMed

    Lewinska, Anna; Miedziak, Beata; Kulak, Klaudia; Molon, Mateusz; Wnuk, Maciej

    2014-06-01

    The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.

  8. The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast.

    PubMed Central

    Dey, B; Caplan, A J; Boschelli, F

    1996-01-01

    Molecular chaperones have been implicated in the formation of active p60v-src tyrosine kinase. In Saccharomyces cerevisiae, expression of p60v-src causes cell death, a phenomenon that requires functional Hsp90. We show here that mutations in a member of a second class of chaperones, the yeast dnaJ homologue YDJ1, suppress the lethality caused by p60v-src. One p60v-src-resistant ydj1 mutant, ydj1-39, which has two point mutations in the highly conserved "J" domain, has reduced levels of v-src mRNA and protein. However, a ydj1 null mutant produces normal quantities of active p60v-src, indicating that Ydj1p facilitates, but is not essential for, the formation of active p60v-src. We also report p60v-src-resistance in a previously identified temperature-sensitive ydj1 mutant, ydj1-151. In this mutant, the level of p60v-src remains unaltered, but the protein is much less active in vivo. In addition, p60v-src immunoprecipitates from the ydj1-151 strain contained Hsp90 and Hsp70 in greater amounts than in wild-type strains. Ydj1 protein was also detected in p60v-src immunoprecipitates from both wild-type and ydj1-151 strains. These results indicate that Ydj1p participates in the formation of active p60v-src via molecular chaperone complexes. Images PMID:8741842

  9. In vitro antifungal activity of fluconazole and voriconazole against non-Candida yeasts and yeast-like fungi clinical isolates.

    PubMed

    Mandras, Narcisa; Roana, Janira; Scalas, Daniela; Fucale, Giacomo; Allizond, Valeria; Banche, Giuliana; Barbui, Anna; Li Vigni, Nicolò; Newell, Vance A; Cuffini, Anna Maria; Tullio, Vivian

    2015-10-01

    The risk of opportunistic infections caused by non-Candida yeasts and yeast-like fungi is increasingly common, mainly in immunocompromised patients. Appropriate first-line therapy has not been defined and standardized, mainly due to the low number of cases reported. To improve empirical treatment guidelines, we describe the susceptibility profile to fluconazole and voriconazole of 176 non-Candida yeasts and yeast-like fungi collected from hospitals in Piedmont, North West Italy from January 2009 to December 2013. The results showed that most isolates are susceptible to voriconazole (94%), but less susceptible to fluconazole (78%), suggesting that voriconazole could be used as first-line therapy in infections caused by these fungi.

  10. Antifungal Activity of Brazilian Propolis Microparticles against Yeasts Isolated from Vulvovaginal Candidiasis

    PubMed Central

    Dota, Kelen Fátima Dalben; Consolaro, Marcia Edilaine Lopes; Svidzinski, Terezinha Inez Estivalet; Bruschi, Marcos Luciano

    2011-01-01

    Propolis, a resinous compound produced by Apis mellifera L. bees, is known to possess a variety of biological activities and is applied in the therapy of various infectious diseases. The aim of this study was to evaluate the in vitro antifungal activity of propolis ethanol extract (PE) and propolis microparticles (PMs) obtained from a sample of Brazilian propolis against clinical yeast isolates of importance in the vulvovaginal candidiasis (VVC). PE was used to prepare the microparticles. Yeast isolates (n = 89), obtained from vaginal exudates of patients with VVC, were exposed to the PE and the PMs. Moreover, the main antifungal drugs used in the treatment of VVC (Fluconazole, Voriconazole, Itraconazole, Ketoconazole, Miconazole and Amphotericin B) were also tested. Minimum inhibitory concentration (MIC) was determined according to the standard broth microdilution method. Some Candida albicans isolates showed resistance or dose-dependent susceptibility for the azolic drugs and Amphotericin B. Non-C. albicans isolates showed more resistance and dose-dependent susceptibility for the azolic drugs than C. albicans. However, all of them were sensitive or dose-dependent susceptible for Amphotericin B. All yeasts were inhibited by PE and PMs, with small variation, independent of the species of yeast. The overall results provided important information for the potential application of PMs in the therapy of VVC and the possible prevention of the occurrence of new symptomatic episodes. PMID:21607012

  11. Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells.

    PubMed

    Safarik, Ivo; Pospiskova, Kristyna; Maderova, Zdenka; Baldikova, Eva; Horska, Katerina; Safarikova, Mirka

    2015-01-01

    An extremely simple procedure has been developed for the immobilization of Saccharomyces cerevisiae cells on magnetic chitosan microparticles. The magnetic carrier was prepared using an inexpensive, simple, rapid, one-pot process, based on the microwave irradiation of chitosan and ferrous sulphate at high pH. Immobilized yeast cells have been used for sucrose hydrolysis, hydrogen peroxide decomposition and the adsorption of selected dyes.

  12. Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

    PubMed Central

    Brand, Ingo L.; Civciristov, Srgjan; Taylor, Nicole L.; Talbo, Gert H.; Pantaki-Eimany, Delara; Levina, Vita; Clem, Rollie J.; Perugini, Matthew A.; Kvansakul, Marc; Hawkins, Christine J.

    2012-01-01

    Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity. PMID:22720082

  13. Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells

    PubMed Central

    Koyama, Sumihiro; Tsubouchi, Taishi; Usui, Keiko; Uematsu, Katsuyuki; Tame, Akihiro; Nogi, Yuichi; Ohta, Yukari; Hatada, Yuji; Kato, Chiaki; Miwa, Tetsuya; Toyofuku, Takashi; Nagahama, Takehiko; Konishi, Masaaki; Nagano, Yuriko; Abe, Fumiyoshi

    2015-01-01

    The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications. PMID:26187908

  14. Classification of yeast cells from image features to evaluate pathogen conditions

    NASA Astrophysics Data System (ADS)

    van der Putten, Peter; Bertens, Laura; Liu, Jinshuo; Hagen, Ferry; Boekhout, Teun; Verbeek, Fons J.

    2007-01-01

    Morphometrics from images, image analysis, may reveal differences between classes of objects present in the images. We have performed an image-features-based classification for the pathogenic yeast Cryptococcus neoformans. Building and analyzing image collections from the yeast under different environmental or genetic conditions may help to diagnose a new "unseen" situation. Diagnosis here means that retrieval of the relevant information from the image collection is at hand each time a new "sample" is presented. The basidiomycetous yeast Cryptococcus neoformans can cause infections such as meningitis or pneumonia. The presence of an extra-cellular capsule is known to be related to virulence. This paper reports on the approach towards developing classifiers for detecting potentially more or less virulent cells in a sample, i.e. an image, by using a range of features derived from the shape or density distribution. The classifier can henceforth be used for automating screening and annotating existing image collections. In addition we will present our methods for creating samples, collecting images, image preprocessing, identifying "yeast cells" and creating feature extraction from the images. We compare various expertise based and fully automated methods of feature selection and benchmark a range of classification algorithms and illustrate successful application to this particular domain.

  15. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  16. Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

    PubMed Central

    Kettenbach, Arminja N.; Deng, Lin; Wu, Youjun; Baldissard, Suzanne; Adamo, Mark E.; Gerber, Scott A.; Moseley, James B.

    2015-01-01

    Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets. PMID:25720772

  17. Modifying dielectrophoretic response of nonviable yeast cells by ionic surfactant treatment.

    PubMed

    Tang, Shi-Yang; Zhang, Wei; Baratchi, Sara; Nasabi, Mahyar; Kalantar-Zadeh, Kourosh; Khoshmanesh, Khashayar

    2013-07-02

    Nonviable cells are essential biosystems, due to the functionalities they offer and their effects on viable cells. Therefore, the separation and immobilization of nonviable cells separately or in the vicinity of viable cells is of great importance for many fundamentals investigations in cell biology. However, most nonviable cells become less polarizable than the surrounding medium at conductivities above 0.01 S/m. This means that in such a medium, dielectrophoresis, despite its great versatilities for manipulation of cells, cannot be employed for immobilizing nonviable cells. Here, we present a novel approach to change the dielectrophoretic (DEP) response of nonviable yeast cells by treating them with low concentrations of ionic surfactants such as sodium dodecyl sulfate. After this treatment, they exhibit a strong positive DEP response, even at high medium conductivities. The capability of this treatment is demonstrated in two proof-of-concept experiments. First, we show the sorting and immobilization of viable and nonviable yeast cells, along consecutive microelectrode arrays. Second, we demonstrate the immobilization of viable and nonviable cells in the vicinity of each other along the same microelectrode array. The proposed technique allows DEP platforms to be utilized for the immobilization and subsequent postanalysis of both viable and nonviable cells with and without the presence of each other.

  18. Ring closure activates yeast γTuRC for species-specific microtubule nucleation

    PubMed Central

    Kollman, Justin M.; Greenberg, Charles H.; Li, Sam; Moritz, Michelle; Zelter, Alex; Fong, Kimberly K.; Fernandez, Jose-Jesus; Sali, Andrej; Kilmartin, John; Davis, Trisha N.; Agard, David A.

    2014-01-01

    The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm its functional importance we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, suggesting this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a profound preference for tubulin from the same species. PMID:25599398

  19. Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

    PubMed

    Fehrmann, Steffen; Bottin-Duplus, Hélène; Leonidou, Andri; Mollereau, Esther; Barthelaix, Audrey; Wei, Wu; Steinmetz, Lars M; Yvert, Gaël

    2013-10-08

    Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

  20. Contactless Investigations of Yeast Cell Cultivation in the 7 GHz and 240 GHz Ranges

    NASA Astrophysics Data System (ADS)

    Wessel, J.; Schmalz, K.; Gastrock, G.; Cahill, B. P.; Meliani, C.

    2013-04-01

    Using a microfluidic system based on PTFE tubes, experimental results of contactless and label-free characterization techniques of yeast cell cultivation are presented. The PTFE tube has an inner diameter of 0.5 mm resulting in a sample volume of 2 μ1 for 1 cm sample length. Two approaches (at frequencies around 7 GHz and 240 GHz) are presented and compared in terms of sensitivity and applicability. These frequency bands are particularly interesting to gain information on the permittivity of yeast cells in Glucose solution. Measurements from 240 GHz to 300 GHz were conducted with a continuous wave spectrometer from Toptica. At 7 GHz band, measurements have been performed using a rat-race based characterizing system realized on a printed circuit board. The conducted experiments demonstrate that by selecting the phase as characterization parameter, the presented contactless and label-free techniques are suitable for cell cultivation monitoring in a PTFE pipe based microfluidic system.

  1. Heavy ion induced DNA-DSB in yeast and mammalian cells

    NASA Technical Reports Server (NTRS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

  2. The fission yeast Schizosaccharomyces pombe as a model to understand how peroxiredoxins influence cell responses to hydrogen peroxide.

    PubMed

    Veal, Elizabeth A; Tomalin, Lewis E; Morgan, Brian A; Day, Alison M

    2014-08-01

    As a more selectively reactive oxygen species, H2O2 (hydrogen peroxide) has been co-opted as a signalling molecule, but high levels can still lead to lethal amounts of cell damage. 2-Cys Prxs (peroxiredoxins) are ubiquitous thioredoxin peroxidases which utilize reversibly oxidized catalytic cysteine residues to reduce peroxides. As such, Prxs potentially make an important contribution to the repertoire of cell defences against oxidative damage. Although the abundance of eukaryotic 2-Cys Prxs suggests an important role in maintaining cell redox, the surprising sensitivity of their thioredoxin peroxidase activity to inactivation by H2O2 has raised questions as to their role as an oxidative stress defence. Indeed, work in model yeast has led the way in revealing that Prxs do much more than simply remove peroxides and have even uncovered circumstances where their thioredoxin peroxidase activity is detrimental. In the present paper, we focus on what we have learned from studies in the fission yeast Schizosaccharomyces pombe about the different roles of 2-Cys Prxs in responses to H2O2 and discuss the general implications of these findings for other systems.

  3. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

    PubMed

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-04-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1 These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1(+)increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of theO-mannosyltransferase encoding gene omh1(+) Loss of omh1(+)abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1(+)resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2(+) or cbf12(+) This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network.

  4. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast

    PubMed Central

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-01-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1+ increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network. PMID:26896331

  5. Inorganic nitrogen assimilation in yeasts: alteration in enzyme activities associated with changes in cultural conditions and growth phase.

    PubMed

    Thomulka, K W; Moat, A G

    1972-01-01

    Ammonia assimilation has been investigated in four strains of Saccharomyces cerevisiae by measuring, at intervals throughout the growth cycle, the activities of several enzymes concerned with inorganic ammonia assimilation. Enzyme activities in extracts of cells were compared after growth in complete and defined media. The effect of shift from growth in a complete to growth in a defined medium (and the reverse) was also determined. The absence of aspartase (EC 4.3.1.1, l-aspartate-ammonia lyase) activity, the low specific activities of alanine dehydrogenase, glutamine synthetase [EC 6.3.1.2, l-glutamate-ammonia ligase (ADP)], and the marked increase in activity of the nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (NADP-GDH) [EC 1.4.1.4, l-glutamate:NADP-oxidoreductase (deaminating)] during the early stages of growth support the conclusion that yeasts assimilate ammonia primarily via glutamate. The NADP-GDH showed a rapid increase in activity just before the initiation of exponential growth, reached a maximum at the mid-exponential stage, and then gradually declined in activity in the stationary phase. The NADP-GDH reached a higher level of activity when the yeasts were grown on the defined medium as compared with complete medium. The nicotinamide adenine dinucleotide-linked glutamate dehydrogenase (NAD-GDH) [EC 1.4.1.2, l-glutamate:NAD-oxidoreductase (deaminating)] showed only slight increases in activity during the exponential phase of growth. There was an inverse relationship in that the NADP-GDH increased in activity as the NAD-GDH decreased. The NAD-GDH activity was higher after growth on the complete medium. The glutamate-oxaloacetate transaminase (EC 2.6.1.1. l-aspartate:2-oxoglutarate aminotransferase) activity rose and fell in parallel with the NADP-GDH, although its specific activity was somewhat lower. Although other ammonia-assimilatory enzymes were demonstrable, it seems unlikely that their combined activities could account

  6. Nitric oxide signaling in yeast.

    PubMed

    Astuti, Rika Indri; Nasuno, Ryo; Takagi, Hiroshi

    2016-11-01

    As a cellular signaling molecule, nitric oxide (NO) is widely conserved from microorganisms, such as bacteria, yeasts, and fungi, to higher eukaryotes including plants and mammals. NO is mainly produced by NO synthase (NOS) or nitrite reductase (NIR) activity. There are several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR). NO homeostasis based on the balance between NO synthesis and degradation is important for the regulation of its physiological functions because an excess level of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds. In yeast, NO may be involved in stress responses, but NO and its signaling have been poorly understood due to the lack of mammalian NOS orthologs in the genome. Even though the activities of NOS and NIR have been observed in yeast cells, the gene encoding NOS and the NO production mechanism catalyzed by NIR remain unclear. On the other hand, yeast cells employ NOD and GSNOR to maintain an intracellular redox balance following endogenous NO production, exogenous NO treatment, or environmental stresses. This article reviews NO metabolism (synthesis, degradation) and its regulation in yeast. The physiological roles of NO in yeast, including the oxidative stress response, are also discussed here. Such investigations into NO signaling are essential for understanding the NO-dependent genetic and physiological modulations. In addition to being responsible for the pathology and pharmacology of various degenerative diseases, NO signaling may be a potential target for the construction and engineering of industrial yeast strains.

  7. Hsp90 mediates the crosstalk between galactose metabolism and cell morphology pathways in yeast.

    PubMed

    Gopinath, Rajaneesh Karimpurath; Leu, Jun-Yi

    2017-02-01

    Galactose metabolism in the yeast Saccharomyces cerevisiae is carried out by a specialized GAL pathway consisting of structural and regulatory proteins. It is known that cells with unbalanced Gal proteins accumulate toxic metabolic intermediates and exhibit severe growth defects. Recently, we found that the molecular chaperone Hsp90 controls the abundance of multiple Gal proteins, possibly to prevent these defects. Hsp90 regulates various cellular processes including cell morphology in response to environmental cues. Yeast cells are known to resort to filamentous growth upon exposure to galactose or other environmental stresses. Our previous and current findings support the "Hsp90 titration model" of Hsp90 buffering, which links the cell morphology and galactose pathways. Our results suggest that, when a large proportion of Hsp90 molecules are used to help Gal proteins, the Hsp90 client proteins in cell morphology pathways are left unattended, leading to filamentous growth. It remains unclear whether this phenomenon serves any biological function or simply reflects a cellular constraint. Nonetheless, it provides an alternative explanation why the GAL pathway is degenerated in some yeast species.

  8. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  9. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.

    PubMed

    Lilly, M; Lambrechts, M G; Pretorius, I S

    2000-02-01

    The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid

  10. [Study on CTP production from CMP by beer yeast cell immobilized in PVA].

    PubMed

    Yang, Hong-Yi; Qian, Shi-Jun; Li, Gao-Wo

    2007-03-01

    With PVA as the carrier, the frozen beer yeast cells were immobilized for production of CTP from CMP. we explored the optimal condition of the immobilization from the aspects of the type, concentration of the PVA, and the immobilizing methods of cells In all 8 continuous batch of fermentation under the reactional condition of the immobilized cells, the conversion rate of CTP were maintained about 85% - 95%. Moreever, the storage stability of immobilized cells were investigated, and the products was also isolated and identifided by HPLC.

  11. A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

    PubMed

    Alver, Bonnie; Jauert, Peter A; Brosnan, Laura; O'Hehir, Melissa; VanderSluis, Benjamin; Myers, Chad L; Kirkpatrick, David T

    2013-04-09

    Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the factors preventing minisatellite alterations. Previously, our laboratory developed a color segregation assay in which a minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that minisatellite alterations that occur in stationary-phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the synthetic genetic array analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different minisatellite tracts: the ade2-min3 construct consisting of three identical 20-bp repeats, and the ade2-h7.5 construct, consisting of seven-and-a-half 28-bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits overlapped both screens, indicating that different factors regulate repeat stability depending upon minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex minisatellites in stationary phase cells. Our work provides insight into the factors regulating minisatellite stability.

  12. Expanding the yeast prion world

    PubMed Central

    Suzuki, Genjiro; Tanaka, Motomasa

    2013-01-01

    Mammalian and fungal prion proteins form self-perpetuating β-sheet-rich fibrillar aggregates called amyloid. Prion inheritance is based on propagation of the regularly oriented amyloid structures of the prion proteins. All yeast prion proteins identified thus far contain aggregation-prone glutamine/asparagine (Gln/Asn)-rich domains, although the mammalian prion protein and fungal prion protein HET-s do not contain such sequences. In order to fill this gap, we searched for novel yeast prion proteins lacking Gln/Asn-rich domains via a genome-wide screen based on cross-seeding between two heterologous proteins and identified Mod5, a yeast tRNA isopentenyltransferase, as a novel non-Gln/Asn-rich yeast prion protein. Mod5 formed self-propagating amyloid fibers in vitro and the introduction of Mod5 amyloids into non-prion yeast induced dominantly and cytoplasmically heritable prion state [MOD+], which harbors aggregates of endogenous Mod5. [MOD+] yeast showed an increased level of membrane lipid ergosterol and acquired resistance to antifungal agents. Importantly, enhanced de novo formation of [MOD+] was observed when non-prion yeast was grown under selective pressures from antifungal drugs. Our findings expand the family of yeast prions to non-Gln/Asn-rich proteins and reveal the acquisition of a fitness advantage for cell survival through active prion conversion. PMID:23117914

  13. Magnetic resonance investigation of magnetic-labeled baker's yeast cells

    NASA Astrophysics Data System (ADS)

    Godoy Morais, J. P. M.; Azevedo, R. B.; Silva, L. P.; Lacava, Z. G. M.; Báo, S. N.; Silva, O.; Pelegrini, F.; Gansau, C.; Buske, N.; Safarik, I.; Safarikova, M.; Morais, P. C.

    2004-05-01

    In this study, the interaction of DMSA-coated magnetite nanoparticles (5 and 10 nm core-size) with Saccharomyces cerevisae was investigated using magnetic resonance (MR) and transmission electron microscopy (TEM). The TEM micrographs revealed magnetite nanoparticles attached externally to the cell wall. The MR data support the strong interaction among the nanoparticles supported by the cells. A remarkable shift in the resonance field was used as signature of particle attachment to the cell wall.

  14. Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast.

    PubMed

    Hampsey, D M; Lewin, A S; Kohlhaw, G B

    1983-03-01

    2-Isopropylmalate synthase (EC 4.1.3.12) of yeast is a mitochondrial enzyme. We now provide evidence showing that a large part of the 2-isopropylmalate synthase activity that is associated with the mitochondria is located in the mitochondrial matrix. In vitro translation of total yeast RNA followed by immunoprecipitation with anti-2-isopropylmalate synthase antibody yields two polypeptides. The larger of these has an apparent molecular weight identical to that of purified 2-isopropylmalate synthase subunit (ca. 65,000). It is incorporated into isolated yeast mitochondria with no detectable change in molecular weight. The import requires energy. The smaller polypeptide migrates to a position corresponding to a molecular weight of 63,000-64,000. It is not taken up by mitochondria. Both polypeptides, which also can be obtained by immunoprecipitation of crude extracts, become labeled when in vitro translation is performed in the presence of N-formyl[35S]methionyl-tRNAf. Mutants with no detectable 2-isopropylmalate synthase activity are deficient in either one or both synthase-related polypeptides. These results are discussed in the light of recent evidence for two 2-isopropylmalate synthase-encoding genes in yeast.

  15. Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

    PubMed Central

    Sheu, Yi-Jun; Santos, Beatriz; Fortin, Nathalie; Costigan, Christine; Snyder, Michael

    1998-01-01

    The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain

  16. Modulating the potency of an activator in a yeast in vitro transcription system.

    PubMed Central

    Ohashi, Y; Brickman, J M; Furman, E; Middleton, B; Carey, M

    1994-01-01

    The intrinsic stimulatory potential or potency of a eukaryotic gene activator is controlled by the interaction between the activation domain and the transcriptional machinery. To further understand this interaction, we undertook a biochemical study to identify parameters that could be used to modulate activator potency. We considered how varying the number of activation domains, their flexibility, and the number of promoter sites affects potency in a yeast nuclear extract. The effects of GAL4 derivatives bearing either one, two, or four herpes simplex virus VP16 activation domains (amino acids 413 to 454) were measured on DNA templates containing one or two GAL4 sites in a Saccharomyces cerevisiae nuclear extract. We found that multimerized VP16 activation domains acted synergistically to increase the potency of the activators. The spacing between the activation domains was critical, such that the increased flexibility imparted by a protein linker contributed to increased activator potency. With highly potent activators, the levels of transcription stimulated on a single site were saturating, whereas the stimulatory effect of weaker activators increased with the number of sites. We discuss how these biochemical studies relate to the mechanism of gene activation and synergy in a yeast in vitro system. Images PMID:8139572

  17. T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium, mercury, and zinc.

    PubMed

    Li, Jian; Liu, Yun; Kong, Dongdong; Ren, Shujuan; Li, Na

    2016-05-01

    In the present study, a two-hybrid yeast bioassay and a T-screen were used to screen for the thyroid receptor (TR)-disrupting activity of select metallic compounds (CdCl2, ZnCl2, HgCl2, CuSO4, MnSO4, and MgSO4). The results reveal that none of the tested metallic compounds showed TR-agonistic activity, whereas ZnCl2, HgCl2, and CdCl2 demonstrated TR antagonism. For the yeast assay, the dose-response relationship of these metallic compounds was established, and the concentrations producing 20 % of the maximum effect of ZnCl2, HgCl2, and CdCl2 were 9.1 × 10(-5), 3.2 × 10(-6), and 1.2 × 10(-6) mol/L, respectively. The T-screen also supported the finding that ZnCl2, HgCl2, and CdCl2 decreased the cell proliferation at concentrations ranging from 10(-6) to 10(-4) mol/L. Furthermore, the thyroid-disrupting activity of metallic compounds in environmental water samples collected from the Guanting Reservoir, Beijing, China was evaluated. Solid-phase extraction was used to separate the organic extracts, and a modified two-hybrid yeast bioassay revealed that the metallic compounds in the water samples could affect thyroid hormone-induced signaling by decreasing the binding of the thyroid hormone. The addition of ethylenediaminetetraacetic acid (30 mg/L) could eliminate the effects. Thus, the cause(s) of the thyroid toxicity in the water samples appeared to be partly related to the metallic compounds.

  18. Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

    PubMed Central

    Moura, Marcelo Victor Holanda; da Silva, Giulia Pontes; Machado, Antônio Carlos de Oliveira; Torres, Fernando Araripe Gonçalves; Freire, Denise Maria Guimarães; Almeida, Rodrigo Volcan

    2015-01-01

    Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase. PMID:26510006

  19. The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures

    PubMed Central

    Davidson, George S.; Joe, Ray M.; Roy, Sushmita; Meirelles, Osorio; Allen, Chris P.; Wilson, Melissa R.; Tapia, Phillip H.; Manzanilla, Elaine E.; Dodson, Anne E.; Chakraborty, Swagata; Carter, Mark; Young, Susan; Edwards, Bruce; Sklar, Larry; Werner-Washburne, Margaret

    2011-01-01

    As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell–specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells. PMID:21289090

  20. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

    PubMed Central

    Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Qin, Jiufu; Siewers, Verena; Nielsen, Jens

    2016-01-01

    Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value. PMID:27222209

  1. Measurements of Myosin-II Motor Activity During Cytokinesis in Fission Yeast.

    PubMed

    Tang, Qing; Pollard, Luther W; Lord, Matthew

    2016-01-01

    Fission yeast myosin-II (Myo2p) represents the critical actin-based motor protein that drives actomyosin ring assembly and constriction during cytokinesis. We detail three different methods to measure Myo2p motor function. Actin-activated ATPases provide a readout of actomyosin ATPase motor activity in a bulk assay; actin filament motility assays reveal the speed and efficiency of myosin-driven actin filament gliding (when motors are anchored); myosin-bead motility assays reveal the speed and efficiency of myosin ensembles traveling along actin filaments (when actin is anchored). Collectively, these methods allow us to combine the standard in vivo approaches common to fission yeast with in vitro biochemical methods to learn more about the mechanistic action of myosin-II during cytokinesis.

  2. Influence of fermentation conditions on specific activity of the enzymes alcohol and aldehyde dehydrogenase from yeasts.

    PubMed

    Mauricio, J C; Ortega, J M

    1993-01-01

    The effects of anaerobic, semi-aerobic and short aeration fermentation conditions and the addition of ergosterol and oleic acid to musts on the specific activity of alcohol and aldehyde dehydrogenase (ADH and ALDH) from two yeast species, Saccharomyces cerevisiae and Torulaspora delbrueckii, were studied. ADH I biosynthesis only occurred during the first few hours of fermentation. ADH II from S. cerevisiae and ALDH-NADP+ from the two yeast species behaved as constitutive enzymes under all fermentation conditions. ADH II from T. delbrueckii was only synthesized in small amounts, and its activity was always lower than in S. cerevisiae, where it was responsible for the termination of alcoholic fermentation during the steady growth phase.

  3. The natural yeast extract isolated by ethanol precipitation inhibits melanin synthesis by modulating tyrosinase activity and downregulating melanosome transfer.

    PubMed

    Lee, Woo Jin; Rhee, Do Young; Bang, Seung Hyun; Kim, Su Yeon; Won, Chong Hyun; Lee, Mi Woo; Choi, Jee Ho; Chang, Sung Eun

    2015-01-01

    This study was conducted to examine the effects of EP-2, a natural yeast extract isolated by ethanol precipitation from Saccharomyces cerevisiae, on melanogenesis and to determine its underlying mechanism of action. Our results show that although EP-2 is not a direct tyrosinase inhibitor, when EP-2 was added to the culture media of B16F10 melanoma cells, intracellular tyrosinase activity was decreased. However, EP-2 had no effect on the expression of microphthalmia-associated transcription factor or tyrosinase. EP-2 was found to inhibit melanogenesis and melanosome transfer when it was added to melanocytes and keratinocytes in coculture. In addition, protease-activated receptor 2, a key protein associated with melanosome transfer from melanocytes to keratinocytes, was downregulated in the presence of EP-2. In conclusion, EP-2 is a potent inhibitor of melanogenesis and its hypomelanogenic effect is related to the inhibition of tyrosinase activity and transfer of melanosomes.

  4. Fully hydrated yeast cells imaged with electron microscopy.

    PubMed

    Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

    2011-05-18

    We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells.

  5. Signaling pathways and posttranslational modifications of tau in Alzheimer's disease: the humanization of yeast cells

    PubMed Central

    Heinisch, Jürgen J.; Brandt, Roland

    2016-01-01

    In the past decade, yeast have been frequently employed to study the molecular mechanisms of human neurodegenerative diseases, generally by means of heterologous expression of genes encoding the relevant hallmark proteins. However, it has become evident that substantial posttranslational modifications of many of these proteins are required for the development and progression of potentially disease relevant changes. This is exemplified by the neuronal tau proteins, which are critically involved in a class of neuro-degenerative diseases collectively called tauopathies and which includes Alz-heimer’s disease (AD) as its most common representative. In the course of the disease, tau changes its phosphorylation state and becomes hyperphosphory-lated, gets truncated by proteolytic cleavage, is subject to O-glycosylation, sumoylation, ubiquitinylation, acetylation and some other modifications. This poses the important question, which of these posttranslational modifications are naturally occurring in the yeast model or can be reconstituted by heterol-ogous gene expression. Here, we present an overview on common modifica-tions as they occur in tau during AD, summarize their potential relevance with respect to disease mechanisms and refer to the native yeast enzyme orthologs capable to perform these modifications. We will also discuss potential approaches to humanize yeast in order to create modification patterns resembling the situation in mammalian cells, which could enhance the value of Saccharomyces cerevisiae and Kluyveromyces lactis as disease models. PMID:28357346

  6. Effect of deoxynivalenol (DON) on growing pigs and its modification by modified yeast cell wall or modified yeast cell wall and bentonite.

    PubMed

    Shehata, S; Richter, W; Schuster, M; Lindermayer, H

    2004-03-01

    The study examined effect of two adsorbents on the toxicity of Deoxynivalenol (DON) in growing pigs in a feeding trial. 24 male growing pigs (average initial body weight 11.5 kg) were assigned to one of six dietary treatments: control (uncontaminated diet); control + 0.5% adsorbent I; DON contaminated diet (1.73 mg/kg); DON contaminated diet + 0.5% adsorbent I; control + 0.5% adsorbent II and DON contaminated diet + 0.5% adsorbent II. Two digestibility trials were conducted on the second and fourth week of the feeding period with a sampling period of 7 days to determine the digestibility of the nutrients and the amounts of DON in faeces and urine. At the end of the experiments, the pigs were slaughtered, followed by blood haematology and biochemi analys. These data suggest that the addition of 0.5% modified yeast cell wall or a combination of modified yeast cell wall and bentonite to the naturally DON - contaminated diets reduce the effect of DON on the immune system of pigs but do not play an significant role in detoxification of DON in growing pigs.

  7. Mitochondrial inheritance in budding yeast.

    PubMed

    Boldogh, I R; Yang, H C; Pon, L A

    2001-06-01

    During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae. A combination of genetics, cell-free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division.

  8. Resistance through inhibition: ectopic expression of serine protease inhibitor offers stress tolerance via delayed senescence in yeast cell.

    PubMed

    Joshi, Rakesh S; Tanpure, Rahul S; Singh, Rajan Kumar; Gupta, Vidya S; Giri, Ashok P

    2014-09-26

    Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains.

  9. Growth promoting effects of prebiotic yeast cell wall products in starter broilers under an immune stress and Clostridium perfringens challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was designed to investigate the growth promoting effects of supplementing different sources and concentrations of prebiotic yeast cell wall (YCW) products containing mannanoligosaccharides in starter broilers under an immune stress and Clostridium perfringens challenge. Through a series ...

  10. Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

    2015-07-01

    High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depletion of arginine caused by decreased activity of the biosynthesis pathway confers sensitivity to HP.

  11. Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay.

    PubMed

    Breinholt, V; Larsen, J C

    1998-06-01

    A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids. Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 microM. Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 microM, whereas the remaining flavonoids were devoid of activity. The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A. With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4'-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin. The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17beta-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation. Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay

  12. Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

    NASA Astrophysics Data System (ADS)

    Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

    2012-10-01

    In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

  13. Active segregation of yeast mitochondria by Myo2 is essential and mediated by Mmr1 and Ypt11.

    PubMed

    Chernyakov, Irina; Santiago-Tirado, Felipe; Bretscher, Anthony

    2013-09-23

    Active segregation of essential organelles is required for successful cell division. The essential budding yeast myosin V Myo2 actively segregates most organelles along polarized actin cables. The mechanism of mitochondrial segregation remains controversial, with movement driven by actin polymerization, movement driven by association with transported cortical endoplasmic reticulum (ER), and direct transport by Myo2 proposed as models. Two nonessential proteins, Mmr1 and the Rab GTPase Ypt11, bind Myo2 and have been implicated in mitochondrial inheritance, although their specific roles are also contended. We generated myo2(sens) mutations that exhibit no overt phenotype but render MMR1 essential and have compromised Ypt11 binding. We then isolated myo2(sens)mmr1(ts) conditional mutants and determined that they have a specific and severe defect in active mitochondrial inheritance, revealing mitochondrial transport by Myo2 as an essential function. ypt11Δ mmr1(ts) cells also have conditional defects in growth and active transport of mitochondria into the bud, both of which are suppressed by artificially forcing mitochondrial inheritance. At the restrictive temperature, cells defective in mitochondrial inheritance give rise to dead buds that go through cytokinesis normally, showing no evidence of a proposed cell-cycle mitochondrial inheritance checkpoint. Thus, active mitochondrial inheritance is an essential process and a function of Myo2 that requires either Mmr1 or Ypt11.

  14. Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

    PubMed

    Bolla, P A; Abraham, A G; Pérez, P F; de Los Angeles Serradell, M

    2016-02-01

    The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the

  15. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM.

    PubMed

    Shen, Yajing; Nakajima, Masahiro; Ahmad, Mohd Ridzuan; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2011-07-01

    A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research.

  16. Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.

  17. Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J.; Marquet, Pierre

    2009-05-01

    Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

  18. Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

    DOE PAGES

    Huang, Xiaojing; Nelson, Johanna; Kirz, Janos; ...

    2009-11-01

    We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below -170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.

  19. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    PubMed Central

    2012-01-01

    Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures. PMID:23131126

  20. [Thermoresistance in Saccharomyces cerevisiae yeasts].

    PubMed

    Kaliuzhin, V A

    2011-01-01

    Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

  1. Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

    PubMed

    Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L

    2014-12-01

    In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

  2. Characteristics of an immobilized yeast cell system using very high gravity for the fermentation of ethanol.

    PubMed

    Ji, Hairui; Yu, Jianliang; Zhang, Xu; Tan, Tianwei

    2012-09-01

    The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280 g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65 h with an average ethanol concentration and ethanol yield of 130.12 g/L and 0.477 g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28 days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75 L. The bioreactor was operated for 26 days at a dilution rate of 0.015 h(-1). The ethanol concentration of the effluent reached 130.77 g/L ethanol while an average 8.18 g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.

  3. Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae

    PubMed Central

    Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I.

    2014-01-01

    A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae. Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific ʺmaster regulatorʺ proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast. PMID:28357241

  4. Reconstruction of a yeast cell from x-ray diffraction data

    DOE PAGES

    Thibault, Pierre; Elser, Veit; Jacobsen, Chris; ...

    2006-06-21

    We provide details of the algorithm used for the reconstruction of yeast cell images in the recent demonstration of diffraction microscopy by Shapiro, Thibault, Beetz, Elser, Howells, Jacobsen, Kirz, Lima, Miao, Nieman & Sayre. Two refinements of the iterative constraint-based scheme are developed to address the current experimental realities of this imaging technique, which include missing central data and noise. A constrained power operator is defined whose eigenmodes allow the identification of a small number of degrees of freedom in the reconstruction that are negligibly constrained as a result of the missing data. To achieve reproducibility in the algorithm's output,more » a special intervention is required for these modes. Weak incompatibility of the constraints caused by noise in both direct and Fourier space leads to residual phase fluctuations. This problem is addressed by supplementing the algorithm with an averaging method. The effect of averaging may be interpreted in terms of an effective modulation transfer function, as used in optics, to quantify the resolution. The reconstruction details are prefaced with simulations of wave propagation through a model yeast cell. These show that the yeast cell is a strong-phase-contrast object for the conditions in the experiment.« less

  5. Reconstruction of a yeast cell from x-ray diffraction data

    SciTech Connect

    Thibault, Pierre; Elser, Veit; Jacobsen, Chris; Shapiro, David; Sayre, David

    2006-06-21

    We provide details of the algorithm used for the reconstruction of yeast cell images in the recent demonstration of diffraction microscopy by Shapiro, Thibault, Beetz, Elser, Howells, Jacobsen, Kirz, Lima, Miao, Nieman & Sayre. Two refinements of the iterative constraint-based scheme are developed to address the current experimental realities of this imaging technique, which include missing central data and noise. A constrained power operator is defined whose eigenmodes allow the identification of a small number of degrees of freedom in the reconstruction that are negligibly constrained as a result of the missing data. To achieve reproducibility in the algorithm's output, a special intervention is required for these modes. Weak incompatibility of the constraints caused by noise in both direct and Fourier space leads to residual phase fluctuations. This problem is addressed by supplementing the algorithm with an averaging method. The effect of averaging may be interpreted in terms of an effective modulation transfer function, as used in optics, to quantify the resolution. The reconstruction details are prefaced with simulations of wave propagation through a model yeast cell. These show that the yeast cell is a strong-phase-contrast object for the conditions in the experiment.

  6. Dynamin-dependent biogenesis, cell cycle regulation and mitochondrial association of peroxisomes in fission yeast.

    PubMed

    Jourdain, Isabelle; Sontam, Dharani; Johnson, Chad; Dillies, Clément; Hyams, Jeremy S

    2008-03-01

    Peroxisomes were visualized for the first time in living fission yeast cells. In small, newly divided cells, the number of peroxisomes was low but increased in parallel with the increase in cell length/volume that accompanies cell cycle progression. In cells grown in oleic acid, both the size and the number of peroxisomes increased. The peroxisomal inventory of cells lacking the dynamin-related proteins Dnm1 or Vps1 was similar to that in wild type. By contrast, cells of the double mutant dnm1Delta vps1Delta contained either no peroxisomes at all or a small number of morphologically aberrant organelles. Peroxisomes exhibited either local Brownian movement or longer-range linear displacements, which continued in the absence of either microtubules or actin filaments. On the contrary, directed peroxisome motility appeared to occur in association with mitochondria and may be an indirect function of intrinsic mitochondrial dynamics. We conclude that peroxisomes are present in fission yeast and that Dnm1 and Vps1 act redundantly in peroxisome biogenesis, which is under cell cycle control. Peroxisome movement is independent of the cytoskeleton but is coupled to mitochondrial dynamics.

  7. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

    PubMed

    Padman, Benjamin S; Bach, Markus; Lucarelli, Giuseppe; Prescott, Mark; Ramm, Georg

    2013-11-01

    Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

  8. Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.

    PubMed

    Miller, D; Wheals, B B; Beresford, N; Sumpter, J P

    2001-02-01

    We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.

  9. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

    PubMed

    Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

    2015-05-01

    Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

  10. The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

    PubMed Central

    Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

    2002-01-01

    Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast. PMID:11916725

  11. Adhesion to the yeast cell surface as a mechanism for trapping pathogenic bacteria by Saccharomyces probiotics.

    PubMed

    Tiago, F C P; Martins, F S; Souza, E L S; Pimenta, P F P; Araujo, H R C; Castro, I M; Brandão, R L; Nicoli, Jacques R

    2012-09-01

    Recently, much attention has been given to the use of probiotics as an adjuvant for the prevention or treatment of gastrointestinal pathology. The great advantage of therapy with probiotics is that they have few side effects such as selection of resistant bacteria or disturbance of the intestinal microbiota, which occur when antibiotics are used. Adhesion of pathogenic bacteria onto the surface of probiotics instead of onto intestinal receptors could explain part of the probiotic effect. Thus, this study evaluated the adhesion of pathogenic bacteria onto the cell wall of Saccharomyces boulardii and Saccharomyces cerevisiae strains UFMG 905, W303 and BY4741. To understand the mechanism of adhesion of pathogens to yeast, cell-wall mutants of the parental strain of Saccharomyces cerevisiae BY4741 were used because of the difficulty of mutating polyploid yeast, as is the case for Saccharomyces cerevisiae and Saccharomyces boulardii. The tests of adhesion showed that, among 11 enteropathogenic bacteria tested, only Escherichia coli, Salmonella Typhimurium and Salmonella Typhi adhered to the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741. The presence of mannose, and to some extent bile salts, inhibited this adhesion, which was not dependent on yeast viability. Among 44 cell-wall mutants of Saccharomyces cerevisiae BY4741, five lost the ability to fix the bacteria. Electron microscopy showed that the phenomenon of yeast-bacteria adhesion occurred both in vitro and in vivo (in the digestive tract of dixenic mice). In conclusion, some pathogenic bacteria were captured on the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741, thus preventing their adhesion to specific receptors on the intestinal epithelium and their subsequent invasion of the host.

  12. Transcription factors of M-phase cyclin CLB2 in the yeast cell wall integrity checkpoint.

    PubMed

    Sekiya, Mizuho; Nogami, Satoru; Ohya, Yoshikazu

    2009-08-01

    The cell wall integrity checkpoint coordinates cell wall synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. It has been reported that this checkpoint arrests the cell cycle at G2/M phase with repression of the M phase cyclin Clb2p at the transcriptional level, under perturbation of cell wall synthesis. We demonstrate that an override of this checkpoint with accumulation of CLB2 mRNA is induced when negative CLB2 transcription factors are deleted or when positive CLB2 transcription factors are overproduced in cell wall-defective cells. Our data imply that transcription factors for CLB2 are involved in the cell wall integrity checkpoint system and suggest that there are multiple regulation pathways of the checkpoint.

  13. Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

    NASA Astrophysics Data System (ADS)

    Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

    Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

  14. Continuous alcohol fermentation by immobilized whole yeast cells

    SciTech Connect

    Hamdy, M.K.; Toledo, R.T.; Kim, K.

    1985-01-01

    Cells of Saccharamoyces cerevisiae strain ASTY-81 were immobilized onto inorganic alumina beads and packed into a reactor column (bioreactor). Feedstock (FS) containing the substrate and nutrients was fed into the bioreactor. Beads with greatest porosity and surface area produced the most ethanol. Factors that affected ethanol productivity included: temperature, pH, flow rate, nutrients, and substrate concentration in FS.

  15. Quantification and characterization of cell wall polysaccharides released by non-Saccharomyces yeast strains during alcoholic fermentation.

    PubMed

    Giovani, Giovanna; Rosi, Iolanda; Bertuccioli, Mario

    2012-11-15

    In order to improve knowledge about the oenological characteristics of non-Saccharomyces yeast strains, and to reconsider their contribution to wine quality, we studied the release of polysaccharides by 13 non-Saccharomyces strains of different species (three wine yeasts, six grape yeasts, and three spoilage yeasts) during alcoholic fermentation in synthetic must. Three Saccharomyces cerevisiae strains were included for comparison. All of the non-Saccharomyces strains released polysaccharides into fermentation medium; the amount released depended on the yeast species, the number of cells formed and their physiological conditions. Normalizing the quantity of macromolecules released to the cell biomass revealed that most non-Saccharomyces strains produced a greater quantity of polysaccharides compared to S. cerevisiae strains after 7 and 14days of fermentation. This capacity was particularly expressed in the studied wine spoilage yeasts (Saccharomycodes ludwigii, Zygosaccharomyces bailii, and Brettanomyces bruxellensis). Chemical characterization of exocellular polysaccharides produced by non-Saccharomyces yeasts revealed them to essentially be mannoproteins with high mannose contents, ranging from 93% for S'codes. ludwigii to 73-74% for Pichia anomala and Starmerella bombicola. Protein contents varied from 9% for P. anomala to 29% for Z. bailii. These compositions were very similar to those of the S. cerevisiae strains, and to the chemical composition of the cell wall mannoproteins of different yeast species. The presence of galactose, in addition to mannose and glucose, in the exocellular polysaccharides released by Schizosaccharomyces pombe, confirmed the parietal nature of the polysaccharides released by non-Saccharomyces yeasts; only this species has a galactomannan located in the outer layer of the cell wall.

  16. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory