WorldWideScience

Sample records for cell networks yeast

  1. Connectivity in the yeast cell cycle transcription network: inferences from neural networks.

    Directory of Open Access Journals (Sweden)

    Christopher E Hart

    2006-12-01

    Full Text Available A current challenge is to develop computational approaches to infer gene network regulatory relationships based on multiple types of large-scale functional genomic data. We find that single-layer feed-forward artificial neural network (ANN models can effectively discover gene network structure by integrating global in vivo protein:DNA interaction data (ChIP/Array with genome-wide microarray RNA data. We test this on the yeast cell cycle transcription network, which is composed of several hundred genes with phase-specific RNA outputs. These ANNs were robust to noise in data and to a variety of perturbations. They reliably identified and ranked 10 of 12 known major cell cycle factors at the top of a set of 204, based on a sum-of-squared weights metric. Comparative analysis of motif occurrences among multiple yeast species independently confirmed relationships inferred from ANN weights analysis. ANN models can capitalize on properties of biological gene networks that other kinds of models do not. ANNs naturally take advantage of patterns of absence, as well as presence, of factor binding associated with specific expression output; they are easily subjected to in silico "mutation" to uncover biological redundancies; and they can use the full range of factor binding values. A prominent feature of cell cycle ANNs suggested an analogous property might exist in the biological network. This postulated that "network-local discrimination" occurs when regulatory connections (here between MBF and target genes are explicitly disfavored in one network module (G2, relative to others and to the class of genes outside the mitotic network. If correct, this predicts that MBF motifs will be significantly depleted from the discriminated class and that the discrimination will persist through evolution. Analysis of distantly related Schizosaccharomyces pombe confirmed this, suggesting that network-local discrimination is real and complements well-known enrichment of

  2. Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

    Directory of Open Access Journals (Sweden)

    Liu Liwen

    2009-09-01

    Full Text Available Abstract Background Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast. Results By integrating genome-wide data from multiple time course cell cycle microarray experiments we reconstructed a gene regulatory network. Based on the network, we discovered in addition to previously known regulatory hubs in M phase, a new putative regulatory hub in the form of the HMG box transcription factor SPBC19G7.04. Further, we inferred periodic activities of several less known transcription factors over the course of the cell cycle, identified over 500 putative regulatory targets and detected many new phase-specific and conserved cis-regulatory motifs. In particular, we show that SPBC19G7.04 has highly significant periodic activity that peaks in early M phase, which is coordinated with the late G2 activity of the forkhead transcription factor fkh2. Finally, using an enhanced Bayesian algorithm to co-cluster the expression data, we obtained 31 clusters of co-regulated genes 1 which constitute regulatory modules from different phases of the cell cycle, 2 whose phase order is coherent across the 10 time course experiments, and 3 which lead to identification of phase-specific control elements at both the transcriptional and post-transcriptional levels in S. pombe. In particular, the ribosome biogenesis clusters expressed in G2 phase reveal new, highly conserved RNA motifs. Conclusion Using a systems-level analysis of the phase-specific nature of the S. pombe cell cycle gene regulation, we have provided new testable evidence for post-transcriptional regulation in the G2 phase of the fission yeast cell cycle

  3. A yeast-based genomic strategy highlights the cell protein networks altered by FTase inhibitor peptidomimetics

    Directory of Open Access Journals (Sweden)

    Porcu Giampiero

    2010-07-01

    Full Text Available Abstract Background Farnesyltransferase inhibitors (FTIs are anticancer agents developed to inhibit Ras oncoprotein activities. FTIs of different chemical structure act via a conserved mechanism in eukaryotic cells. They have low toxicity and are active on a wide range of tumors in cellular and animal models, independently of the Ras activation state. Their ultimate mechanism of action, however, remains undetermined. FTase has hundred of substrates in human cells, many of which play a pivotal role in either tumorigenesis or in pro-survival pathways. This lack of knowledge probably accounts for the failure of FTIs at clinical stage III for most of the malignancies treated, with the notable exception of haematological malignancies. Understanding which cellular pathways are the ultimate targets of FTIs in different tumor types and the basis of FTI resistance is required to improve the efficacy of FTIs in cancer treatment. Results Here we used a yeast-based cellular assay to define the transcriptional changes consequent to FTI peptidomimetic administration in conditions that do not substantially change Ras membrane/cytosol distribution. Yeast and cancer cell lines were used to validate the results of the network analysis. The transcriptome of yeast cells treated with FTase inhibitor I was compared with that of untreated cells and with an isogenic strain genetically inhibited for FTase activity (Δram1. Cells treated with GGTI-298 were analyzed in a parallel study to validate the specificity of the FTI response. Network analysis, based on gene ontology criteria, identified a cell cycle gene cluster up-regulated by FTI treatment that has the Aurora A kinase IPL1 and the checkpoint protein MAD2 as hubs. Moreover, TORC1-S6K-downstream effectors were found to be down-regulated in yeast and mammalian FTI-treated cells. Notably only FTIs, but not genetic inhibition of FTase, elicited up-regulation of ABC/transporters. Conclusions This work provides a view

  4. TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network.

    Science.gov (United States)

    Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L; Costanzo, Michael; Andrews, Brenda; Boone, Charles

    2017-05-05

    Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. Copyright © 2017 Usaj et al.

  5. Endoplasmic reticulum involvement in yeast cell death

    International Nuclear Information System (INIS)

    Nicanor Austriaco, O.

    2012-01-01

    Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

  6. Synchronization of glycolytic oscillations in a yeast cell population

    DEFF Research Database (Denmark)

    Dano, S.; Hynne, F.; De Monte, Silvia

    2001-01-01

    The mechanism of active phase synchronization in a suspension of oscillatory yeast cells has remained a puzzle for almost half a century. The difficulty of the problem stems from the fact that the synchronization phenomenon involves the entire metabolic network of glycolysis and fermentation, and...

  7. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...... causing significant DNA damage was 20 μM for H2O2 and 200 mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA...

  8. Live Cell Imaging in Fission Yeast.

    Science.gov (United States)

    Mulvihill, Daniel P

    2017-10-03

    Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast. © 2017 Cold Spring Harbor Laboratory Press.

  9. Immobilization of yeast cells by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Fujimura, T.; Kaetsu, I.

    1982-01-01

    Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system. (orig.)

  10. Sorption of grape proanthocyanidins and wine polyphenols by yeasts, inactivated yeasts, and yeast cell walls.

    Science.gov (United States)

    Mekoue Nguela, J; Sieczkowski, N; Roi, S; Vernhet, A

    2015-01-21

    Inactivated yeast fractions (IYFs) can be used in enology to improve the stability and mouthfeel of red wines. However, information concerning the mechanisms involved and the impact of the IYF characteristics is scarce. Adsorption isotherms were used to investigate interactions between grape proanthocyanidin fractions (PAs) or wine polyphenols (WP) and a commercial yeast strain (Y), the inactivated yeast (IY), the yeast submitted to autolyzis and inactivation (A-IY), and the cell walls obtained by mechanical disruption (CW). High affinity isotherms and high adsorption capacities were observed for grape PAs and whole cells (Y, IY, and A-IY). Affinity and adsorbed amount were lower with wine PAs, due to chemical changes occurring during winemaking. By contrast to whole cells, grape PAs and WP adsorption on CW remained very low. This raises the issue of the part played by cell walls in the interactions between yeast and proanthocyanidins and suggests the passage of the latter through the wall pores and their interaction with the plasma membrane.

  11. Yeast fuel cell: Application for desalination

    Science.gov (United States)

    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.

  12. Networking for proteins : A yeast two-hybrid and RNAi profiling approach to uncover C. elegans cell polarity regulators

    NARCIS (Netherlands)

    Koorman, T.

    2016-01-01

    Cell polarity is a near universal trait of life and guides many aspects of animal development. Although a number of key polarity proteins have been identified, many interactions with proteins acting downstream likely remain to be elucidated. Mutations in polarity proteins or deregulation of polarity

  13. Extraction of proteins from yeast cell wall

    African Journals Online (AJOL)

    USER

    2010-05-24

    May 24, 2010 ... Figure 2. The UV absorption spectrum of extracted proteins. Startup Foundation of Chongqing Normal University (No. 07XLB025), and Natural Science Foundation Project of. CQ CSTC (No. CSTC, 2009BB5238) China. REFERENCES. Cabib E, Roh DH, Schmidt M, Crotti LB, Varma A (2001). The yeast cell.

  14. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  15. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

    Science.gov (United States)

    Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

    2015-01-01

    The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.

  16. Guidelines and recommendations on yeast cell death nomenclature

    NARCIS (Netherlands)

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J; Breitenbach, Michael; Burhans, William C; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W; Grant, Chris M; Greenwood, Michael T; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D; Outeiro, Tiago F; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F; Sharon, Amir; Sigrist, Stephan J; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B; Tuite, Mick; Vögtle, F-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J; Zhao, Richard Y; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely

  17. The core regulation module of stress-responsive regulatory networks in yeast

    Science.gov (United States)

    Kim, Dongsan; Kim, Man-Sun; Cho, Kwang-Hyun

    2012-01-01

    How does a cell respond to numerous external stresses with a limited number of internal molecular components? It has been observed that there are some common responses of yeast to various stresses, but most observations were based on gene-expression profiles and only some part of the common responses were intensively investigated. So far there has been no system-level analysis to identify commonly responsive or regulated genes against various stresses. In this study, we identified a core regulation module (CRM), a commonly involved regulation structure in the regulatory networks of yeast, which cells reuse in response to an array of environmental stresses. We found that regulators in the CRM constitute a hierarchical backbone of the yeast regulatory network and that the CRM is evolutionarily well conserved, stable against genetic variations and crucial for cell growth. All these findings were consistently held up to considerable noise levels that we introduced to address experimental noise and the resulting false positives of regulatory interactions. We conclude that the CRM of yeast might be an evolutionarily conserved information processing unit that endows a cell with enhanced robustness and efficiency in dealing with numerous environmental stresses with a limited number of internal elements. PMID:22784859

  18. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C; knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

  19. Effect of salt hyperosmotic stress on yeast cell viability

    Directory of Open Access Journals (Sweden)

    Logothetis Stelios

    2007-01-01

    Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

  20. Yeast systems biology to unravel the network of life

    DEFF Research Database (Denmark)

    Mustacchi, Roberta; Hohmann, S; Nielsen, Jens

    2006-01-01

    Systems biology focuses on obtaining a quantitative description of complete biological systems, even complete cellular function. In this way, it will be possible to perform computer-guided design of novel drugs, advanced therapies for treatment of complex diseases, and to perform in silico design....... Furthermore, it serves as an industrial workhorse for production of a wide range of chemicals and pharmaceuticals. Systems biology involves the combination of novel experimental techniques from different disciplines as well as functional genomics, bioinformatics and mathematical modelling, and hence no single...... laboratory has access to all the necessary competences. For this reason the Yeast Systems Biology Network (YSBN) has been established. YSBN will coordinate research efforts, in yeast systems biology and, through the recently obtained EU funding for a Coordination Action, it will be possible to set...

  1. The ART-Rsp5 ubiquitin ligase network comprises a plasma membrane quality control system that protects yeast cells from proteotoxic stress

    Science.gov (United States)

    Zhao, Yingying; MacGurn, Jason A; Liu, Max; Emr, Scott

    2013-01-01

    Secretory cargo that cannot fold properly in the ER are selectively targeted for removal by a well-studied ER-associated degradation pathway, or ERAD. In contrast, very little is known about post-ER quality control mechanisms for damaged or misfolded integral membrane proteins. Here we describe a quality control function of the Rsp5-ART ubiquitin ligase adaptor network that functions to protect plasma membrane (PM) integrity. Failure to mediate this protective response during heat stress leads to toxic accumulation of misfolded integral membrane proteins at the cell surface, which causes loss of PM integrity and cell death. Thus, the Rsp5-ART network comprises a PM quality control system that works together with sequential quality control pathways in the ER and Golgi to (i) target the degradation of proteins that have exceeded their functional lifetime due to damage and/or misfolding and (ii) limit the toxic accumulation of specific proteins at the cell surface during proteotoxic stress. DOI: http://dx.doi.org/10.7554/eLife.00459.001 PMID:23599894

  2. Construction and application of a protein and genetic interaction network (yeast interactome).

    Science.gov (United States)

    Stuart, Gregory R; Copeland, William C; Strand, Micheline K

    2009-04-01

    Cytoscape is a bioinformatic data analysis and visualization platform that is well-suited to the analysis of gene expression data. To facilitate the analysis of yeast microarray data using Cytoscape, we constructed an interaction network (interactome) using the curated interaction data available from the Saccharomyces Genome Database (www.yeastgenome.org) and the database of yeast transcription factors at YEASTRACT (www.yeastract.com). These data were formatted and imported into Cytoscape using semi-automated methods, including Linux-based scripts, that simplified the process while minimizing the introduction of processing errors. The methods described for the construction of this yeast interactome are generally applicable to the construction of any interactome. Using Cytoscape, we illustrate the use of this interactome through the analysis of expression data from a recent yeast diauxic shift experiment. We also report and briefly describe the complex associations among transcription factors that result in the regulation of thousands of genes through coordinated changes in expression of dozens of transcription factors. These cells are thus able to sensitively regulate cellular metabolism in response to changes in genetic or environmental conditions through relatively small changes in the expression of large numbers of genes, affecting the entire yeast metabolome.

  3. Lipid raft involvement in yeast cell growth and death

    Directory of Open Access Journals (Sweden)

    Faustino eMollinedo

    2012-10-01

    Full Text Available 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 Crytococcus 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.

  4. Lipid raft involvement in yeast cell growth and death

    International Nuclear Information System (INIS)

    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.

  5. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in ... that the 50% yeast SCP fed fish had the highest percentage of body protein (55.35%), but with a lower amount of fat at the end of the feeding trial compared to the control.

  6. Evolution of protein-protein interaction networks in yeast.

    Directory of Open Access Journals (Sweden)

    Andrew Schoenrock

    Full Text Available Interest in the evolution of protein-protein and genetic interaction networks has been rising in recent years, but the lack of large-scale high quality comparative datasets has acted as a barrier. Here, we carried out a comparative analysis of computationally predicted protein-protein interaction (PPI networks from five closely related yeast species. We used the Protein-protein Interaction Prediction Engine (PIPE, which uses a database of known interactions to make sequence-based PPI predictions, to generate high quality predicted interactomes. Simulated proteomes and corresponding PPI networks were used to provide null expectations for the extent and nature of PPI network evolution. We found strong evidence for conservation of PPIs, with lower than expected levels of change in PPIs for about a quarter of the proteome. Furthermore, we found that changes in predicted PPI networks are poorly predicted by sequence divergence. Our analyses identified a number of functional classes experiencing fewer PPI changes than expected, suggestive of purifying selection on PPIs. Our results demonstrate the added benefit of considering predicted PPI networks when studying the evolution of closely related organisms.

  7. Yeast cell factories on the horizon

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2015-01-01

    been engineered to make chemicals at industrial scale (e.g., succinic acid, lactic acid, resveratrol) and advanced biofuels (e.g., isobutanol) (1). On page 1095 of this issue, Galanie et al. (2) demonstrate that yeast can now be engineered to produce opioids (2), a major class of compounds used...... for treating severe pain. Their study represents a tour de force in the metabolic engineering of yeast, as it involved the expression of genes for more than 20 enzymatic activities from plants, mammals, bacteria, and yeast itself. It clearly represents a breakthrough advance for making complex natural products...

  8. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    DEFF Research Database (Denmark)

    Usaite, Renata; Jewett, Michael Christopher; Soberano de Oliveira, Ana Paula

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite...... levels in wild type, Deltasnf1, Deltasnf4, and Deltasnf1Deltasnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time...

  9. Oxidative Stress and Programmed Cell Death in Yeast

    International Nuclear Information System (INIS)

    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.

  10. Guidelines and recommendations on yeast cell death nomenclature

    Directory of Open Access Journals (Sweden)

    Didac Carmona-Gutierrez

    2018-01-01

    Full Text Available Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

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

    Czech Academy of Sciences Publication Activity Database

    Pálková, Z.; Váchová, Libuše

    2016-01-01

    Roč. 57, SEP (2016), s. 110-119 ISSN 1084-9521 R&D Projects: GA ČR GA13-08605S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Pathogenic yeasts * Biofilms and colonies * Cell differentiation Subject RIV: EE - Microbiology, Virology Impact factor: 6.614, year: 2016

  12. Monitoring of yeast cell concentration using a micromachined impedance sensor

    NARCIS (Netherlands)

    Krommenhoek, E.E.; Gardeniers, Johannes G.E.; Bomer, Johan G.; van den Berg, Albert; Li, X.; Ottens, M.; van der Wielen, L.A.M.; van Dedem, G.W.K.; van Leeuwen, M.; van Gulik, W.M.; Heijnen, J.J.

    2005-01-01

    The paper describes the design, modelling and experimental characterization of a micromachined impedance sensor for on-line monitoring of the viable yeast cell concentration (biomass) in a miniaturized cell assay. Measurements in a Saccharomyces cerevisiae cell culture show that the permittivity of

  13. Yeast cell wall chitin reduces wine haze formation.

    Science.gov (United States)

    Ndlovu, Thulile; Divol, Benoit; Bauer, Florian F

    2018-04-27

    Protein haze formation in bottled wines is a significant concern for the global wine industry and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels indicating differences in haze protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli -produced GFP-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and propose a strategy for optimizing wine yeast strains to improve wine clarification. Importance In this study, we establish a new mechanism by which wine yeast strains can impact on the protein haze formation of wines, and demonstrate that yeast cell wall chitin binds grape chitinase in a chitin-concentration dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also

  14. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

    OpenAIRE

    Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

    2015-01-01

    The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin ...

  15. Reconstruction and validation of RefRec: a global model for the yeast molecular interaction network.

    Directory of Open Access Journals (Sweden)

    Tommi Aho

    2010-05-01

    Full Text Available Molecular interaction networks establish all cell biological processes. The networks are under intensive research that is facilitated by new high-throughput measurement techniques for the detection, quantification, and characterization of molecules and their physical interactions. For the common model organism yeast Saccharomyces cerevisiae, public databases store a significant part of the accumulated information and, on the way to better understanding of the cellular processes, there is a need to integrate this information into a consistent reconstruction of the molecular interaction network. This work presents and validates RefRec, the most comprehensive molecular interaction network reconstruction currently available for yeast. The reconstruction integrates protein synthesis pathways, a metabolic network, and a protein-protein interaction network from major biological databases. The core of the reconstruction is based on a reference object approach in which genes, transcripts, and proteins are identified using their primary sequences. This enables their unambiguous identification and non-redundant integration. The obtained total number of different molecular species and their connecting interactions is approximately 67,000. In order to demonstrate the capacity of RefRec for functional predictions, it was used for simulating the gene knockout damage propagation in the molecular interaction network in approximately 590,000 experimentally validated mutant strains. Based on the simulation results, a statistical classifier was subsequently able to correctly predict the viability of most of the strains. The results also showed that the usage of different types of molecular species in the reconstruction is important for accurate phenotype prediction. In general, the findings demonstrate the benefits of global reconstructions of molecular interaction networks. With all the molecular species and their physical interactions explicitly modeled, our

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

    Science.gov (United States)

    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. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. A yeast synthetic network for in vivo assessment of reverse-engineering and modeling approaches.

    Science.gov (United States)

    Cantone, Irene; Marucci, Lucia; Iorio, Francesco; Ricci, Maria Aurelia; Belcastro, Vincenzo; Bansal, Mukesh; Santini, Stefania; di Bernardo, Mario; di Bernardo, Diego; Cosma, Maria Pia

    2009-04-03

    Systems biology approaches are extensively used to model and reverse engineer gene regulatory networks from experimental data. Conversely, synthetic biology allows "de novo" construction of a regulatory network to seed new functions in the cell. At present, the usefulness and predictive ability of modeling and reverse engineering cannot be assessed and compared rigorously. We built in the yeast Saccharomyces cerevisiae a synthetic network, IRMA, for in vivo "benchmarking" of reverse-engineering and modeling approaches. The network is composed of five genes regulating each other through a variety of regulatory interactions; it is negligibly affected by endogenous genes, and it is responsive to small molecules. We measured time series and steady-state expression data after multiple perturbations. These data were used to assess state-of-the-art modeling and reverse-engineering techniques. A semiquantitative model was able to capture and predict the behavior of the network. Reverse engineering based on differential equations and Bayesian networks correctly inferred regulatory interactions from the experimental data.

  20. Immobilization of yeast cells with various porous carriers by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Fujimura, Takashi; Kaetsu, Isao

    1982-06-01

    Yeast cells were immobilized by radiation-induced polymerization in twice. Various kinds of porous polymer carriers were prepared by radiation-induced polymerization of glass-forming monomers at a low temperature. Precultured yeast cells were incubated aerobically at 30 0 C with these porous carriers for 24 h. Porous carriers with yeast cells were immersed in low concentration monomer solution. Yeast cells were immobilized by radiation-induced polymerization. The maximum ethanol productivity in immobilized yeast system was around 10 times as much as that in free yeast cell system. High activity of immobilized yeast cells was maintained more than 480 h. The growth of yeast cells in immobilized yeast cells during aerobical incubation was indicated. Immobilized yeast cells thus grown were incubated for fermentation reaction. In this immobilized system, 100% of glucose was converted to ethanol, that is 100% ethanol yield was obtained, within 180 min. In free cell system, only 15% ethanol yield was obtained within 180 min. These results indicates clearly the superiority of immobilized growing cell. Yeast cells were also immobilized with non woven material as carrier by radiation-induced polymerization. The relationship between pore size of non woven material and activity in immobilized yeast cells was made clear. (author)

  1. Magnetically responsive yeast cells: methods of preparation and applications

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Maděrová, Zdeňka; Pospišková, K.; Horská, Kateřina; Šafaříková, Miroslava

    2015-01-01

    Roč. 32, č. 1 (2015), s. 227-237 ISSN 0749-503X R&D Projects: GA MŠk(CZ) LD13023; GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : yeast cells * Saccharomyces * Kluyveromyces * Rhodotorula * Yarrowia * magnetic modification Subject RIV: CE - Biochemistry Impact factor: 2.259, year: 2015

  2. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    Jane

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  3. Sorption of volatile phenols by yeast cell walls

    Directory of Open Access Journals (Sweden)

    Nerea Jiménez-Moreno

    2009-01-01

    Full Text Available Nerea Jiménez-Moreno, Carmen Ancín-AzpilicuetaDepartment of Applied Chemistry, Universidad Pública de Navarra, Pamplona, SpainAbstract: Yeast walls can retain different wine compounds and so its use is interesting in order to eliminate harmful substances from the must which affect alcoholic fermentation (medium chain fatty acids or which affect wine quality in a negative way (ethyl phenols, ochratoxin A. The aim of this study was to examine the capacity of commercial yeast cell walls in eliminating volatile phenols (4-ethylphenol and 4-ethylguaiacol from a synthetic wine that contained 1 mg/L of each one of these compounds. The binding of these compounds to the wall was quite fast which would seem to indicate that the yeast wall-volatile compound union is produced in the outer surface layers of this enological additive. The cell walls used reduced the concentration of 4-ethylphenol and 4-ethylguaiacol, although it would seem that on modifying the matrix of the wine the number of free binding sites on the walls is also modified.Keywords: volatile phenols, yeast cell walls, wine, sorption

  4. Virtual mutagenesis of the yeast cyclins genetic network reveals complex dynamics of transcriptional control networks.

    Directory of Open Access Journals (Sweden)

    Eliska Vohradska

    Full Text Available Study of genetic networks has moved from qualitative description of interactions between regulators and regulated genes to the analysis of the interaction dynamics. This paper focuses on the analysis of dynamics of one particular network--the yeast cyclins network. Using a dedicated mathematical model of gene expression and a procedure for computation of the parameters of the model from experimental data, a complete numerical model of the dynamics of the cyclins genetic network was attained. The model allowed for performing virtual experiments on the network and observing their influence on the expression dynamics of the genes downstream in the regulatory cascade. Results show that when the network structure is more complicated, and the regulatory interactions are indirect, results of gene deletion are highly unpredictable. As a consequence of quantitative behavior of the genes and their connections within the network, causal relationship between a regulator and target gene may not be discovered by gene deletion. Without including the dynamics of the system into the network, its functional properties cannot be studied and interpreted correctly.

  5. Immobilized yeast cell systems for continuous fermentation applications.

    Science.gov (United States)

    Verbelen, Pieter J; De Schutter, David P; Delvaux, Filip; Verstrepen, Kevin J; Delvaux, Freddy R

    2006-10-01

    In several yeast-related industries, continuous fermentation systems offer important economical advantages in comparison with traditional systems. Fermentation rates are significantly improved, especially when continuous fermentation is combined with cell immobilization techniques to increase the yeast concentration in the fermentor. Hence the technique holds a great promise for the efficient production of fermented beverages, such as beer, wine and cider as well as bio-ethanol. However, there are some important pitfalls, and few industrial-scale continuous systems have been implemented. Here, we first review the various cell immobilization techniques and reactor setups. Then, the impact of immobilization on cell physiology and fermentation performance is discussed. In a last part, we focus on the practical use of continuous fermentation and cell immobilization systems for beer production.

  6. Effects of gamma radiation on Sporothrix schenckii yeast cells

    International Nuclear Information System (INIS)

    Lacerda, Camila M. de Sousa; Martins, Estefania Mara Nascimento; Andrade, Antero S.R.; Resende, Maria Aparecida de

    2009-01-01

    Sporotrichosis is a subacute or chronic infection caused by the fungus Sporothrix schenckii. Zoonotic transmission can occur after scratches or bites of animals, mainly cats, rodents, and armadillos. Up to the moment, no approved vaccine was reported for S. schenckii or to any important pathogenic fungi infection in humans, indicating the need to expand the research in this field and to explore new alternatives. The aim of this study was to evaluate the effects of gamma radiation in the viability, metabolic activity and reproductive ability of S. schenckii yeast cells for further studies on the development of a vaccine for immunization of cats and dogs. The culture of S. schenckii, in solid medium, was irradiated at doses ranging from 1.0 to 9.0 kGy. After each dose the reproductive capacity, viability and protein synthesis were estimated. The results showed that a reduction of 6 log 10 cycles in the number of colonies was achieved at 6.0 kGy and after 8.0 kGy no colonies could be recovered. The viability analysis indicated that yeast cells remained viable up to 9.0 kGy. The results of protein synthesis analysis showed that the yeast cells, irradiated up to 9.0 kGy, were able to synthesize proteins. Our preliminary results indicated that for the yeast cells of S. schenckii, it is possible to find an absorbed dose in which the pathogen loses its reproductive ability, while retaining its viability, a necessary condition for the development of a radioattenuated yeast vaccine. (author)

  7. Effects of gamma radiation on Sporothrix schenckii yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacerda, Camila M. de Sousa; Martins, Estefania Mara Nascimento; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: cmsl@cdtn.br, e-mail: estefaniabio@yahoo.com.br, e-mail: antero@cdtn.br; Resende, Maria Aparecida de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: maressend@mono.icb.ufmg.br

    2009-07-01

    Sporotrichosis is a subacute or chronic infection caused by the fungus Sporothrix schenckii. Zoonotic transmission can occur after scratches or bites of animals, mainly cats, rodents, and armadillos. Up to the moment, no approved vaccine was reported for S. schenckii or to any important pathogenic fungi infection in humans, indicating the need to expand the research in this field and to explore new alternatives. The aim of this study was to evaluate the effects of gamma radiation in the viability, metabolic activity and reproductive ability of S. schenckii yeast cells for further studies on the development of a vaccine for immunization of cats and dogs. The culture of S. schenckii, in solid medium, was irradiated at doses ranging from 1.0 to 9.0 kGy. After each dose the reproductive capacity, viability and protein synthesis were estimated. The results showed that a reduction of 6 log{sub 10} cycles in the number of colonies was achieved at 6.0 kGy and after 8.0 kGy no colonies could be recovered. The viability analysis indicated that yeast cells remained viable up to 9.0 kGy. The results of protein synthesis analysis showed that the yeast cells, irradiated up to 9.0 kGy, were able to synthesize proteins. Our preliminary results indicated that for the yeast cells of S. schenckii, it is possible to find an absorbed dose in which the pathogen loses its reproductive ability, while retaining its viability, a necessary condition for the development of a radioattenuated yeast vaccine. (author)

  8. Hubs with network motifs organize modularity dynamically in the protein-protein interaction network of yeast.

    Science.gov (United States)

    Jin, Guangxu; Zhang, Shihua; Zhang, Xiang-Sun; Chen, Luonan

    2007-11-21

    It has been recognized that modular organization pervades biological complexity. Based on network analysis, 'party hubs' and 'date hubs' were proposed to understand the basic principle of module organization of biomolecular networks. However, recent study on hubs has suggested that there is no clear evidence for coexistence of 'party hubs' and 'date hubs'. Thus, an open question has been raised as to whether or not 'party hubs' and 'date hubs' truly exist in yeast interactome. In contrast to previous studies focusing on the partners of a hub or the individual proteins around the hub, our work aims to study the network motifs of a hub or interactions among individual proteins including the hub and its neighbors. Depending on the relationship between a hub's network motifs and protein complexes, we define two new types of hubs, 'motif party hubs' and 'motif date hubs', which have the same characteristics as the original 'party hubs' and 'date hubs' respectively. The network motifs of these two types of hubs display significantly different features in spatial distribution (or cellular localizations), co-expression in microarray data, controlling topological structure of network, and organizing modularity. By virtue of network motifs, we basically solved the open question about 'party hubs' and 'date hubs' which was raised by previous studies. Specifically, at the level of network motifs instead of individual proteins, we found two types of hubs, motif party hubs (mPHs) and motif date hubs (mDHs), whose network motifs display distinct characteristics on biological functions. In addition, in this paper we studied network motifs from a different viewpoint. That is, we show that a network motif should not be merely considered as an interaction pattern but be considered as an essential function unit in organizing modules of networks.

  9. Combining inferred regulatory and reconstructed metabolic networks enhances phenotype prediction in yeast.

    Science.gov (United States)

    Wang, Zhuo; Danziger, Samuel A; Heavner, Benjamin D; Ma, Shuyi; Smith, Jennifer J; Li, Song; Herricks, Thurston; Simeonidis, Evangelos; Baliga, Nitin S; Aitchison, John D; Price, Nathan D

    2017-05-01

    Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM's enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells.

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

    Science.gov (United States)

    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

  11. Networks in Cell Biology

    Science.gov (United States)

    Buchanan, Mark; Caldarelli, Guido; De Los Rios, Paolo; Rao, Francesco; Vendruscolo, Michele

    2010-05-01

    Introduction; 1. Network views of the cell Paolo De Los Rios and Michele Vendruscolo; 2. Transcriptional regulatory networks Sarath Chandra Janga and M. Madan Babu; 3. Transcription factors and gene regulatory networks Matteo Brilli, Elissa Calistri and Pietro Lió; 4. Experimental methods for protein interaction identification Peter Uetz, Björn Titz, Seesandra V. Rajagopala and Gerard Cagney; 5. Modeling protein interaction networks Francesco Rao; 6. Dynamics and evolution of metabolic networks Daniel Segré; 7. Hierarchical modularity in biological networks: the case of metabolic networks Erzsébet Ravasz Regan; 8. Signalling networks Gian Paolo Rossini; Appendix 1. Complex networks: from local to global properties D. Garlaschelli and G. Caldarelli; Appendix 2. Modelling the local structure of networks D. Garlaschelli and G. Caldarelli; Appendix 3. Higher-order topological properties S. Ahnert, T. Fink and G. Caldarelli; Appendix 4. Elementary mathematical concepts A. Gabrielli and G. Caldarelli; References.

  12. Gamma irradiation induced ultrastructural changes in Paracoccidioides brasiliensis yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Demicheli, Marina C.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)]. E-mails: marinademicheli@yahoo.com.br; antero@cdtn.br; Goes, Alfredo Miranda [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia]. E-mail: goes@mono.icb.ufmg.br

    2007-07-01

    Paracoccidioides brasiliensis is a thermally dimorphic fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans with high prevalence in Latin America. Up to the moment no vaccine has still been reported. Ionizing radiation can be used to attenuate pathogens for vaccine development and we have successfully attenuated yeast cells of P. brasiliensis by gamma irradiation. The aim of the present study was to examine at ultrastructural level the effects of gamma irradiation attenuation on the morphology of P. brasiliensis yeast cells. P. brasiliensis (strain Pb-18) cultures were irradiated with a dose of 6.5 kGy. The irradiated cells were examined by scanning and also transmission electron microscopy. When examined two hours after the irradiation by scanning electron microscopy the 6.5 kGy irradiated cells presented deep folds or were collapsed. These lesions were reversible since examined 48 hours after irradiation the yeast have recovered the usual morphology. The transmission electron microscopy showed that the irradiated cells plasma membrane and cell wall were intact and preserved. Remarkable changes were found in the nucleus that was frequently in a very electrodense form. A extensive DNA fragmentation was produced by the gamma irradiation treatment. (author)

  13. Yeast 5 – an expanded reconstruction of the Saccharomyces cerevisiae metabolic network

    Directory of Open Access Journals (Sweden)

    Heavner Benjamin D

    2012-06-01

    Full Text Available Abstract Background Efforts to improve the computational reconstruction of the Saccharomyces cerevisiae biochemical reaction network and to refine the stoichiometrically constrained metabolic models that can be derived from such a reconstruction have continued since the first stoichiometrically constrained yeast genome scale metabolic model was published in 2003. Continuing this ongoing process, we have constructed an update to the Yeast Consensus Reconstruction, Yeast 5. The Yeast Consensus Reconstruction is a product of efforts to forge a community-based reconstruction emphasizing standards compliance and biochemical accuracy via evidence-based selection of reactions. It draws upon models published by a variety of independent research groups as well as information obtained from biochemical databases and primary literature. Results Yeast 5 refines the biochemical reactions included in the reconstruction, particularly reactions involved in sphingolipid metabolism; updates gene-reaction annotations; and emphasizes the distinction between reconstruction and stoichiometrically constrained model. Although it was not a primary goal, this update also improves the accuracy of model prediction of viability and auxotrophy phenotypes and increases the number of epistatic interactions. This update maintains an emphasis on standards compliance, unambiguous metabolite naming, and computer-readable annotations available through a structured document format. Additionally, we have developed MATLAB scripts to evaluate the model’s predictive accuracy and to demonstrate basic model applications such as simulating aerobic and anaerobic growth. These scripts, which provide an independent tool for evaluating the performance of various stoichiometrically constrained yeast metabolic models using flux balance analysis, are included as Additional files 1, 2 and 3. Additional file 1 Function testYeastModel.m.m. Click here for file Additional file 2 Function model

  14. Mechanical characterization of yeast cells: effects of growth conditions.

    Science.gov (United States)

    Overbeck, A; Kampen, I; Kwade, A

    2015-10-01

    Industrial biotechnology uses microbiological cells to produce a wide range of products. While the organisms in question are well understood regarding their genetic and molecular properties, less is known about their mechanical properties. Previous work has established a testing procedure for single Saccharomyces cerevisiae cells using a Nanoindenter equipped with a Flat Punch probe, allowing the compression between two parallel surfaces. The resulting force-displacement curves clearly showed the bursting of the cells and served to determine characteristic values such as the bursting force, bursting energy and relative deformation. This study examined the mechanical characteristics of yeast cells under the influence of varying cultivation parameters, namely the pH value, temperature, aeration rate, stirrer speed and culture medium composition. It was observed that only temperature and medium composition showed significant effect on the mechanical properties of the cells. Higher temperatures during cultivation caused lower bursting forces and energies. Further analysis of the data showed that the mechanical characteristics of the cells were only influenced by parameters which also had an influence on the growth rate. In conclusion, higher growth rates result in a lower mechanical strength of the yeast cells. This study provides data on the influence of growth conditions on the mechanical properties of yeast cells. Single cell compression tests on Saccharomyces cerevisiae cells indicate that higher growth rates result in a lower mechanical strength of the cells. As in biotechnological processes mechanical degradation is often part of the downstream process to release the product from the micro-organisms, the knowledge about the mechanical properties of the cells is relevant for process optimization. © 2015 The Society for Applied Microbiology.

  15. High power density yeast catalyzed microbial fuel cells

    Science.gov (United States)

    Ganguli, Rahul

    Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density

  16. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    Science.gov (United States)

    Usaite, Renata; Jewett, Michael C; Oliveira, Ana Paula; Yates, John R; Olsson, Lisbeth; Nielsen, Jens

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases. PMID:19888214

  17. Relationship Between the Sterol Content of Yeast Cells and Their Fermentation Activity in Grape Must

    OpenAIRE

    Larue, F.; Lafon-Lafourcade, S.; Ribereau-Gayon, P.

    1980-01-01

    In grape must of high sugar concentration, yeast growth, the viability rate of “resting” yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols add...

  18. A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology

    DEFF Research Database (Denmark)

    Herrgard, Markus; Swainston, Neil; Dobson, Paul

    2008-01-01

    a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously...... of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms....

  19. [Yeast urinary tract infections. Multicentre study in 14 hospitals belonging to the Buenos Aires City Mycology Network].

    Science.gov (United States)

    Maldonado, Ivana; Arechavala, Alicia; Guelfand, Liliana; Relloso, Silvia; Garbasz, Claudia

    2016-01-01

    Urinary tract infections are a frequent ailment in patients in intensive care units. Candida and other yeasts cause 5-12% of these infections. The value of the finding of any yeast is controversial, and there is no consensus about which parameters are adequate for differentiating urinary infections from colonization or contamination. To analyse the epidemiological characteristics of patients with funguria, to determine potential cut-off points in cultures (to distinguish an infection from other conditions), to identify the prevalent yeast species, and to determine the value of a second urine sample. A multicentre study was conducted in intensive care units of 14 hospitals in the Buenos Aires City Mycology Network. The first and second samples of urine from every patient were cultured. The presence of white cells and yeasts in direct examination, colony counts, and the identification of the isolated species, were evaluated. Yeasts grew in 12.2% of the samples. There was no statistical correlation between the number of white cells and the fungal colony-forming units. Eighty five percent of the patients had indwelling catheters. Funguria was not prevalent in women or in patients over the age of 65. Candida albicans, followed by Candida tropicalis, were the most frequently isolated yeasts. Candida parapsilosis and Candida glabrata appeared less frequently. The same species were isolated in 70% of second samples, and in 23% of the cases the second culture was negative. It was not possible to determine a useful cut-off point for colony counts to help in the diagnosis of urinary infections. As in other publications, C. albicans, followed by C. tropicalis, were the most prevalent species. Copyright © 2015 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  20. Evaluation of a recombinant yeast cell estrogen screening assay.

    OpenAIRE

    Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

    1997-01-01

    A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estroge...

  1. Content of endogenous thiols and radioresistance of gemmating cells of Saccharomyces ellipsoideus and Saccharomyces cerevisiale yeasts

    International Nuclear Information System (INIS)

    Simonyan, N.V.; Avakyan, Ts.M.; Dzhanpoladyan, N.L.; Stepanyan, L.G.

    1983-01-01

    It has been shown that gemmating cells of ''wild type'' yeasts are more radioresistant and contain more endogenous thiols, than resting cells. Gemmating cells of Saccharomyces cerevisial yeasts, carrying the mutation rad 51, as to radioresistance and content of SH groups do not differ from resting cells. The results obtained testify to a connec-- tion between increased radioresistance of the yeast gemmating cells and increased content of endogenous thiols in them

  2. Network Thermodynamic Curation of Human and Yeast Genome-Scale Metabolic Models

    Science.gov (United States)

    Martínez, Verónica S.; Quek, Lake-Ee; Nielsen, Lars K.

    2014-01-01

    Genome-scale models are used for an ever-widening range of applications. Although there has been much focus on specifying the stoichiometric matrix, the predictive power of genome-scale models equally depends on reaction directions. Two-thirds of reactions in the two eukaryotic reconstructions Homo sapiens Recon 1 and Yeast 5 are specified as irreversible. However, these specifications are mainly based on biochemical textbooks or on their similarity to other organisms and are rarely underpinned by detailed thermodynamic analysis. In this study, a to our knowledge new workflow combining network-embedded thermodynamic and flux variability analysis was used to evaluate existing irreversibility constraints in Recon 1 and Yeast 5 and to identify new ones. A total of 27 and 16 new irreversible reactions were identified in Recon 1 and Yeast 5, respectively, whereas only four reactions were found with directions incorrectly specified against thermodynamics (three in Yeast 5 and one in Recon 1). The workflow further identified for both models several isolated internal loops that require further curation. The framework also highlighted the need for substrate channeling (in human) and ATP hydrolysis (in yeast) for the essential reaction catalyzed by phosphoribosylaminoimidazole carboxylase in purine metabolism. Finally, the framework highlighted differences in proline metabolism between yeast (cytosolic anabolism and mitochondrial catabolism) and humans (exclusively mitochondrial metabolism). We conclude that network-embedded thermodynamics facilitates the specification and validation of irreversibility constraints in compartmentalized metabolic models, at the same time providing further insight into network properties. PMID:25028891

  3. Understanding gene essentiality by finely characterizing hubs in the yeast protein interaction network.

    Science.gov (United States)

    Pang, Kaifang; Sheng, Huanye; Ma, Xiaotu

    2010-10-08

    The centrality-lethality rule, i.e., high-degree proteins or hubs tend to be more essential than low-degree proteins in the yeast protein interaction network, reveals that a protein's central position indicates its important function, but whether and why hubs tend to be more essential have been heavily debated. Here, we integrated gene expression and functional module data to classify hubs into four types: non-co-expressed non-co-cluster hubs, non-co-expressed co-cluster hubs, co-expressed non-co-cluster hubs and co-expressed co-cluster hubs. We found that all the four hub types are more essential than non-hubs, but they also show different enrichments in essential proteins. Non-co-expressed non-co-cluster hubs play key role in organizing different modules formed by the other three hub types, but they are less important to the survival of the yeast cell. Among the four hub types, co-expressed co-cluster hubs, which likely correspond to the core components of stable protein complexes, are the most essential. These results demonstrated that our classification of hubs into four types could better improve the understanding of gene essentiality. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. A systems-level approach for metabolic engineering of yeast cell factories.

    Science.gov (United States)

    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. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. A Comparative Study of the Cell Wall Structure of Basidiomycetous and Related Yeasts

    NARCIS (Netherlands)

    Kreger-van Rij, N.J.W.; Veenhuis, M.

    1971-01-01

    The wall of basidiomycetous and related yeasts showed a lamellar structure in sections of both budding cells and hyphae fixed with potassium permanganate. The yeasts also had a typical way of bud formation and septation. These features differ from those recorded for ascomycetous yeasts. In the

  6. A study of ethanol production of yeast cells immobilized with polymer carrier produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu Zhaoxin; Fujimura, Takashi

    1993-01-01

    Polymer carriers, poly(hydroxyethyl acrylate(HEA)-methoxy polyethylene glycol methylacrylate (M-23G)) and poly(hydroxyethyl acrylate(HEA)-glycidyl methylacrylate (GMA)) used for the immobilization of yeast cells were prepared by radiation polymerization at low temperature. Yeast cells were immobilized through adhesion and multiplication of yeast cells. The ethanol productivity of immobilized yeast cells with these carriers was related to the monomer composition of polymers and the optimum monomer composition was 20%:10% in poly(HEA-M-23G) and 17%:6% in poly(HEA-GMA). In this case, the ethanol productivity of immobilized yeast cells was about 4 times that of cells in free system. The relationship between the activity of immobilized yeast cells and the water content of the polymer carrier were also discussed. (author)

  7. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall...... with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...

  8. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  9. Possibility for simultaneous electricity generation and bioremediation by using Candida melibiosica yeast in biofuel cell

    International Nuclear Information System (INIS)

    Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

    2013-01-01

    Recently, we have proved that Candida melibiosica 2491 yeast strain possesses electrogenic properties and could be used as a biocatalyst in yeast-based biofuel cells. In this paper we demonstrate that when the yeast is cultivated under polarization conditions in a biofuel cell its phytase activity exceeds that obtained during cultivation in a conventional bioreactor. Furthermore, there is a correlation between the yeast phytase activity and the electrical characteristic of the biofuel cell during the different yeast growth phases. The obtained results reveal a possibility for application of C.melibiosica for simultaneous electricity generation and bioremediation of hardly degradable polyphosphates, especially in the regions with intensive stock-farming. Keywords: Biofuel cells, yeast, Candida melibiosica, electricity generation, bioremediation

  10. Systematic Analysis of the DNA Damage Response Network in Telomere Defective Budding Yeast

    Directory of Open Access Journals (Sweden)

    Eva-Maria Holstein

    2017-07-01

    Full Text Available Functional telomeres are critically important to eukaryotic genetic stability. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. Genetic interactions were examined in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA binding complex that binds telomeric DNA. For comparison, genetic interactions were also examined in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. In more complex experiments, genetic interactions were measured in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response, and containing a cdc13-1 induced telomere defect. Comparing fitness profiles across these data sets helps build a picture of the specific responses to different types of dysfunctional telomeres. The experiments show that each context reveals different genetic interactions, consistent with the idea that each genetic defect causes distinct molecular defects. To help others engage with the large volumes of data, the data are made available via two interactive web-based tools: Profilyzer and DIXY. One particularly striking genetic interaction observed was that the chk1∆ mutation improved fitness of cdc13-1 exo1∆ cells more than other checkpoint mutations (ddc1∆, rad9∆, rad17∆, and rad24∆, whereas, in cdc13-1 cells, the effects of all checkpoint mutations were similar. We show that this can be explained by Chk1 stimulating resection—a new function for Chk1 in the eukaryotic DNA damage response network.

  11. Exploring hierarchical and overlapping modular structure in the yeast protein interaction network

    Directory of Open Access Journals (Sweden)

    Zhao Yi

    2010-12-01

    Full Text Available Abstract Background Developing effective strategies to reveal modular structures in protein interaction networks is crucial for better understanding of molecular mechanisms of underlying biological processes. In this paper, we propose a new density-based algorithm (ADHOC for clustering vertices of a protein interaction network using a novel subgraph density measurement. Results By statistically evaluating several independent criteria, we found that ADHOC could significantly improve the outcome as compared with five previously reported density-dependent methods. We further applied ADHOC to investigate the hierarchical and overlapping modular structure in the yeast PPI network. Our method could effectively detect both protein modules and the overlaps between them, and thus greatly promote the precise prediction of protein functions. Moreover, by further assaying the intermodule layer of the yeast PPI network, we classified hubs into two types, module hubs and inter-module hubs. Each type presents distinct characteristics both in network topology and biological functions, which could conduce to the better understanding of relationship between network architecture and biological implications. Conclusions Our proposed algorithm based on the novel subgraph density measurement makes it possible to more precisely detect hierarchical and overlapping modular structures in protein interaction networks. In addition, our method also shows a strong robustness against the noise in network, which is quite critical for analyzing such a high noise network.

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

    Directory of Open Access Journals (Sweden)

    Ping Liu

    2015-10-01

    Full Text Available 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.

  13. Microbiology and epidemiology of oral yeast colonization in hemopoietic progenitor cell transplant recipients.

    Science.gov (United States)

    Westbrook, Steven D; Kirkpatrick, William R; Wiederhold, Nathan P; Freytes, Cesar O; Toro, Juan J; Patterson, Thomas F; Redding, Spencer W

    2013-03-01

    We monitored the epidemiology and microbiology of oral yeast colonization in patients undergoing hemopoietic progenitor cell transplantation (HPCT) to examine associations between yeast colonization and oral mucositis. One hundred twenty-one consecutive HPCT patients were sampled for oral yeasts prior to fluconazole (FLC) prophylaxis, at transplantation, and weekly until discharge. Clinical oral mucositis screenings were performed triweekly. Yeast colonization was evident at 216 of 510 total visits. Candida albicans and Candida glabrata were the predominant organisms. Eight patients showed elevated minimal inhibitory concentrations to FLC. One patient developed fungal septicemia. Patients with oral mucositis assessment scale scores oral yeasts in HPCT recipients. FLC-resistant yeasts do emerge and can be the source of fungal sepsis. A positive association was not shown between yeast colonization and the presence or severity of oral mucositis. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Effects of Selenium on Morphological Changes in Candida utilis ATCC 9950 Yeast Cells.

    Science.gov (United States)

    Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna; Kurcz, Agnieszka

    2016-02-01

    This paper presents the results of microscopic examinations of the yeast cells cultured in yeast extract-peptone-dextrose (YPD) media supplemented with sodium selenite(IV). The analysis of the morphological changes in yeast cells aimed to determine whether the selected selenium doses and culturing time may affect this element accumulation in yeast cell structures in a form of inorganic or organic compounds, as a result of detoxification processes. The range of characteristic morphological changes in yeasts cultivated in experimental media with sodium selenite(IV) was observed, including cell shrinkage and cytoplasm thickening of the changes within vacuole structure. The processes of vacuole disintegration were observed in aging yeast cells in culturing medium, which may indicate the presence of so-called ghost cells lacking intracellular organelles The changes occurring in the morphology of yeasts cultured in media supplemented with sodium selenite were typical for stationary phase of yeast growth. From detailed microscopic observations, larger surface area of the cell (6.03 μm(2)) and yeast vacuole (2.17 μm(2)) were noticed after 24-h culturing in the medium with selenium of 20 mg Se(4+)/L. The coefficient of shape of the yeast cells cultured in media enriched with sodium selenite as well as in the control YPD medium ranged from 1.02 to 1.22. Elongation of cultivation time (up to 48 and 72 h) in the media supplemented with sodium selenite caused a reduction in the surface area of the yeast cell and vacuole due to detoxification processes.

  15. Solving the influence maximization problem reveals regulatory organization of the yeast cell cycle.

    Directory of Open Access Journals (Sweden)

    David L Gibbs

    2017-06-01

    Full Text Available The Influence Maximization Problem (IMP aims to discover the set of nodes with the greatest influence on network dynamics. The problem has previously been applied in epidemiology and social network analysis. Here, we demonstrate the application to cell cycle regulatory network analysis for Saccharomyces cerevisiae. Fundamentally, gene regulation is linked to the flow of information. Therefore, our implementation of the IMP was framed as an information theoretic problem using network diffusion. Utilizing more than 26,000 regulatory edges from YeastMine, gene expression dynamics were encoded as edge weights using time lagged transfer entropy, a method for quantifying information transfer between variables. By picking a set of source nodes, a diffusion process covers a portion of the network. The size of the network cover relates to the influence of the source nodes. The set of nodes that maximizes influence is the solution to the IMP. By solving the IMP over different numbers of source nodes, an influence ranking on genes was produced. The influence ranking was compared to other metrics of network centrality. Although the top genes from each centrality ranking contained well-known cell cycle regulators, there was little agreement and no clear winner. However, it was found that influential genes tend to directly regulate or sit upstream of genes ranked by other centrality measures. The influential nodes act as critical sources of information flow, potentially having a large impact on the state of the network. Biological events that affect influential nodes and thereby affect information flow could have a strong effect on network dynamics, potentially leading to disease. Code and data can be found at: https://github.com/gibbsdavidl/miergolf.

  16. Solving the influence maximization problem reveals regulatory organization of the yeast cell cycle

    Science.gov (United States)

    Shmulevich, Ilya

    2017-01-01

    The Influence Maximization Problem (IMP) aims to discover the set of nodes with the greatest influence on network dynamics. The problem has previously been applied in epidemiology and social network analysis. Here, we demonstrate the application to cell cycle regulatory network analysis for Saccharomyces cerevisiae. Fundamentally, gene regulation is linked to the flow of information. Therefore, our implementation of the IMP was framed as an information theoretic problem using network diffusion. Utilizing more than 26,000 regulatory edges from YeastMine, gene expression dynamics were encoded as edge weights using time lagged transfer entropy, a method for quantifying information transfer between variables. By picking a set of source nodes, a diffusion process covers a portion of the network. The size of the network cover relates to the influence of the source nodes. The set of nodes that maximizes influence is the solution to the IMP. By solving the IMP over different numbers of source nodes, an influence ranking on genes was produced. The influence ranking was compared to other metrics of network centrality. Although the top genes from each centrality ranking contained well-known cell cycle regulators, there was little agreement and no clear winner. However, it was found that influential genes tend to directly regulate or sit upstream of genes ranked by other centrality measures. The influential nodes act as critical sources of information flow, potentially having a large impact on the state of the network. Biological events that affect influential nodes and thereby affect information flow could have a strong effect on network dynamics, potentially leading to disease. Code and data can be found at: https://github.com/gibbsdavidl/miergolf. PMID:28628618

  17. Solving the influence maximization problem reveals regulatory organization of the yeast cell cycle.

    Science.gov (United States)

    Gibbs, David L; Shmulevich, Ilya

    2017-06-01

    The Influence Maximization Problem (IMP) aims to discover the set of nodes with the greatest influence on network dynamics. The problem has previously been applied in epidemiology and social network analysis. Here, we demonstrate the application to cell cycle regulatory network analysis for Saccharomyces cerevisiae. Fundamentally, gene regulation is linked to the flow of information. Therefore, our implementation of the IMP was framed as an information theoretic problem using network diffusion. Utilizing more than 26,000 regulatory edges from YeastMine, gene expression dynamics were encoded as edge weights using time lagged transfer entropy, a method for quantifying information transfer between variables. By picking a set of source nodes, a diffusion process covers a portion of the network. The size of the network cover relates to the influence of the source nodes. The set of nodes that maximizes influence is the solution to the IMP. By solving the IMP over different numbers of source nodes, an influence ranking on genes was produced. The influence ranking was compared to other metrics of network centrality. Although the top genes from each centrality ranking contained well-known cell cycle regulators, there was little agreement and no clear winner. However, it was found that influential genes tend to directly regulate or sit upstream of genes ranked by other centrality measures. The influential nodes act as critical sources of information flow, potentially having a large impact on the state of the network. Biological events that affect influential nodes and thereby affect information flow could have a strong effect on network dynamics, potentially leading to disease. Code and data can be found at: https://github.com/gibbsdavidl/miergolf.

  18. Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

    Science.gov (United States)

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-02-01

    In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sifontes, Ángela B., E-mail: asifonte@ivic.gob.ve [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); González, Gema [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Tovar, Leidy M.; Méndez, Franklin J. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Gomes, Maria E. [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Cañizales, Edgar [Área de Análisis Químico Inorgánico, PDVSA, INTEVEP, Los Teques 1070-A (Venezuela, Bolivarian Republic of); Niño-Vega, Gustavo; Villalobos, Hector [Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Brito, Joaquin L. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    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}.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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 2 g −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 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 2 g −1 .

  1. Cell cycle commitment in budding yeast emerges from the cooperation of multiple bistable switches

    Science.gov (United States)

    Zhang, Tongli; Schmierer, Bernhard; Novák, Béla

    2011-01-01

    The start-transition (START) in the G1 phase marks the point in the cell cycle at which a yeast cell initiates a new round of cell division. Once made, this decision is irreversible and the cell is committed to progressing through the entire cell cycle, irrespective of arrest signals such as pheromone. How commitment emerges from the underlying molecular interaction network is poorly understood. Here, we perform a dynamical systems analysis of an established cell cycle model, which has never been analysed from a commitment perspective. We show that the irreversibility of the START transition and subsequent commitment can be consistently explained in terms of the interplay of multiple bistable molecular switches. By applying an existing mathematical model to a novel problem and by expanding the model in a self-consistent manner, we achieve several goals: we bring together a large number of experimental findings into a coherent theoretical framework; we increase the scope and the applicability of the original model; we give a systems level explanation of how the START transition and the cell cycle commitment arise from the dynamical features of the underlying molecular interaction network; and we make clear, experimentally testable predictions. PMID:22645649

  2. Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

    Science.gov (United States)

    Moreno-García, J; Coi, A L; Zara, G; García-Martínez, T; Mauricio, J C; Budroni, M

    2018-03-01

    Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

  3. Synthetic yeast based cell factories for vanillin-glucoside production

    DEFF Research Database (Denmark)

    Strucko, Tomas

    and controlled expression/overexpression of genes of interest. De novo biosynthetic pathway for vanillin-β-glucoside production was employed as a model system for several case studies in this project. In order to construct yeast cell factories fulfilling current demands of industrial biotechnology, methods...... method was further applied for balancing vanillin-β-glucoside production in S. cerevisiae. It was previously demonstrated that de novo biosynthetic pathway is not capable to efficiently convert its precursor metabolite into vanillin-β-glucoside, which resulted in significant accumulation of several...... intermediates. Here, the gene amplification system was used to systematically overexpress individual genes or gene combinations of the biosynthetic pathway. Using this strategy, metabolic bottlenecks were identified and the production yield of vanillin-β-glucoside was 6-fold improved. Several S. cerevisiae...

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

    Directory of Open Access Journals (Sweden)

    Vanessa Palermo

    2012-01-01

    Full Text Available 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.

  5. Electricity production from microbial fuel cell by using yeast

    International Nuclear Information System (INIS)

    Vorasingha, A.; Souvakon, C.; Boonchom, K.

    2006-01-01

    The continuous search for methods to generate electricity from renewable sources such as water, solar energy, wind, nuclear or chemicals was discussed with particular focus on attaining the full power of the microbial fuel cell (MFC). Under ideal environmental conditions, the only byproducts of a biofuel cell would be water and carbon dioxide (CO 2 ). The production of energy from renewables such as biomass is important for sustainable development and reducing global emissions of CO 2 . Hydrogen can also be an important component of an energy infrastructure that reduces CO 2 emissions if the hydrogen is produced from renewable sources and used in fuel cells. Hydrogen gas can be biologically produced at high concentration from the fermentation of high sugar substrates such as glucose and sucrose. Some of the issues of MFC design were addressed, including the use of cheap substrates to derive microbial electricity. In the MFC, yeast donates electrons to a chemical electron mediator, which in turn transfers the electrons to an electrode, producing electricity. Experimental results showed that glucose yielded the highest peak voltage, but a semi-processed sugar and molasses were similar to glucose in the electricity production pattern. It was noted that this technology is only at the research stages, and more research is needed before household microbial fuel cells can be made available for producing power for prolonged periods of time. Future research efforts will focus on increasing the efficiency, finding alternatives to hazardous electron mediators and finding new microbes. 12 refs., 6 figs

  6. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  7. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    International Nuclear Information System (INIS)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D'Urso, Gennaro; Huberman, Joel A.

    2003-01-01

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication

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

    Directory of Open Access Journals (Sweden)

    Weitao Chen

    2016-07-01

    Full Text Available 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

  9. Virtual Mutagenesis of the Yeast Cyclins Genetic Network Reveals Complex Dynamics of Transcriptional Control Networks

    Czech Academy of Sciences Publication Activity Database

    Vohradská, E.; Vohradský, Jiří

    2011-01-01

    Roč. 6, č. 4 (2011), s. 1-9 E-ISSN 1932-6203 R&D Projects: GA ČR GA310/07/1009; GA ČR GAP302/11/0229 Institutional research plan: CEZ:AV0Z50200510 Keywords : REGULATORY NETWORKS * SACCHAROMYCES-CEREVISIAE * CELL-CYCLE Subject RIV: EE - Microbiology, Virology Impact factor: 4.092, year: 2011

  10. Performance of a Yeast-mediated Biological Fuel Cell

    Directory of Open Access Journals (Sweden)

    Filip To

    2008-10-01

    Full Text Available 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.

  11. Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

    Science.gov (United States)

    Bodenberger, Nicholas; Kubiczek, Dennis; Paul, Patrick; Preising, Nico; Weber, Lukas; Bosch, Ramona; Hausmann, Rudolf; Gottschalk, Kay-Eberhard; Rosenau, Frank

    2017-03-01

    Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 μm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 μM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.

  12. Comparative Analysis of Yeast Metabolic Network Models Highlights Progress, Opportunities for Metabolic Reconstruction.

    Directory of Open Access Journals (Sweden)

    Benjamin D Heavner

    2015-11-01

    Full Text Available We have compared 12 genome-scale models of the Saccharomyces cerevisiae metabolic network published since 2003 to evaluate progress in reconstruction of the yeast metabolic network. We compared the genomic coverage, overlap of annotated metabolites, predictive ability for single gene essentiality with a selection of model parameters, and biomass production predictions in simulated nutrient-limited conditions. We have also compared pairwise gene knockout essentiality predictions for 10 of these models. We found that varying approaches to model scope and annotation reflected the involvement of multiple research groups in model development; that single-gene essentiality predictions were affected by simulated medium, objective function, and the reference list of essential genes; and that predictive ability for single-gene essentiality did not correlate well with predictive ability for our reference list of synthetic lethal gene interactions (R = 0.159. We conclude that the reconstruction of the yeast metabolic network is indeed gradually improving through the iterative process of model development, and there remains great opportunity for advancing our understanding of biology through continued efforts to reconstruct the full biochemical reaction network that constitutes yeast metabolism. Additionally, we suggest that there is opportunity for refining the process of deriving a metabolic model from a metabolic network reconstruction to facilitate mechanistic investigation and discovery. This comparative study lays the groundwork for developing improved tools and formalized methods to quantitatively assess metabolic network reconstructions independently of any particular model application, which will facilitate ongoing efforts to advance our understanding of the relationship between genotype and cellular phenotype.

  13. Effects of metal salt catalysts on yeast cell growth in ethanol conversion

    Science.gov (United States)

    Chung-Yun Hse; Yin Lin

    2009-01-01

    The effects of the addition of metal salts and metal salt-catalyzed hydrolyzates on yeast cell growth in ethanol fermentation were investigated. Four yeast strains (Saccharomyces cerevisiae WT1, Saccharomyces cerevisiae MT81, Candida sp. 1779, and Klumaromyces fragilis), four metal salts (CuCl2, FeCl3, AgNO3, and I2), two metal salt-catalyzed hydrolyzates (...

  14. Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-Treated Yeast Cells

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Weinert, Brian Tate; Choudhary, Chuna Ram

    2014-01-01

    , phosphorylation, and proteome changes in rapamycin-treated yeast cells. Our data constitutes a detailed proteomic analysis of rapamycin-treated yeast with 3,590 proteins, 8,961 phosphorylation sites, and 2,498 di-Gly modified lysines (putative ubiquitylation sites) quantified. The phosphoproteome was extensively...

  15. Time scale and dimension analysis of a budding yeast cell cycle model

    Directory of Open Access Journals (Sweden)

    Novák Béla

    2006-11-01

    Full Text Available Abstract Background The progress through the eukaryotic cell division cycle is driven by an underlying molecular regulatory network. Cell cycle progression can be considered as a series of irreversible transitions from one steady state to another in the correct order. Although this view has been put forward some time ago, it has not been quantitatively proven yet. Bifurcation analysis of a model for the budding yeast cell cycle has identified only two different steady states (one for G1 and one for mitosis using cell mass as a bifurcation parameter. By analyzing the same model, using different methods of dynamical systems theory, we provide evidence for transitions among several different steady states during the budding yeast cell cycle. Results By calculating the eigenvalues of the Jacobian of kinetic differential equations we have determined the stability of the cell cycle trajectories of the Chen model. Based on the sign of the real part of the eigenvalues, the cell cycle can be divided into excitation and relaxation periods. During an excitation period, the cell cycle control system leaves a formerly stable steady state and, accordingly, excitation periods can be associated with irreversible cell cycle transitions like START, entry into mitosis and exit from mitosis. During relaxation periods, the control system asymptotically approaches the new steady state. We also show that the dynamical dimension of the Chen's model fluctuates by increasing during excitation periods followed by decrease during relaxation periods. In each relaxation period the dynamical dimension of the model drops to one, indicating a period where kinetic processes are in steady state and all concentration changes are driven by the increase of cytoplasmic growth. Conclusion We apply two numerical methods, which have not been used to analyze biological control systems. These methods are more sensitive than the bifurcation analysis used before because they identify those

  16. A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology.

    Science.gov (United States)

    Herrgård, Markus J; Swainston, Neil; Dobson, Paul; Dunn, Warwick B; Arga, K Yalçin; Arvas, Mikko; Blüthgen, Nils; Borger, Simon; Costenoble, Roeland; Heinemann, Matthias; Hucka, Michael; Le Novère, Nicolas; Li, Peter; Liebermeister, Wolfram; Mo, Monica L; Oliveira, Ana Paula; Petranovic, Dina; Pettifer, Stephen; Simeonidis, Evangelos; Smallbone, Kieran; Spasić, Irena; Weichart, Dieter; Brent, Roger; Broomhead, David S; Westerhoff, Hans V; Kirdar, Betül; Penttilä, Merja; Klipp, Edda; Palsson, Bernhard Ø; Sauer, Uwe; Oliver, Stephen G; Mendes, Pedro; Nielsen, Jens; Kell, Douglas B

    2008-10-01

    Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.

  17. Toxicity of Aromatic Ketone to Yeast Cell and Improvement of the Asymmetric Reduction of Aromatic Ketone Catalyzed by Yeast Cell with the Introduction of Resin Adsorption

    Directory of Open Access Journals (Sweden)

    Zhong-Hua Yang

    2008-01-01

    Full Text Available Asymmetric reduction of the prochiral aromatic ketone catalyzed by yeast cells is one of the most promising routes to produce its corresponding enantiopure aromatic alcohol, but the space-time yield does not meet people’s expectations. Therefore, the toxicity of aromatic ketone and aromatic alcohol to the yeast cell is investigated in this work. It has been found that the aromatic compounds are poisonous to the yeast cell. The activity of yeast cell decreases steeply when the concentration of acetophenone (ACP is higher than 30.0 mmol/L. Asymmetric reduction of acetophenone to chiral S-α-phenylethyl alcohol (PEA catalyzed by the yeast cell was chosen as the model reaction to study in detail the promotion effect of the introduction of the resin adsorption on the asymmetric reduction reaction. The resin acts as the substrate reservoir and product extraction agent in situ. It has been shown that this reaction could be remarkably improved with this technique when the appropriate kind of resin is applied. The enantioselectivity and yield are acceptable even though the initial ACP concentration reaches 72.2 mmol/L.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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...... and challenges involved in obtaining superior industrial yeasts with improved ethanol tolerance....

  19. Timing robustness in the budding and fission yeast cell cycles.

    KAUST Repository

    Mangla, Karan

    2010-02-01

    Robustness of biological models has emerged as an important principle in systems biology. Many past analyses of Boolean models update all pending changes in signals simultaneously (i.e., synchronously), making it impossible to consider robustness to variations in timing that result from noise and different environmental conditions. We checked previously published mathematical models of the cell cycles of budding and fission yeast for robustness to timing variations by constructing Boolean models and analyzing them using model-checking software for the property of speed independence. Surprisingly, the models are nearly, but not totally, speed-independent. In some cases, examination of timing problems discovered in the analysis exposes apparent inaccuracies in the model. Biologically justified revisions to the model eliminate the timing problems. Furthermore, in silico random mutations in the regulatory interactions of a speed-independent Boolean model are shown to be unlikely to preserve speed independence, even in models that are otherwise functional, providing evidence for selection pressure to maintain timing robustness. Multiple cell cycle models exhibit strong robustness to timing variation, apparently due to evolutionary pressure. Thus, timing robustness can be a basis for generating testable hypotheses and can focus attention on aspects of a model that may need refinement.

  20. Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

    Science.gov (United States)

    Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

    2018-02-08

    Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nishimura, Akira; Kawahara, Nobuhiro [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan); Takagi, Hiroshi, E-mail: hiro@bs.naist.jp [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan)

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

    Science.gov (United States)

    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.

  3. Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display.

    Science.gov (United States)

    Li, Xiaobo; Jin, Xiaobao; Lu, Xuemei; Chu, Fujiang; Shen, Juan; Ma, Yan; Liu, Manyu; Zhu, Jiayong

    2014-10-01

    To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.

  4. Construction and evaluation of yeast expression networks by database-guided predictions

    Directory of Open Access Journals (Sweden)

    Katharina Papsdorf

    2016-05-01

    Full Text Available DNA-Microarrays are powerful tools to obtain expression data on the genome-wide scale. We performed microarray experiments to elucidate the transcriptional networks, which are up- or down-regulated in response to the expression of toxic polyglutamine proteins in yeast. Such experiments initially generate hit lists containing differentially expressed genes. To look into transcriptional responses, we constructed networks from these genes. We therefore developed an algorithm, which is capable of dealing with very small numbers of microarrays by clustering the hits based on co-regulatory relationships obtained from the SPELL database. Here, we evaluate this algorithm according to several criteria and further develop its statistical capabilities. Initially, we define how the number of SPELL-derived co-regulated genes and the number of input hits influences the quality of the networks. We then show the ability of our networks to accurately predict further differentially expressed genes. Including these predicted genes into the networks improves the network quality and allows quantifying the predictive strength of the networks based on a newly implemented scoring method. We find that this approach is useful for our own experimental data sets and also for many other data sets which we tested from the SPELL microarray database. Furthermore, the clusters obtained by the described algorithm greatly improve the assignment to biological processes and transcription factors for the individual clusters. Thus, the described clustering approach, which will be available through the ClusterEx web interface, and the evaluation parameters derived from it represent valuable tools for the fast and informative analysis of yeast microarray data.

  5. Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, K.; Ueda, M. [Lab. of Applied Biological Chemistry, Kyoto Univ., Yoshida, Kyoto (Japan)

    2004-07-01

    The Cd{sup 2+}-chelating abilities of yeast metallothionein (YMT) and hexa-His displayed on the yeast-cell surface were compared. Display of YMT and hexa-His by {alpha}-agglutinin-based cell-surface engineering was confirmed by immunofluorescent labeling. Surface-engineered yeast cells with YMT and hexa-His fused in tandem showed superior cell-surface adsorption and recovery of Cd{sup 2+} under EDTA treatment on the cell surface than hexa-His-displaying cells. YMT was demonstrated to be more effective than hexa-His for the adsorption of Cd{sup 2+}. Yeast cells displaying YMT and/or hexa-His exhibited a higher potential for the adsorption of Cd{sup 2+} than Escherichia coli cells displaying these molecules. In order to investigate the effect of the displayed YMT and hexa-His on sensitivity to toxic Cd{sup 2+}, growth in Cd{sup 2+}-containing liquid medium was monitored. Unlike hexa-His-displaying cells, cells displaying YMT and hexa-His fused in tandem induced resistance to Cd{sup 2+} through active and enhanced adsorption of toxic Cd{sup 2+}. These results indicate that YMT-displaying yeast cells are a unique bioadsorbent with a functional chelating ability superior to that of E. coli. (orig.)

  6. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  7. Metabolic diversification of cells during the development of yeast colonies

    Czech Academy of Sciences Publication Activity Database

    Váchová, Libuše; Kučerová, Helena; Devaux, F.; Úlehlová, M.; Palková, Z.

    2009-01-01

    Roč. 11, č. 2 (2009), s. 494-504 ISSN 1462-2912 R&D Projects: GA ČR GA204/05/0294; GA ČR GA204/08/0718; GA MŠk(CZ) LC531 Grant - others:GB(GB) Howard Hughes Medical Institute International Research Award Institutional research plan: CEZ:AV0Z50200510 Keywords : yeast * yeast colonies * saccharomyces cerevisiae Subject RIV: EE - Microbiology , Virology Impact factor: 4.909, year: 2009

  8. A Network of Multiple Regulatory Layers Shapes Gene Expression in Fission Yeast

    OpenAIRE

    Lackner, Daniel H.; Beilharz, Traude H.; Marguerat, Samuel; Mata, Juan; Watt, Stephen; Schubert, Falk; Preiss, Thomas; B?hler, J?rg

    2007-01-01

    Summary Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered wide...

  9. Effect of selenium on the Hg, Zn, Fe and Co content of yeast cells

    International Nuclear Information System (INIS)

    Czauderna, M.; Peplowski, A.; Smolinski, S.

    1992-01-01

    The yeast cells, Saccharomyces cerevisiae, were exposed to Hg 2+ ions (10 -4 M) and SeO 2 (2x10 -4 -10 -2 M) or Se-methionine (2x10 -4 M). Instrumental neutron activation analysis (INAA) was used to analyze changes in the Hg, Zn,Fe and Co levels in these cells. When the yeast was incubated in a medium containing 10 -3 M and 10 -2 M Se) 2 , the Hg content of the yeast markedly increased. It was also found that the uptake of Se and Hg influenced the levels of Zn, Fe and Co found in the cells. While the presence of Se-methionine (Se-Met), SeO 2 or Hg 2+ ions caused increases in the intracellular Zn levels, the combined presence of Hg 2+ and SeO 2 and their assumed interaction, reduced the efficiency of Se for increasing the Zn content of yeast. (author) 17 refs.; 3 tabs

  10. Detection and quantitative determination by PIXE of the mutagen Sn{sup 2+} in yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Viau, C.M. [Departamento de Biofisica/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, UFRGS (Brazil); Yoneama, M.-L. [Instituto de Fisica, UFRGS, Av. Bento Goncalves 9500, CEP 91501-970, CP 15051, Porto Alegre, RS (Brazil)]. E-mail: jfdias@if.ufrgs.br; Dias, J.F. [Instituto de Fisica, UFRGS, Av. Bento Goncalves 9500, CEP 91501-970, CP 15051, Porto Alegre, RS (Brazil); Pungartnik, C. [Departamento de Ciencias Biologicas, Universidade Estadual de Santa Cruz, UESC, Ilheus, BA (Brazil); Brendel, M. [Departamento de Biofisica/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, UFRGS (Brazil); Departamento de Ciencias Biologicas, Universidade Estadual de Santa Cruz, UESC, Ilheus, BA (Brazil); Henriques, J.A.P. [Departamento de Biofisica/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, UFRGS (Brazil); Faculdade de Farmacia, Universidade Luterana do Brasil, ULBRA, Porto Alegre, RS (Brazil)

    2006-08-15

    The main goal of this work was to determine the concentration of Sn{sup 2+} ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl{sub 2}. To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl{sub 2} and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn{sup 2+} induces DNA lesions and that the absorption depends on the genetic background of each strain.

  11. Detection and quantitative determination by PIXE of the mutagen Sn2+ in yeast cells

    International Nuclear Information System (INIS)

    Viau, C.M.; Yoneama, M.-L.; Dias, J.F.; Pungartnik, C.; Brendel, M.; Henriques, J.A.P.

    2006-01-01

    The main goal of this work was to determine the concentration of Sn 2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl 2 . To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl 2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn 2+ induces DNA lesions and that the absorption depends on the genetic background of each strain

  12. Surface-modified yeast cells: A novel eukaryotic carrier for oral application.

    Science.gov (United States)

    Kenngott, Elisabeth E; Kiefer, Ruth; Schneider-Daum, Nicole; Hamann, Alf; Schneider, Marc; Schmitt, Manfred J; Breinig, Frank

    2016-02-28

    The effective targeting and subsequent binding of particulate carriers to M cells in Peyer's patches of the gut is a prerequisite for the development of oral delivery systems. We have established a novel carrier system based on cell surface expression of the β1-integrin binding domain of invasins derived from Yersinia enterocolitica and Yersinia pseudotuberculosis on the yeast Saccharomyces cerevisiae. All invasin derivatives were shown to be effectively expressed on the cell surface and recombinant yeast cells showed improved binding to both human HEp-2 cells and M-like cells in vitro. Among the different derivatives tested, the integrin-binding domain of Y. enterocolitica invasin proved to be the most effective and was able to target Peyer's patches in vivo. In conclusion, cell surface-modified yeasts might provide a novel bioadhesive, eukaryotic carrier system for efficient and targeted delivery of either antigens or drugs via the oral route. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Genetics of single-cell protein abundance variation in large yeast populations

    Science.gov (United States)

    Albert, Frank W.; Treusch, Sebastian; Shockley, Arthur H.; Bloom, Joshua S.; Kruglyak, Leonid

    2014-02-01

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power. Consequently, many eQTL are probably missed, especially those with smaller effects. Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes, but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in `hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell

  14. The sensitivity of yeast and yeast-like cells to new lysosomotropic agents

    Czech Academy of Sciences Publication Activity Database

    Krasowska, A.; Chmielewska, L.; Adamski, R.; Luszynski, J.; Witek, S.; Sigler, Karel

    2004-01-01

    Roč. 9, 4A (2004), s. 675-683 ISSN 1425-8153 R&D Projects: GA AV ČR IBS5020202; GA MŠk ME 577 Grant - others:GA MŠk(CZ) KONTAKT 01-032; Polish-Czech Treaty on Scientific and Scientific-Technical Cooperation(XX) 2 Institutional research plan: CEZ:AV0Z5020903 Keywords : lysosomotropic agents * yeast * quinacrine Subject RIV: EE - Microbiology, Virology Impact factor: 0.495, year: 2004

  15. Asymmetric bioreduction of acetophenones by Baker's yeast and its cell-free extract encapsulated in sol-gel silica materials

    Science.gov (United States)

    Kato, Katsuya; Nakamura, Hitomi; Nakanishi, Kazuma

    2014-02-01

    Baker's yeast (BY) encapsulated in silica materials was synthesized using a yeast cell suspension and its cell-free extract during a sol-gel reaction of tetramethoxysilane with nitric acid as a catalyst. The synthesized samples were fully characterized using various methods, such as scanning electron microscopy, nitrogen adsorption-desorption, Fourier transform infrared spectroscopy, thermogravimetry, and differential thermal analysis. The BY cells were easily encapsulated inside silica-gel networks, and the ratio of the cells in the silica gel was approximately 75 wt%, which indicated that a large volume of BY was trapped with a small amount of silica. The enzyme activity (asymmetric reduction of prochiral ketones) of BY and its cell-free extract encapsulated in silica gel was investigated in detail. The activities and enantioselectivities of free and encapsulated BY were similar to those of acetophenone and its fluorine derivatives, which indicated that the conformation structure of BY enzymes inside silica-gel networks did not change. In addition, the encapsulated BY exhibited considerably better solvent (methanol) stability and recyclability compared to free BY solution. We expect that the development of BY encapsulated in sol-gel silica materials will significantly impact the industrial-scale advancement of high-efficiency and low-cost biocatalysts for the synthesis of valuable chiral alcohols.

  16. Using network component analysis to dissect regulatory networks mediated by transcription factors in yeast.

    Directory of Open Access Journals (Sweden)

    Chun Ye

    2009-03-01

    Full Text Available Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request.

  17. Edge usage, motifs, and regulatory logic for cell cycling genetic networks

    Science.gov (United States)

    Zagorski, M.; Krzywicki, A.; Martin, O. C.

    2013-01-01

    The cell cycle is a tightly controlled process, yet it shows marked differences across species. Which of its structural features follow solely from the ability to control gene expression? We tackle this question in silico by examining the ensemble of all regulatory networks which satisfy the constraint of producing a given sequence of gene expressions. We focus on three cell cycle profiles coming from baker's yeast, fission yeast, and mammals. First, we show that the networks in each of the ensembles use just a few interactions that are repeatedly reused as building blocks. Second, we find an enrichment in network motifs that is similar in the two yeast cell cycle systems investigated. These motifs do not have autonomous functions, yet they reveal a regulatory logic for cell cycling based on a feed-forward cascade of activating interactions.

  18. Information processing in the transcriptional regulatory network of yeast: Functional robustness

    Directory of Open Access Journals (Sweden)

    Dehmer Matthias

    2009-03-01

    Full Text Available Abstract Background Gene networks are considered to represent various aspects of molecular biological systems meaningfully because they naturally provide a systems perspective of molecular interactions. In this respect, the functional understanding of the transcriptional regulatory network is considered as key to elucidate the functional organization of an organism. Results In this paper we study the functional robustness of the transcriptional regulatory network of S. cerevisiae. We model the information processing in the network as a first order Markov chain and study the influence of single gene perturbations on the global, asymptotic communication among genes. Modification in the communication is measured by an information theoretic measure allowing to predict genes that are 'fragile' with respect to single gene knockouts. Our results demonstrate that the predicted set of fragile genes contains a statistically significant enrichment of so called essential genes that are experimentally found to be necessary to ensure vital yeast. Further, a structural analysis of the transcriptional regulatory network reveals that there are significant differences between fragile genes, hub genes and genes with a high betweenness centrality value. Conclusion Our study does not only demonstrate that a combination of graph theoretical, information theoretical and statistical methods leads to meaningful biological results but also that such methods allow to study information processing in gene networks instead of just their structural properties.

  19. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

    Science.gov (United States)

    Chen, Xianzhong

    2017-03-04

    The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

  20. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

    Science.gov (United States)

    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Sandip Chakraborty

    2016-01-01

    Full Text Available Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins are expected to be “seen” by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes’ adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another.

  2. Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks.

    Science.gov (United States)

    Chakraborty, Sandip; Alvarez-Ponce, David

    2016-01-01

    Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins) are expected to be "seen" by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons) tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes' adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another.

  3. Tolerance of yeast biofilm cells towards systemic antifungals

    DEFF Research Database (Denmark)

    Bojsen, Rasmus Kenneth

    of this thesis has been to explore the tolerance mechanisms of yeast biofilms to systemic antifungal agents and to identify the molecular target of a novel peptidomimetic with anti-biofilm activity. The genetic tractable S. cerevisiae was used as biofilm model system for the pathogenic Candida species...

  4. Copper Biosorption on Magnetically Modified Yeast Cells Under Magnetic Field

    Czech Academy of Sciences Publication Activity Database

    Uzun, L.; Saglam, N.; Šafaříková, Miroslava; Šafařík, Ivo; Denizli, A.

    2011-01-01

    Roč. 46, č. 6 (2011), s. 1045-1051 ISSN 0149-6395 Institutional research plan: CEZ:AV0Z60870520 Keywords : copper removal * heavy metal removal * magnetic biosorbents * yeast Subject RIV: CE - Biochemistry Impact factor: 1.088, year: 2011

  5. Biosorption of mercury on magnetically modified yeast cells

    Czech Academy of Sciences Publication Activity Database

    Yavuz, H.; Denizli, A.; Gungunes, H.; Šafaříková, Miroslava; Šafařík, Ivo

    2006-01-01

    Roč. 52, - (2006), s. 253-260 ISSN 1383-5866 R&D Projects: GA MŠk(CZ) OC 108 Institutional research plan: CEZ:AV0Z60870520 Keywords : mercury removal * magnetic biosorbents * yeast Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.497, year: 2006

  6. Immobilised Sarawak Malaysia yeast cells for production of bioethanol.

    Science.gov (United States)

    Zain, Masniroszaime Mohd; Kofli, Noorhisham Tan; Rozaimah, Siti; Abdullah, Sheikh

    2011-05-01

    Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.

  7. Yeast Biological Networks Unfold the Interplay of Antioxidants, Genome and Phenotype, and Reveal a Novel Regulator of the Oxidative Stress Response

    DEFF Research Database (Denmark)

    Otero, José Manuel; Papadakis, M.A.; Udatha, D.B.R.K.G.

    2010-01-01

    Background: Identifying causative biological networks associated with relevant phenotypes is essential in the field of systems biology. We used ferulic acid (FA) as a model antioxidant to characterize the global expression programs triggered by this small molecule and decipher the transcriptional...... network controlling the phenotypic adaptation of the yeast Saccharomyces cerevisiae. Methodology/Principal Findings: By employing a strict cut off value during gene expression data analysis, 106 genes were found to be involved in the cell response to FA, independent of aerobic or anaerobic conditions....... Network analysis of the system guided us to a key target node, the FMP43 protein, that when deleted resulted in marked acceleration of cellular growth (similar to 15% in both minimal and rich media). To extend our findings to human cells and identify proteins that could serve as drug targets, we replaced...

  8. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel

    2006-01-01

    protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol...... that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent...

  9. Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

    Science.gov (United States)

    Smith, I M; Baker, A; Arneborg, N; Jespersen, L

    2015-11-01

    The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast

  10. Yeast Killer Toxin K28: Biology and Unique Strategy of Host Cell Intoxication and Killing

    Directory of Open Access Journals (Sweden)

    Björn Becker

    2017-10-01

    Full Text Available The initial discovery of killer toxin-secreting brewery strains of Saccharomyces cerevisiae (S. cerevisiae in the mid-sixties of the last century marked the beginning of intensive research in the yeast virology field. So far, four different S. cerevisiae killer toxins (K28, K1, K2, and Klus, encoded by cytoplasmic inherited double-stranded RNA viruses (dsRNA of the Totiviridae family, have been identified. Among these, K28 represents the unique example of a yeast viral killer toxin that enters a sensitive cell by receptor-mediated endocytosis to reach its intracellular target(s. This review summarizes and discusses the most recent advances and current knowledge on yeast killer toxin K28, with special emphasis on its endocytosis and intracellular trafficking, pointing towards future directions and open questions in this still timely and fascinating field of killer yeast research.

  11. The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast.

    Science.gov (United States)

    Leitao, Ricardo M; Kellogg, Douglas R

    2017-11-06

    The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast. © 2017 Leitao and Kellogg.

  12. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

    DEFF Research Database (Denmark)

    Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

    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 p...

  13. Expression of death receptor 4 induces caspase-independent cell death in MMS-treated yeast.

    Science.gov (United States)

    Kang, Mi-Sun; Lee, Sung-Keun; Park, Chang-Shin; Kang, Ju-Hee; Bae, Sung-Ho; Yu, Sung-Lim

    2008-11-14

    DR4, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, is a key element in the extrinsic pathway of TRAIL/TRAIL receptor-related apoptosis that exerts a preferential toxic effect against tumor cells. However, TRAIL and DR4 are expressed in various normal cells, and recent studies indicate that DR4 has a number of non-apoptotic functions. In this study, we evaluated the effects of human DR4 expression in yeast to determine the function of DR4 in normal cells. The expression of DR4 in yeast caused G1 arrest, which resulted in transient growth inhibition. Moreover, treatment of DR4-expressing yeast with a DNA damaging agent, MMS, elicited drastic, and sustained cell growth inhibition accompanied with massive apoptotic cell death. Further analysis revealed that cell death in the presence of DNA damage and DR4 expression was not dependent on the yeast caspase, YCA1. Taken together, these results indicate that DR4 triggers caspase-independent programmed cell death during the response of normal cells to DNA damage.

  14. Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

    Science.gov (United States)

    Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

    2018-04-02

    During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Ethanol production potential from fermented rice noodle wastewater treatment using entrapped yeast cell sequencing batch reactor

    Science.gov (United States)

    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.

  16. Yeast CUP1 protects HeLa cells against copper-induced stress

    Energy Technology Data Exchange (ETDEWEB)

    Xie, X.X. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China); College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou (China); Ma, Y.F.; Wang, Q.S.; Chen, Z.L.; Liao, R.R.; Pan, Y.C. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China)

    2015-06-12

    As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. YeastCUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study,CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

  17. Kinetic analysis of ethanol production from glucose fermentation by yeast cells immobilized onto ceramic supports.

    Science.gov (United States)

    Zhang, Y; Kennedy, J F; Knill, C J

    1996-01-01

    Porous ceramic supports have been developed and utilized for the immobilization of yeast cells to produce ethanol by the fermentation of glucose. The relationship between the porous structure of the ceramic support and the quantity of yeast cells immobilized and the production of ethanol by the fermentation have been investigated. A comparison of the properties of the ceramic supports with those of a calcium alginate gel indicated that the ceramics are the better of the two types of material and have potential for industrial application.

  18. Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica.

    Directory of Open Access Journals (Sweden)

    Pengcheng Pan

    Full Text Available With the emergence of energy scarcity, the use of renewable energy sources such as biodiesel is becoming increasingly necessary. Recently, many researchers have focused their minds on Yarrowia lipolytica, a model oleaginous yeast, which can be employed to accumulate large amounts of lipids that could be further converted to biodiesel. In order to understand the metabolic characteristics of Y. lipolytica at a systems level and to examine the potential for enhanced lipid production, a genome-scale compartmentalized metabolic network was reconstructed based on a combination of genome annotation and the detailed biochemical knowledge from multiple databases such as KEGG, ENZYME and BIGG. The information about protein and reaction associations of all the organisms in KEGG and Expasy-ENZYME database was arranged into an EXCEL file that can then be regarded as a new useful database to generate other reconstructions. The generated model iYL619_PCP accounts for 619 genes, 843 metabolites and 1,142 reactions including 236 transport reactions, 125 exchange reactions and 13 spontaneous reactions. The in silico model successfully predicted the minimal media and the growing abilities on different substrates. With flux balance analysis, single gene knockouts were also simulated to predict the essential genes and partially essential genes. In addition, flux variability analysis was applied to design new mutant strains that will redirect fluxes through the network and may enhance the production of lipid. This genome-scale metabolic model of Y. lipolytica can facilitate system-level metabolic analysis as well as strain development for improving the production of biodiesels and other valuable products by Y. lipolytica and other closely related oleaginous yeasts.

  19. Fluorescence emission spectra of calcofluor stained yeast cell suspensions: heuristic assessment of basis spectra for their linear unmixing.

    Science.gov (United States)

    Plášek, Jaromír; Dostál, Marek; Gášková, Dana

    2012-07-01

    Fluorescence emission spectra of yeast cell suspensions stained with calcofluor have recently been identified as promising markers of variations in the quality of yeast cell wall. It is shown in this paper how the raw fluorescence spectra of calcofluor can be transformed to reliable spectral signatures of cell wall quality, which are independent of actual dye-to-cell concentrations of examined cell suspensions. Moreover, the presented approach makes it possible to assess basis fluorescence spectra that allows for the spectral unmixing of raw fluorescence spectra in terms of respective fluorescence contributions of calcofluor solvated in the suspension medium and bound to yeast cell walls.

  20. A living vector field reveals constraints on galactose network induction in yeast.

    Science.gov (United States)

    Stockwell, Sarah R; Rifkin, Scott A

    2017-01-30

    When a cell encounters a new environment, its transcriptional response can be constrained by its history. For example, yeast cells in galactose induce GAL genes with a speed and unanimity that depends on previous nutrient conditions. Cellular memory of long-term glucose exposure delays GAL induction and makes it highly variable with in a cell population, while other nutrient histories lead to rapid, uniform responses. To investigate how cell-level gene expression dynamics produce population-level phenotypes, we built living vector fields from thousands of single-cell time courses of the proteins Gal3p and Gal1p as cells switched to galactose from various nutrient histories. We show that, after sustained glucose exposure, the lack of these GAL transducers leads to induction delays that are long but also variable; that cellular resources constrain induction; and that bimodally distributed expression levels arise from lineage selection-a subpopulation of cells induces more quickly and outcompetes the rest. Our results illuminate cellular memory in this important model system and illustrate how resources and randomness interact to shape the response of a population to a new environment. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  1. An integrated microfluidic device for the sorting of yeast cells using image processing.

    Science.gov (United States)

    Yu, Bo Yang; Elbuken, Caglar; Shen, Chong; Huissoon, Jan Paul; Ren, Carolyn L

    2018-02-23

    The process of detection and separation of yeast cells based on their morphological characteristics is critical to the understanding of cell division cycles, which is of vital importance to the understanding of some diseases such as cancer. The traditional process of manual detection is usually tedious and inconsistent. This paper presents a microfluidic device integrated with microvalves for fluid control for the sorting of yeast cells using image processing algorithms and confirmation based on their fluorescent tag. The proposed device is completely automated, low cost and easy to implement in an academic research setting. Design details of the integrated microfluidic system are highlighted in this paper, along with experimental validation. Real time cell sorting was demonstrated with a cell detection rate of 12 cells per minute.

  2. An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells.

    Science.gov (United States)

    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

  3. Dynamic expression of cell surface hydrophobicity during initial yeast cell growth and before germ tube formation of Candida albicans.

    OpenAIRE

    Hazen, B W; Hazen, K C

    1988-01-01

    Expression of cell surface hydrophobicity (CSH) during initial growth of Candida albicans was monitored. CSH of hydrophobic and hydrophilic yeast cells changed within 30 min upon subculture into fresh medium. Morphologic evidence of germination was preceded by expression of CSH. These results indicate that CSH expression is important in C. albicans growth.

  4. Systematic identification of yeast cell cycle transcription factors using multiple data sources

    Directory of Open Access Journals (Sweden)

    Li Wen-Hsiung

    2008-12-01

    Full Text Available Abstract Background Eukaryotic cell cycle is a complex process and is precisely regulated at many levels. Many genes specific to the cell cycle are regulated transcriptionally and are expressed just before they are needed. To understand the cell cycle process, it is important to identify the cell cycle transcription factors (TFs that regulate the expression of cell cycle-regulated genes. Results We developed a method to identify cell cycle TFs in yeast by integrating current ChIP-chip, mutant, transcription factor binding site (TFBS, and cell cycle gene expression data. We identified 17 cell cycle TFs, 12 of which are known cell cycle TFs, while the remaining five (Ash1, Rlm1, Ste12, Stp1, Tec1 are putative novel cell cycle TFs. For each cell cycle TF, we assigned specific cell cycle phases in which the TF functions and identified the time lag for the TF to exert regulatory effects on its target genes. We also identified 178 novel cell cycle-regulated genes, among which 59 have unknown functions, but they may now be annotated as cell cycle-regulated genes. Most of our predictions are supported by previous experimental or computational studies. Furthermore, a high confidence TF-gene regulatory matrix is derived as a byproduct of our method. Each TF-gene regulatory relationship in this matrix is supported by at least three data sources: gene expression, TFBS, and ChIP-chip or/and mutant data. We show that our method performs better than four existing methods for identifying yeast cell cycle TFs. Finally, an application of our method to different cell cycle gene expression datasets suggests that our method is robust. Conclusion Our method is effective for identifying yeast cell cycle TFs and cell cycle-regulated genes. Many of our predictions are validated by the literature. Our study shows that integrating multiple data sources is a powerful approach to studying complex biological systems.

  5. Mice immunization with radioattenuated Paracoccidioides brasiliensis yeast cells: protective immunity induction evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear CDTN/CNEN-MG, Belo Horizonte, MG (Brazil)]. E-mail: estefaniabio@yahoo.com.br; antero@cdtn.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia]. E-mail: brsgarbi@mono.icb.ufmg.br; goes@mono.icb.ufmg.br

    2007-07-01

    Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a chronic systemic disease prevalent in Latin America. To date, there is no effective vaccine. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory was developed radioattenuated yeast cells of P. brasiliensis and the aim of this work was to evaluate the protection elicited by the immunization with this cells. To check the protector effect BALB/c mice were divided in two groups. The mice of group 1 were immunized once and those of group 2 twice, at two weeks intervals, using 10{sup 5} radioattenuated yeast cells. The mice were sacrificed 30 and 90 days after challenge. The removed organs were used for colony-forming units (CFUs) recover and histopathologic analysis. The gamma irradiated yeast loses its virulence since fails in producing infection in BALB/c mice. An efficient protection against highly infective forms of P. brasiliensis was developed in the group of mice immunized two times. The immunization was able to reduce the initial infection and elicited a long lasted protection. We concluded that the radioattenuated yeast cells are a valuable tool for the protective immunity study in the PCM and for vaccine research. (author)

  6. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    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…

  7. Effect of Growth Conditions on Flocculation and Cell Surface Hydrophobicity of Brewing Yeast

    Czech Academy of Sciences Publication Activity Database

    Kopecká, J.; Němec, M.; Matoulková, D.; Čejka, P.; Jelínková, Markéta; Felsberg, Jürgen; Sigler, Karel

    2015-01-01

    Roč. 73, č. 2 (2015), s. 143-150 ISSN 0361-0470 Institutional support: RVO:61388971 Keywords : Ale and lager yeast * Cell surface hydrophobicity * FLO genes Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.492, year: 2015

  8. Study of the inhibition of respiration/fermentation in yeast cells by sodium fluoride

    Directory of Open Access Journals (Sweden)

    López Pérez, José Pedro

    2013-01-01

    Full Text Available This paper presents the necessary guidelines to achieve the observation of the inhibition of sugar metabolism in yeast cells by means of the compound sodium fluoride. This activity is appropriate for the subject of Biology in Secondary Education as well as High School.

  9. Study of the inhibition of respiration/fermentation in yeast cells by sodium fluoride

    OpenAIRE

    López Pérez, José Pedro; Boronat Gil, Raquel

    2013-01-01

    This paper presents the necessary guidelines to achieve the observation of the inhibition of sugar metabolism in yeast cells by means of the compound sodium fluoride. This activity is appropriate for the subject of Biology in Secondary Education as well as High School.

  10. Yeast single cell protein in the diet of Oreochromis niloticus (L ...

    African Journals Online (AJOL)

    use

    Diets D10 to D50 had fish meal replaced systematically with yeast single cell protein (SCP) in the order 10, 20, 30, 40 and 50%, respectively. Trial feeding was ... Key word: microbial protein, Oreochromis niloticus, feeding, cost benefit, aquaculture. .... Supplementary Feeding for Production of Nile Tilapia, Silver Carp.

  11. Yeast cell wall supplementation alters the performance and health of beef heifers during the receiving period

    Science.gov (United States)

    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...

  12. Magnetic resonance investigation of magnetically-labeled bakerŽs yeast cells

    Czech Academy of Sciences Publication Activity Database

    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.; Šafařík, Ivo; Šafaříková, Miroslava; Morais, P. C.

    272-276, - (2004), s. 2400-2401 ISSN 0304-8853. [International Conference on Magnetism. Roma, 27.07.2003-01.08.2003] R&D Projects: GA AV ČR IBS6087204 Keywords : magnetic fluid * yeast cells * magnetic resonance Subject RIV: EE - Microbiology, Virology Impact factor: 1.031, year: 2004

  13. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    Directory of Open Access Journals (Sweden)

    Arjen J. Jakobi

    2016-03-01

    Full Text Available 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.

  14. Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  16. Reconstruction of the yeast protein-protein interaction network involved in nutrient sensing and global metabolic regulation

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Jouhten, Paula; Nielsen, Jens

    2010-01-01

    BACKGROUND: Several protein-protein interaction studies have been performed for the yeast Saccharomyces cerevisiae using different high-throughput experimental techniques. All these results are collected in the BioGRID database and the SGD database provide detailed annotation of the different......-sensing and metabolic regulatory signal transduction pathways (STP) operating in Saccharomyces cerevisiae. The reconstructed STP network includes a full protein-protein interaction network including the key nodes Snf1, Tor1, Hog1 and Pka1. The network includes a total of 623 structural open reading frames (ORFs...

  17. Microencapsulation of yeast cells and their use as a biocatalyst in organic solvents.

    Science.gov (United States)

    Green, K D; Gill, I S; Khan, J A; Vulfson, E N

    1996-03-05

    Stable, semipermeable polyamide microcapsules were prepared by interfacial polymerization from a mixture of 1,6-hexanediamine and poly(allylamine) crosslinked with di-acid chlorides and were used to encapsulate baker's yeast. The size and distribution of cells within the capsules were investigated by a combination of laser confocal, electron scanning, and transmission electron microscopy. The encapsulated cells were studied as a biocatalyst for the model reduction of 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenyl-1-propanone in a number of organic solvents. The polymerization conditions were extensively investigated and were found to greatly influence the product yield. Microencapsulated yeast cells, prepared under optimized conditions, carried out the reduction more efficiently than free cells as well as those immobilized in alginate and kappa-carrageenan beads. The developed methodology should be broadly applicable to other biotransformations of interest.

  18. Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

    Energy Technology Data Exchange (ETDEWEB)

    Procházka, Václav, E-mail: prochazkav@fzu.cz [Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague (Czech Republic); Institute of Physics, The Czech Academy of Sciences, Cukrovarnická 10/112, 162 00 Prague (Czech Republic); Cifra, Michal [Institute of Photonics and Electronics, The Czech Academy of Sciences, Chaberská 57, 182 51 Prague (Czech Republic); Kulha, Pavel [Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague (Czech Republic); Institute of Physics, The Czech Academy of Sciences, Cukrovarnická 10/112, 162 00 Prague (Czech Republic); Ižák, Tibor [Institute of Physics, The Czech Academy of Sciences, Cukrovarnická 10/112, 162 00 Prague (Czech Republic); Rezek, Bohuslav [Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague (Czech Republic); Institute of Physics, The Czech Academy of Sciences, Cukrovarnická 10/112, 162 00 Prague (Czech Republic); Kromka, Alexander [Institute of Physics, The Czech Academy of Sciences, Cukrovarnická 10/112, 162 00 Prague (Czech Republic); Faculty of Civil Engineering, Czech Technical University in Prague, Thákurova 7, 16629 Prague (Czech Republic)

    2017-02-15

    Highlights: • Interaction of non-adherent yeast cells with H-terminated diamond described. • Effect of cell culture solutions on H-diamond SGFET (positive potential shifts). • H-diamond sensitive to metabolic activity of yeast cells (negative potential shift). - Abstract: Diamond thin films provide unique features as substrates for cell cultures and as bio-electronic sensors. Here we employ solution-gated field effect transistors (SGFET) based on nanocrystalline diamond thin films with H-terminated surface which exhibits the sub-surface p-type conductive channel. We study an influence of yeast cells (Saccharomyces cerevisiae) on electrical characteristics of the diamond SGFETs. Two different cell culture solutions (sucrose and yeast peptone dextrose–YPD) are used, with and without the cells. We have found that transfer characteristics of the SGFETs exhibit a negative shift of the gate voltage by −26 mV and −42 mV for sucrose and YPD with cells in comparison to blank solutions without the cells. This effect is attributed to a local pH change in close vicinity of the H-terminated diamond surface due to metabolic processes of the yeast cells. The pH sensitivity of the diamond-based SGFETs, the role of cell and protein adhesion on the gate surface and the role of negative surface charge of yeast cells on the SGFETs electrical characteristics are discussed as well.

  19. One-step magnetic modification of yeast cells by microwave-synthesized iron oxide microparticles

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Procházková, G.; Šafařík, Ivo

    2013-01-01

    Roč. 56, č. 6 (2013), s. 456-461 ISSN 0266-8254 R&D Projects: GA MŠk(CZ) LD13023; GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : invert sugar formation * magnetic iron oxide microparticles * magnetic modification * magnetic separation * microwave-assisted synthesis * whole-cell biocatalyst * yeast cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.749, year: 2013

  20. Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

    Science.gov (United States)

    Holland, David O; Johnson, Margaret E

    2018-03-01

    Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module

  1. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yajing, E-mail: shen@robo.mein.nagoya-u.ac.jp [Department of Micro-Nano Systems Engineering, Nagoya University, Nagoya 464-8603 (Japan); Nakajima, Masahiro [Department of Micro-Nano Systems Engineering, Nagoya University, Nagoya 464-8603 (Japan); Ridzuan Ahmad, Mohd [Department of Mechatronics and Robotics, Faculty of Electrical Engineering, Universiti Teknologi Malaysia, Skudai 81310 (Malaysia); Kojima, Seiji; Homma, Michio [Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602 (Japan); Fukuda, Toshio [Department of Micro-Nano Systems Engineering, Nagoya University, Nagoya 464-8603 (Japan)

    2011-07-15

    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. -- Research highlights: {yields} A nanorobotic manipulation system was developed inside an ESEM. {yields} A micro-puller was designed for single yeast cell adhesion force measurement. {yields} Yeast cells were still alive after being observed and manipulated inside ESEM. {yields} Yeast cell adhesion force to substrate is smaller at high humidity condition than at low humidity condition.

  2. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM

    International Nuclear Information System (INIS)

    Shen, Yajing; Nakajima, Masahiro; Ridzuan Ahmad, Mohd; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2011-01-01

    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. -- Research highlights: → A nanorobotic manipulation system was developed inside an ESEM. → A micro-puller was designed for single yeast cell adhesion force measurement. → Yeast cells were still alive after being observed and manipulated inside ESEM. → Yeast cell adhesion force to substrate is smaller at high humidity condition than at low humidity condition.

  3. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard

    2015-02-01

    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  4. A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation.

    Directory of Open Access Journals (Sweden)

    Camille Duc

    Full Text Available Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation.

  5. A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation.

    Science.gov (United States)

    Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine; Blondin, Bruno

    2017-01-01

    Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation.

  6. Inferring the effective TOR-dependent network: a computational study in yeast.

    Science.gov (United States)

    Mohammadi, Shahin; Subramaniam, Shankar; Grama, Ananth

    2013-08-30

    Calorie restriction (CR) is one of the most conserved non-genetic interventions that extends healthspan in evolutionarily distant species, ranging from yeast to mammals. The target of rapamycin (TOR) has been shown to play a key role in mediating healthspan extension in response to CR by integrating different signals that monitor nutrient-availability and orchestrating various components of cellular machinery in response. Both genetic and pharmacological interventions that inhibit the TOR pathway exhibit a similar phenotype, which is not further amplified by CR. In this paper, we present the first comprehensive, computationally derived map of TOR downstream effectors, with the objective of discovering key lifespan mediators, their crosstalk, and high-level organization. We adopt a systematic approach for tracing information flow from the TOR complex and use it to identify relevant signaling elements. By constructing a high-level functional map of TOR downstream effectors, we show that our approach is not only capable of recapturing previously known pathways, but also suggests potential targets for future studies.Information flow scores provide an aggregate ranking of relevance of proteins with respect to the TOR signaling pathway. These rankings must be normalized for degree bias, appropriately interpreted, and mapped to associated roles in pathways. We propose a novel statistical framework for integrating information flow scores, the set of differentially expressed genes in response to rapamycin treatment, and the transcriptional regulatory network. We use this framework to identify the most relevant transcription factors in mediating the observed transcriptional response, and to construct the effective response network of the TOR pathway. This network is hypothesized to mediate life-span extension in response to TOR inhibition. Our approach, unlike experimental methods, is not limited to specific aspects of cellular response. Rather, it predicts transcriptional

  7. Mass spectrometry-based metabolomics of single yeast cells.

    Science.gov (United States)

    Ibáñez, Alfredo J; Fagerer, Stephan R; Schmidt, Anna Mareike; Urban, Pawel L; Jefimovs, Konstantins; Geiger, Philipp; Dechant, Reinhard; Heinemann, Matthias; Zenobi, Renato

    2013-05-28

    Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study, we demonstrate that, with the microarrays for mass spectrometry platform, we are able to observe this variability. We monitor environmentally (2-deoxy-D-glucose) and genetically (ΔPFK2) perturbed Saccharomyces cerevisiae cells at the single-cell, few-cell, and population levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge, give biological insight that is not accessible from population-level metabolomic data.

  8. Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast.

    Science.gov (United States)

    Strelnikova, Natalja; Sauter, Nora; Guizar-Sicairos, Manuel; Göllner, Michael; Diaz, Ana; Delivani, Petrina; Chacón, Mariola; Tolić, Iva M; Zaburdaev, Vasily; Pfohl, Thomas

    2017-10-23

    Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting.

  9. Studies on regulation of the cell cycle in fission yeast.

    Directory of Open Access Journals (Sweden)

    Miroslava Požgajová

    2015-05-01

    Full Text Available All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to understand multicellular organism we need to understand the life cycle of individual cells from which the organism comprises. The cell cycle is the life cycle of a single cell in the plant or animal body. It involves series of events in which components of the cell doubles and afterwards equally segregate into daughter cells. Such process ensures growth of the organism, and specialized reductional cell division which leads to production of gamets, assures sexual reproduction. Cell cycle is divided in the G1, S, G2 and M phase. Two gap-phases (G1 and G2 separate S phase (or synthesis and M phase which stays either for mitosis or meiosis. Essential for normal life progression and reproduction is correct chromosome segregation during mitosis and meiosis. Defects in the division program lead to aneuploidy, which in turn leads to birth defects, miscarriages or cancer. Even thou, researchers invented much about the regulation of the cell cycle, there is still long way to understand the complexity of the regulatory machineries that ensure proper segregation of chromosomes. In this paper we would like to describe techniques and materials we use for our studies on chromosome segregation in the model organism Schizosaccharomyces pombe.

  10. Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

    International Nuclear Information System (INIS)

    Braun, Ralf J.

    2012-01-01

    Mitochondrial damage and dysfunction are common hallmarks for neurodegenerative disorders, including Alzheimer, Parkinson, Huntington diseases, and the motor neuron disorder amyotrophic lateral sclerosis. Damaged mitochondria pivotally contribute to neurotoxicity and neuronal cell death in these disorders, e.g., due to their inability to provide the high energy requirements for neurons, their generation of reactive oxygen species (ROS), and their induction of mitochondrion-mediated cell death pathways. Therefore, in-depth analyses of the underlying molecular pathways, including cellular mechanisms controlling the maintenance of mitochondrial function, is a prerequisite for a better understanding of neurodegenerative disorders. The yeast Saccharomyces cerevisiae is an established model for deciphering mitochondrial quality control mechanisms and the distinct mitochondrial roles during apoptosis and programmed cell death. Cell death upon expression of various human neurotoxic proteins has been characterized in yeast, revealing neurotoxic protein-specific differences. This review summarizes how mitochondria are affected in these neurotoxic yeast models, and how they are involved in the execution and prevention of cell death. I will discuss to which extent this mimics the situation in other neurotoxic model systems, and how this may contribute to a better understanding of the mitochondrial roles in the human disorders.

  11. Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

    Directory of Open Access Journals (Sweden)

    Passos Geraldo AS

    2006-08-01

    Full Text Available Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM, a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein, bgl (encoding for a 1,3-β-glucosidase in mycelium cells; and ags (an α-1,3-glucan synthase, cda (a chitin deacetylase and vrp (a verprolin in yeast cells; (ii ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken

  12. The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures.

    Science.gov (United States)

    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

    2011-04-01

    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.

  13. Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides

    DEFF Research Database (Denmark)

    Cerantola, Vanessa; Guillas, Isabelle; Roubaty, Carole

    2009-01-01

    , 2Delta.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1, yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die...... in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303lag1Delta lac1Delta ypc1Delta ydc1Delta cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous...

  14. Influence the oxidant action of selenium in radiosensitivity induction and cell death in yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Porto, Barbara Abranches de Araujo

    2012-01-01

    Ionizing radiations are from both natural sources such as from anthropogenic sources. Recently, radiotherapy has emerged as one of the most common therapies against cancer. Co-60 irradiators (cobalt-60 linear accelerators) are used to treat of malignant tumors routinely in hospitals around the world. Exposure to ionizing radiation can induce changes in cellular macromolecules and affect its functions, because they cause radiolysis of the water molecule generating reactive oxygen species, which can cause damage to virtually all organelles and cell components known as oxidative damage that can culminate in oxidative stress. Oxidative stress is a situation in which the balance between oxidants and antioxidants is broken resulting in excessive production of reactive species, it is not accompanied by the increase in antioxidant capacity, making it impossible to neutralize them. Selenium is a micronutrient considered as antioxidant, antiinflammatory, which could prevent cancer. Selenium in biological system exists as seleno proteins. Nowadays, 25 human seleno proteins have been identified, including glutathione peroxidase, an antioxidant enzyme. Yeasts have the ability to incorporate various metals such as iron, cadmium, zinc and selenium, as well as all biological organisms. The yeast Saccharomyces cerevisiae, unlike mammalian cells is devoid of seleno proteins, being considered as a practical model for studies on the toxicity of selenium, without any interference from the metabolism of seleno proteins. Moreover, yeast cells proliferate through the fermentation, the microbial equivalent of aerobic glycolysis in mammals and the process is also used by tumors. Several reports show that the pro-oxidante effects and induced toxic selenium compounds occur at lower doses and in malignant cells compared with benign cells. Therefore selenium giving a great therapeutic potential in cancer treatment .Our objective was to determine whether selenium is capable to sensitize yeasts

  15. Magnetic resonance investigation of magnetic-labeled baker's yeast cells

    Science.gov (United States)

    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.

  16. Relationship between sensitivity to ultraviolet light and budding in yeast cells of different culture ages

    International Nuclear Information System (INIS)

    Atsuta, J.; Okajima, S.

    1976-01-01

    Subpopulations of yeast cells, consisting of cells of different sizes and different percentages of budding cells, were prepared by centrifugation through sucrose solutions with linear density gradients of cultures at different phases of the growth cycle. Ultraviolet survival of these cells was determined by colony counting, and the survival rate was compared with the cells' respiratory rates. Individual budding cells and interdivisional cells, and also mother cells and daughter cells derived from irradiated budding cells, were isolated by the micromanipulation technique. The number of divisions in each cell was measured during a 21-hr incubation period immediately after irradiation. In the population in the logarithmic phase consisting of homogeneous cells of middle size, no difference in uv sensitivity was observed between mother cells and daughter cells, irrespective of mutual adhesion. Budding cell resistance was observed in the population in the transitional phase; this was due to the lesser uv sensitivity of daughter cells in the fresh medium. In the stationary phase, daughter cells were rather more sensitive than mother cells or interdivisional cells, so there was little difference in uv sensitivity between budding cells and interdivisional cells

  17. New vector for transfer of yeast artificial chromosomes to mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Markie, D.; Ragoussis, J.; Senger, G.; Rowan, A.; Trowsdale, J.; Sheer, D.; Bodmer, W.F. (Imperial Cancer Research Fund, London (United Kingdom)); Sansom, D. (Bath Inst. for Rheumatic Diseases, Avon (United Kingdom))

    1993-03-01

    A modification vector has been constructed to facilitate the transfer of yeast artificial chromosomes (YACs) to mammalian cells in culture by targeting a dominant selectable marker (G418 resistance) to the right arm of pYAC4 clones. The ADE2 gene is used for yeast selection with consequent disruption of the URA3 gene, allowing direct modification of YACs within the common host strain AB1380, and providing a simple test for correct targeting. This vector has been tested by modification of a 550-kb YAC containing part of the human MHC class II region and transfer to CHO cells by protoplast fusion. Analysis of 15 independent G418-resistant CHO lines obtained following fusion suggests the majority contain a complete YAC with moderate amplification in some lines. 24 refs., 4 figs.

  18. Calorie restriction-mediated replicative lifespan extension in yeast is non-cell autonomous.

    Directory of Open Access Journals (Sweden)

    Szu-Chieh Mei

    2015-01-01

    Full Text Available In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA, are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.

  19. Mitogen-activated protein kinase (MAPK) dynamics determine cell fate in the yeast mating response.

    Science.gov (United States)

    Li, Yang; Roberts, Julie; AkhavanAghdam, Zohreh; Hao, Nan

    2017-12-15

    In the yeast Saccharomyces cerevisiae , the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Production of α-keto acids with alginate-entrapped whole cells of the yeastTrigonopsis variabilis.

    Science.gov (United States)

    Nilsson, K; Brodelius, P; Mosbach, K

    1982-01-01

    The yeast,Trigonopsis variabilis, was immobilized by entrapment in alginate. The immobilized cells containing high amounts of D-amino acid oxidase were used to convert D-amino acids to their corresponding α-keto acids.

  1. Effect of inactive yeast cell wall on growth performance, survival rate and immune parameters in Pacific White Shrimp (Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Rutchanee Chotikachinda

    2008-10-01

    Full Text Available Effects of dietary inactive yeast cell wall on growth performance, survival rate, and immune parameters in pacific white shrimp (Litopenaeus vannamei was investigated. Three dosages of inactive yeast cell wall (0, 1, and 2 g kg-1 were tested in three replicate groups of juvenile shrimps with an average initial weight of 7.15±0.05 g for four weeks. There was no significant difference in final weight, survival rate, specific growth rate, feed conversion ratio, feed intake, protein efficiency ratio, and apparent net protein utilization of each treatments. However, different levels of inactive yeast cell wall showed an effect on certain immune parameters (p<0.05. Total hemocyte counts, granular hemocyte count, and bacterial clearance were better in shrimp fed diets supplemented with 1 and 2 g kg-1 inactive yeast cell wall as compared with thecontrol group.

  2. Development of a yeast cell factory for production of aromatic secondary metabolites

    DEFF Research Database (Denmark)

    Rodriguez Prado, Edith Angelica

    Aromatic secondary metabolites are compounds mainly synthesized by plants and fungi as a response to predators and environmental stresses. These compounds have a broad range of natural properties such as reduction of oxidative damage in cells, antibacterial effects and UV protection. Many of thes...... six different types of flavonoids, and some of the engineered strains produced significant titers of flavonoid compounds such as kaempferol and quercetin. Moreover, for the first time, we synthesized the flavonoids liquiritigenin, resokaempferol and fisetin in yeast....

  3. Extraction of proteins from yeast cell wall | Huang | African Journal of ...

    African Journals Online (AJOL)

    The proteins from yeast cell wall were extracted by autolysis and depositing in turn. The results show that the change of pH value greatly affects the yield of the final product. The content of obtained crude proteins is maximal (more than 66%) when the autolysis time is 3 h, and pH value is 6 - 7. The extracted proteins can be ...

  4. A high-throughput method for quantifying metabolically active yeast cells

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander

    2015-01-01

    equivalent, displaying reduction curves that interrelated directly with CFU counts. For growth rate estimation, the methylene blue reduction test (MBRT) proved superior, since the discriminatory nature of the method allowed for the quantification of metabolically active cells only, excluding dead cells....... The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e...

  5. Effect of NaCl and KCl on irradiated diploid yeast cells

    International Nuclear Information System (INIS)

    Amirtaev, K.G.; Lobachevskij, P.N.; Lyu Gvan Son

    1984-01-01

    Irradiated dipload yeast Saccharomyces cerevisiae kept in NaCl and KCl solutions died more readily than nonirradiated cells: the death rate was a functaon of radiation Jose and temperature of exposure. It was suggested that the radiation-induced injury to mass cell structures was responsible for the death rate. It was shown that the postirradiataon recovery of cells from radiation damages proceeded in KCl solution two-three times slower than mn water, and it was inhibited completely in NaCl solution

  6. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

    Science.gov (United States)

    Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

    2017-07-25

    We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected

  7. Sorption of 4-ethylguaiacol and 4-ethylphenol on yeast cell walls, using a synthetic wine.

    Science.gov (United States)

    Nieto-Rojo, Rodrigo; Ancín-Azpilicueta, Carmen; Garrido, Julián J

    2014-01-01

    4-Ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) are the identified volatile phenolic compounds associated with off-odour in wine. The aim of this work was to investigate the kinetics and thermodynamics of sorption of 4-EG and 4-EP by yeast cell walls, using a synthetic wine. Results showed that the sorption capacity by yeast cell walls for 4-EG was greater than that for 4-EP and that the kinetics of 4-EG were quicker, although the unions were weaker than in the case of 4-EP. The retention of these compounds was by means of specific chemical sorption. The process of sorption of these compounds to the yeast walls could be due to their binding to the residual lipids, as well as to interaction of 4-EP and 4-EG (positively charged compounds), with the functional groups of the mannoproteins and the free amino acids of the surface of the cell walls. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Yashiroda, Yoko, E-mail: ytyy@riken.jp [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Okamoto, Reika [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-8073 (Japan); Hatsugai, Kaori [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550 (Japan); Division of Chemotherapy, Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo 105-8512 (Japan); Takemoto, Yasushi [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Goshima, Naoki [National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo 135-0064 (Japan); Saito, Tamio [Chemical Biology Core Facility/Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Hamamoto, Makiko [Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571 (Japan); Sugimoto, Yoshikazu [Division of Chemotherapy, Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo 105-8512 (Japan); Osada, Hiroyuki [Chemical Biology Core Facility/Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Seimiya, Hiroyuki [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550 (Japan); Yoshida, Minoru [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Saitama 332-0012 (Japan)

    2010-04-09

    The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

  9. The role of the Parkinson's disease gene PARK9 in essential cellular pathways and the manganese homeostasis network in yeast.

    Directory of Open Access Journals (Sweden)

    Alessandra Chesi

    Full Text Available YPK9 (Yeast PARK9; also known as YOR291W is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

  10. Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities.

    Science.gov (United States)

    Peng, Bingyin; Williams, Thomas C; Henry, Matthew; Nielsen, Lars K; Vickers, Claudia E

    2015-06-26

    Predictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of "constitutive" and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter. The "constitutive" promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (P(HXT7), P(SSA1) and P(ADH2)) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the "constitutive" promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several "constitutive" promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift. The data provides a foundation for predictable and optimised control of gene expression levels on different carbon sources and throughout batch

  11. Fabrication of cyclo olefin polymer microfluidic devices for trapping and culturing of yeast cells.

    Science.gov (United States)

    Puza, Sevde; Gencturk, Elif; Odabasi, Irem E; Iseri, Emre; Mutlu, Senol; Ulgen, Kutlu O

    2017-06-01

    A microfluidic platform is designed and fabricated to investigate the role of uncharacterized YOR060C (Sld7) protein in aging in yeast cells for the first time. Saccharomyces cerevisiae yeast cells are trapped in the series of C-shaped regions (0.5 nL) of COP (cyclo olefin polymer), PMMA (poly methylmethacrylate), or PS (polystyrene) microbioreactors. The devices are fabricated using hot embossing and thermo-compression bonding methods. Photolithography and electrochemical etching are used to form the steel mold needed for hot embossing. The cell cycle processes are investigated by monitoring green fluorescent protein (GFP) tagged Sld7 expressions under normal as well as calorie restricted conditions. The cells are loaded at 1 μL/min flowrate and trapped successfully within each chamber. The medium is continuously fed at 0.1 μL/min throughout the experiments. Fluorescent signals of the low abundant Sld7 proteins could be distinguished only on COP devices. The background fluorescence of COP is found 1.22 and 7.24 times lower than that of PMMA, and PS, respectively. Hence, experiments are continued with COP, and lasted for more than 40 h without any contamination. The doubling time of the yeast cells are found as 72 min and 150 min, and the growth rates as 9.63 × 10 -3  min -1 and 4.62 × 10 -3  min -1 , in 2% glucose containing YPD and YNB medium, respectively. The product concentration (Sld7p:GFP) increased in accordance with cell growth. The dual role of Sld7 protein in both cell cycle and chronological aging needs to be further investigated following the preliminary experimental results.

  12. Metabolic network analysis on Phaffia rhodozyma yeast using C-13-labeled glucose and gas chromatography-mass spectrometry

    DEFF Research Database (Denmark)

    Cannizzaro, C.; Christensen, B.; Nielsen, Jens

    2004-01-01

    high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar......Carotenoid production by micro organisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for 'all natural' products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement....... In order to have a better understanding of its metabolism, labeling experiments with [1-C-13]glucose were conducted with the wildtype strain (CBS5905T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes. Amino acid...

  13. Model of fission yeast cell shape driven by membrane-bound growth factors and the cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Tyler Drake

    Full Text Available Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular

  14. Quantification and characterization of cell wall polysaccharides released by non-Saccharomyces yeast strains during alcoholic fermentation.

    Science.gov (United States)

    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. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Biostimulation effects of low-energy laser radiation on yeast cell suspensions

    Science.gov (United States)

    Anghel, Sorin; Stanescu, Constantin S.; Giosanu, Dana; Neagu, Ionica; Savulescu, Geta; Iorga-Siman, Ion

    2000-02-01

    This paper presents work to determine the effects produced by low energy laser radiation on the metabolism and growth of a yeast cell suspension. As experimental material, we used young yeast culture in liquid medium, then distributed on a solid medium, to obtain isolated colonies. As laser source, we used a He-Ne laser, and the irradiation was made with different exposure times. Form each irradiated material, a sample of white grape sterile must was sowed, that has fermented at 18 divided by 20 degrees C for 10 divided by 15 days, after that some properties was tested. Some microscopic studies were also made. The results prove some influence of low energy laser irradiation, which can induce mutations, with new properties of the irradiated material. These mutations can be obtained in a positive sense, with new and important perspectives in wine industry. Also, we observed an inhibitory effect of the laser radiation on the yeast cell growth, due, probably to the too high values of the exposure.

  16. Novel and improved yeast cell factories for biosustainable processes

    DEFF Research Database (Denmark)

    Workman, Mhairi

    2014-01-01

    The utilization of an increasingly diverse range of cheap waste substrates will be an ongoing challenge for the bio-based economy, where the mobilization of nutrients from a variety of waste products will be necessary for realization of biosustainability on an industrial scale. Bioprocesses...... utilizing traditionally applied cell factories are generally based on a limited range of substrates (mainly glucose). However, a wider diversity in substrate range is highly desirable in developing biorefinery scenarios where feed-stocks containing a number of carbon sources are typically employed...

  17. Asymmetric bioreduction of acetophenones by Baker's yeast and its cell-free extract encapsulated in sol–gel silica materials

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Katsuya, E-mail: katsuya-kato@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, 463-8560 (Japan); Nakamura, Hitomi [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, 463-8560 (Japan); Nakanishi, Kazuma [Department of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie, 514-8570 (Japan)

    2014-02-28

    Baker's yeast (BY) encapsulated in silica materials was synthesized using a yeast cell suspension and its cell-free extract during a sol–gel reaction of tetramethoxysilane with nitric acid as a catalyst. The synthesized samples were fully characterized using various methods, such as scanning electron microscopy, nitrogen adsorption–desorption, Fourier transform infrared spectroscopy, thermogravimetry, and differential thermal analysis. The BY cells were easily encapsulated inside silica-gel networks, and the ratio of the cells in the silica gel was approximately 75 wt%, which indicated that a large volume of BY was trapped with a small amount of silica. The enzyme activity (asymmetric reduction of prochiral ketones) of BY and its cell-free extract encapsulated in silica gel was investigated in detail. The activities and enantioselectivities of free and encapsulated BY were similar to those of acetophenone and its fluorine derivatives, which indicated that the conformation structure of BY enzymes inside silica-gel networks did not change. In addition, the encapsulated BY exhibited considerably better solvent (methanol) stability and recyclability compared to free BY solution. We expect that the development of BY encapsulated in sol–gel silica materials will significantly impact the industrial-scale advancement of high-efficiency and low-cost biocatalysts for the synthesis of valuable chiral alcohols.

  18. Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

    Directory of Open Access Journals (Sweden)

    Stefania Castagnetti

    2010-10-01

    Full Text Available Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

  19. Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

    2013-05-01

    Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

  20. High-resolution transcription atlas of the mitotic cell cycle in budding yeast

    DEFF Research Database (Denmark)

    Granovskaia, Marina V; Jensen, Lars J; Ritchie, Matthew E

    2010-01-01

    Extensive transcription of non-coding RNAs has been detected in eukaryotic genomes and is thought to constitute an additional layer in the regulation of gene expression. Despite this role, their transcription through the cell cycle has not been studied; genome-wide approaches have only focused...... on protein-coding genes. To explore the complex transcriptome architecture underlying the budding yeast cell cycle, we used 8 bp tiling arrays to generate a 5 minute-resolution, strand-specific expression atlas of the whole genome....

  1. Lighting up yeast cell factories by transcription factor-based biosensors

    DEFF Research Database (Denmark)

    D'ambrosio, Vasil; Jensen, Michael Krogh

    2017-01-01

    process. For this purpose, there is a need to develop new techniques for screening and selection of best-performing cell factory designs in multiplex. Here we review the current status of the sourcing, design and engineering of biosensors derived from allosterically regulated transcription factors applied...... to the biotechnology work-horse budding yeast Saccharomyces cerevisiae. We conclude by providing a perspective on the most important challenges and opportunities lying ahead in order to harness the full potential of biosensor development for increasing both the throughput of cell factory development and robustness...

  2. STUDY ON ALCOHOLIC FERMENTATION IN A STATIONARY BASKET BIOREACTOR WITH IMMOBILIZED YEAST CELLS

    Directory of Open Access Journals (Sweden)

    Dan Caşcaval

    2011-02-01

    Full Text Available The use of a stationary basket bioreactor with immobilized S. cerevisiae cells indicated the possibility to extend the number of alcoholic fermentation cycles that can be carried out with the same biocatalysts to over nine. Although the rates of glucose consumption and ethanol production were lower than those recorded for the mobile beds of immobilized yeast cells, the mechanical lysis of the biocatalysts is avoided in the case of basket bed. Due to the substrate and product accumulation inside the basket bed, the fermentation process can be improved by washing out the biocatalysts bed over two or four cycles.

  3. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions.

    Directory of Open Access Journals (Sweden)

    Laia Castells-Roca

    Full Text Available Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon. In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.

  4. [Signaling network-based functional cell design].

    Science.gov (United States)

    Ju, Jianqi; Wei, Ping

    2017-03-25

    Cellular signaling networks act as the central processor to deal with environmental signals and regulate cell function, and determine cell fate. Using synthetic biology approach to engineer cell signaling networks is crucial for ultimately constructing man-made "cell machines". Cellular signaling networks can encode sophisticated cell information by processing quantitatively signaling dynamics, which enables multi-dimensional regulation of functional sub-circuits. Here, we first review the research progresses on the signaling coding mechanisms; and then elaborate the methodologies and applications of cells signaling engineering; finally, we envision that signaling-based cell engineering are important for the increasingly-complicated next generation synthetic biology.

  5. Application of Environmental Scanning Electron Microscope-Nanomanipulation System on Spheroplast Yeast Cells Surface Observation.

    Science.gov (United States)

    Rad, Maryam Alsadat; Ahmad, Mohd Ridzuan; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2017-01-01

    The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD 800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl 2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD 800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.

  6. Reduced photoinactivation of 10-dodecyl acridine orange-sensitized yeast cells at high fluence rates measurements and computer simulations

    NARCIS (Netherlands)

    Keij, J.F.; Jansen, J.Th.M.; Schultz, F.W.; Visser, J.W.M.

    1994-01-01

    During the development of a photodamage cell sorter several photosensitizers were tested for their ability to photoinactivate more than 90% of the sensitized cells after a brief irradiation with a fluence of 10 kJ/m2. In pilot experiments, yeast cells sensitized with 10-dodecyl acridine orange (DAO)

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis.

    Science.gov (United States)

    Grimm, T; Grimm, M; Klat, R; Neubauer, A; Palela, M; Neubauer, P

    2012-05-01

    The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10-15% and an increased glucose release by increasing the enzyme content to 15 U L(-1). The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD(600)) of approximately 40 AU (corresponding to over 60 g L(-1) wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.

  9. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  10. Comparison of methods used for assessing the viability and vitality of yeast cells.

    Science.gov (United States)

    Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata

    2014-11-01

    Determination of cell viability is the most commonly used method for assessing the impact of various types of stressors in toxicity research and in industrial microbiology studies. Viability is defined as a percentage of live cells in a whole population. Although cell death is one of the consequences of toxicity, chemical or physical factors may exert their toxic effects through a number of cellular alterations that may compromise cell ability to divide without necessarily leading to cell death. This aspect represents the term 'cell vitality' defined as physiological capabilities of cells. It is important to note that cell viability and cell vitality represent two different aspects of cell functions, and both are required for the estimation of the physiological state of a cell after exposure to various types of stressors and chemical or physical factors. In this paper, we introduced a classification of available methods for estimating both viability and vitality in Saccharomyces cerevisiae yeast cells (wild-type and Δsod1 mutant) in which the effects of selected oxidants causing oxidative stress is evaluated. We present the advantages as well as disadvantages of the selected methods and assess their usefulness in different types of research. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

    Science.gov (United States)

    2018-01-01

    The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368

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

    Energy Technology Data Exchange (ETDEWEB)

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Monti, Paola; Fronza, Gilberto [Mutagenesis Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa (Italy); Pereira, Clara [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Saraiva, Lucília, E-mail: lucilia.saraiva@ff.up.pt [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal)

    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.

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

    International Nuclear Information System (INIS)

    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

  14. Influence of intracellular adenosine-triphosphate concentration of yeast cells on survival following X-irradiation

    International Nuclear Information System (INIS)

    Reinhard, R.D.; Pohlit, W.

    1975-01-01

    The effect of D-glucose, 2-deoxy-D-glucose and starvation in buffer on the ATP-concentration of yeast cells has been studied. In both the wild-type and a respiratory-deficient mutant strain 2-deoxy-D-glucose decreases the value for ATP, while it is enhanced by glucose only in the mutant strain. Populations with different ATP-concentrations have been irradiated. The results suggest that ATP may be an essential factor in the system that determines the length of the shoulder of the dose effect curves. (orig.) [de

  15. The respiration of cells and mitochondria of porin deficient yeast mutants is coupled.

    Science.gov (United States)

    Michejda, J; Guo, X J; Lauquin, G J

    1990-08-31

    Several mutants of yeast lacking the porin gene have been found stable and viable on glucose or glycerol media. Ethanol-supported respiration of porin-free mutant and wild cells appeared equally coupled in vivo being similarly depressed by inhibitors of ADP/ATP translocase or of ATP synthase and stimulated by the uncoupler FCCP. The absence of porin in isolated mutant mitochondria hardly impaired the electron flux but increased the requirement for Mg2+ (or Ca2+) and for ADP and carboxyatractylate concentrations necessary to drive effectively state 3 - state 4 and state 4 - state 3 transitions, respectively. The existence of another porin species, possibly controlled by bivalent cations, is postulated.

  16. Effects of radiation on the cell division cycle. Using yeasts as models

    International Nuclear Information System (INIS)

    Mann, C.; Marsolier, M.C.

    2000-01-01

    The living organisms, since the appearance on earth of the simplest of them, are submitted to numerous attacks having different origin. They use response systems to the DNA damages coming from these attacks and especially radiations. The cell knows how to take stock of the situation, at different moment of its life, to slow down, eventually to stop its cycle before continuing, after repairing of its DNA and divided itself. These mechanisms have kept a remarkable similarity during the evolution. The study of these systems among yeasts is a precious help to understand the corresponding systems for man and to evaluate the limits but also the possibilities, particularly, in oncology. (N.C.)

  17. Yeast cell based feed additives: Studies on aflatoxin B1 and zearalenone

    OpenAIRE

    2011-01-01

    Abstract Thirty commercially available yeast cell wall products and two reference bentonites were tested for their ability to bind aflatoxin B1 (AFB1) and zearalenone (ZON) in buffer solutions at pH 3 and pH 6.5 as well as in real gastric juice. For most products, the binding efficacy of AFB1 correlated with the ash content which was between 2.6 and 89% and constituted the inorganic non-volatile components, like mineral clays, of the samples. Samples with smectite as main ash compo...

  18. The effects of 'cell age' upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Parry, J.M.

    1976-01-01

    Yeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52 0 C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C. Exponential phase cells showed maximum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD 50 gene. Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth. (orig.) [de

  19. Direct conversion of inulin into cell lipid by an inulinase-producing yeast Rhodosporidium toruloides 2F5.

    Science.gov (United States)

    Wang, Zhi-Peng; Fu, Wen-Juan; Xu, Hong-Mei; Chi, Zhen-Ming

    2014-06-01

    In this study, an inulinase-producing yeast strain 2F5 of Rhodosporidium toruloides was obtained. It was found that the yeast strain 2F5 could produce higher amount of oil from inulin and larger lipid bodies in its cells than any other yeast strains tested in this study. Under the optimal conditions, 62.14% (w/w) of lipid based on cell dry weight and 15.82g/l of the dry cell mass were produced from 6.0% (w/v) inulin at flask level, leaving 0.92% (w/v) of total sugar in the fermented medium. During 2-l fermentation, 70.36% (w/w) of lipid based on cell dry weight and 15.64g/l of the dry cell mass were produced from 6.0% (w/v) inulin. Over 99.09% of the fatty acids from the yeast strain 2F5 grown on inulin was C16:0, C18:0, C18:1 and C18:2, especially C18:1 (52.2%). The biodiesel prepared using the lipids produced by the yeast strain 2F5 could be burnt well. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

    Science.gov (United States)

    Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

    2015-01-01

    The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd.

  1. The indentation of pressurized elastic shells: from polymeric capsules to yeast cells

    KAUST Repository

    Vella, D.

    2011-08-10

    Pressurized elastic capsules arise at scales ranging from the 10 m diameter pressure vessels used to store propane at oil refineries to the microscopic polymeric capsules that may be used in drug delivery. Nature also makes extensive use of pressurized elastic capsules: plant cells, bacteria and fungi have stiff walls, which are subject to an internal turgor pressure. Here, we present theoretical, numerical and experimental investigations of the indentation of a linearly elastic shell subject to a constant internal pressure. We show that, unlike unpressurized shells, the relationship between force and displacement demonstrates two linear regimes. We determine analytical expressions for the effective stiffness in each of these regimes in terms of the material properties of the shell and the pressure difference. As a consequence, a single indentation experiment over a range of displacements may be used as a simple assay to determine both the internal pressure and elastic properties of capsules. Our results are relevant for determining the internal pressure in bacterial, fungal or plant cells. As an illustration of this, we apply our results to recent measurements of the stiffness of baker\\'s yeast and infer from these experiments that the internal osmotic pressure of yeast cells may be regulated in response to changes in the osmotic pressure of the external medium.

  2. Rapid and serial quantification of adhesion forces of yeast and Mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eva Potthoff

    Full Text Available Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM. In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

  3. Molecular tools and protocols for engineering the acid-tolerant yeast Zygosaccharomyces bailii as a potential cell factory.

    Science.gov (United States)

    Branduardi, Paola; Dato, Laura; Porro, Danilo

    2014-01-01

    Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

  4. [The effect of succinate on respiration, transamination, and pyruvate formation in cells of the yeast Dipodascus magnusii].

    Science.gov (United States)

    Il'chenko, A P; Ogorelyshev, D I; Shishkanova, N V; Sokolov, A P; Finogenova, T V; Kondrashova, M N

    2005-01-01

    The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.

  5. Fuel cells for telephone networks

    International Nuclear Information System (INIS)

    Wells, J.D.; Scott, D.S.

    1993-01-01

    Critical telephone network systems are currently protected from electric utility power failures by a backup system consisting of lead-acid batteries and an engine-alternator. It is considered here an alternate power system where less expensive off-peak commercial electricity electrolyses water, while fuel cells draw continuously on the stored gas products to provide direct current for the protected equipment. The lead acid batteries are eliminated. The benefits and costs of the existing and alternate systems in scenarios with various system efficiencies, capital costs, and electric utility rates and incentives, are compared. In today's conditions, the alternate system is not economical; however, cost and performance feasibility domains are identified. 2 figs., 4 tabs., 12 refs

  6. Microarray studies on lager brewer's yeasts reveal cell status in the process of autolysis.

    Science.gov (United States)

    Xu, Weina; Wang, Jinjing; Li, Qi

    2014-08-01

    In this work, we performed DNA microarray studies on lager brewer's yeast Saccharomyces pastorianus to investigate changes in gene expression in the process of autolysis. The two strains we used were Qing2 and 5-2. Strain 5-2 is a mutant of Qing2 and autolyzes much more slowly than its parent strain. Four samples of these two strains during different autolysis stages (0% and 15%) were tested using DNA microarray containing > 10,000 yeast's genes. Analysis of genes with the same transcription pattern (up- or down-regulated in both strains) showed that the same 99 genes were up-regulated (transcription levels were increased), and the same 97 genes were down-regulated (transcription levels were decreased) by fivefold or more during autolysis. Genes involved in energy production/utilization, protein anabolism, and stress response were down-regulated. Genes related to cell wall organization and biogenesis, starvation response and DNA damage response were up-regulated. Analysis of genes with opposite transcription patterns (up-regulated in one strain and down-regulated in the other one) showed that 246 genes were up-regulated in 5-2 (autolyzes slowly) and down-regulated in Qing2 (autolyzes rapidly). Another 18 genes had opposite transcription levels, indicating that the strain which autolyzes slowly had better cell vitality despite the same autolysis stage. These findings might further promote the global understanding of autolysis in yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  8. Cell‐to‐cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community

    Science.gov (United States)

    Campbell, Kate; Vowinckel, Jakob

    2016-01-01

    Abstract 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 (H2O2, 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. PMID:27312776

  9. Proteomics analysis for asymmetric inheritance of preexisting proteins between mother and daughter cells in budding yeast.

    Science.gov (United States)

    Okada, Mitsuhiro; Kusunoki, Shunta; Ishibashi, Yuko; Kito, Keiji

    2017-06-01

    In budding yeast, a mother cell can produce a finite number of daughter cells over its life. The accumulation of a variety of types of damaged components has an impact on the aging process. Asymmetrical inheritance during cell division causes these aberrant intracellular constituents to be retained in mother cells and prevents them from segregating to daughter cells. However, the understanding of asymmetrical inheritance of individual proteins that are damaged or old age, and their relevance to the aging process, has been limited. The aim of this study is to propose a proteomics strategy for asymmetrical inheritance of preexisting proteins between mother and daughter cells. During synchronous culture for one generation, newly synthesized proteins were labeled with stable isotope amino acids to discriminate preexisting proteins originally expressed in mother cells, followed by separation of mother and daughter cells using a conventional method based on biotin labeling. Isotope incorporation ratios for individual proteins were quantified using mass spectrometry. We successfully identified 21 proteins whose preexisting versions were asymmetrically inherited in mother cells, including plasma membrane transporter involved in the aging process and organelle-anchoring proteins related to the stress response to misfolded proteins. Thus, our approach would be useful for making catalog of asymmetrically inherited proteins. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  10. UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1

    International Nuclear Information System (INIS)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2013-01-01

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D 3 or vitamin D 2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D 2 was produced without additional vitamin D 2 . Endogenous ergosterol was likely converted into vitamin D 2 by UV irradiation and thermal isomerization, and then the resulting vitamin D 2 was converted to 25-hydroxyvitamin D 2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D 2 without a substrate could be useful for practical purposes

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

    Science.gov (United States)

    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

  12. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio, E-mail: y_mukai@nagahama-i-bio.ac.jp

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  13. DEA1, a circadian- and cold-regulated tomato gene, protects yeast cells from freezing death.

    Science.gov (United States)

    Weyman, Philip D; Pan, Zhiqiang; Feng, Qin; Gilchrist, David G; Bostock, Richard M

    2006-11-01

    Cold and freezing damage to plants can be mitigated by inducible factors during an acclimation period. DEA1 is a circadian-regulated tomato (Solanum lycopersicum) gene with sequence similarity to EARLI1, an Arabidopsis thaliana gene that confers cold protection. To investigate whether DEA1 was responsive to environmental variables such as cold, cold-treated tomatoes were analyzed for DEA1 expression. DEA1 transcript accumulated in response to cold, and the rapidity of the cold-induced transcript accumulation was regulated by the circadian rhythm. To test whether DEA1 could protect cells from freezing damage, we transformed the yeast, Pichia pastoris, with an inducible DEA1 construct. Yeast cells transformed with the gene survived freezing at a significantly higher rate than control strains and a strain expressing the LacZ gene. Transgenic tomato plants over-expressing or knocking down DEA1 transcript levels did not have an altered phenotype with respect to cold- or pathogen-susceptibility relative to control plants.

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

    Science.gov (United States)

    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.

  15. Drying of immobilized yeast cells in a spouted bed dryer with a moving draft tube

    Directory of Open Access Journals (Sweden)

    Dragan Povrenović

    2010-07-01

    Full Text Available Brewery yeast cells immobilized in Ca-alginate were dried in a laboratory scale spouted bed with a draft tube.The experiment was conducted under variable temperatures and air flow rates. The temperature and air velocity at the bottom of the column have been varied in the range from 30 to 60 °C and from 6 to 10 m/s in a duration of 60 min. The moisture of dryied particles was in the interval of 10.00 to 21.00 g/g, while the water activity was in the range of 0.40 to 0.45 what ensures the preservation of immobilized yeast as a starter and provides the biological activity of dried particles. A rehidration process of dryied particles proved that dried particles could completely restore their original shape and starting volume, while the mechanical resistance is somewhat reduced. The cells preserved in this way completely restore their catalytical activity after the rehidration.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Direct lifts of coupled cell networks

    Science.gov (United States)

    Dias, A. P. S.; Moreira, C. S.

    2018-04-01

    In networks of dynamical systems, there are spaces defined in terms of equalities of cell coordinates which are flow-invariant under any dynamical system that has a form consistent with the given underlying network structure—the network synchrony subspaces. Given a network and one of its synchrony subspaces, any system with a form consistent with the network, restricted to the synchrony subspace, defines a new system which is consistent with a smaller network, called the quotient network of the original network by the synchrony subspace. Moreover, any system associated with the quotient can be interpreted as the restriction to the synchrony subspace of a system associated with the original network. We call the larger network a lift of the smaller network, and a lift can be interpreted as a result of the cellular splitting of the smaller network. In this paper, we address the question of the uniqueness in this lifting process in terms of the networks’ topologies. A lift G of a given network Q is said to be direct when there are no intermediate lifts of Q between them. We provide necessary and sufficient conditions for a lift of a general network to be direct. Our results characterize direct lifts using the subnetworks of all splitting cells of Q and of all split cells of G. We show that G is a direct lift of Q if and only if either the split subnetwork is a direct lift or consists of two copies of the splitting subnetwork. These results are then applied to the class of regular uniform networks and to the special classes of ring networks and acyclic networks. We also illustrate that one of the applications of our results is to the lifting bifurcation problem.

  18. Small cell networks deployment, management, and optimization

    CERN Document Server

    Claussen, Holger; Ho, Lester; Razavi, Rouzbeh; Kucera, Stepan

    2018-01-01

    Small Cell Networks: Deployment, Management, and Optimization addresses key problems of the cellular network evolution towards HetNets. It focuses on the latest developments in heterogeneous and small cell networks, as well as their deployment, operation, and maintenance. It also covers the full spectrum of the topic, from academic, research, and business to the practice of HetNets in a coherent manner. Additionally, it provides complete and practical guidelines to vendors and operators interested in deploying small cells. The first comprehensive book written by well-known researchers and engineers from Nokia Bell Labs, Small Cell Networks begins with an introduction to the subject--offering chapters on capacity scaling and key requirements of future networks. It then moves on to sections on coverage and capacity optimization, and interference management. From there, the book covers mobility management, energy efficiency, and small cell deployment, ending with a section devoted to future trends and applicat...

  19. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

    Directory of Open Access Journals (Sweden)

    Andy Hesketh

    2017-07-01

    Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

  20. Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

    OpenAIRE

    Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

    2008-01-01

    The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

  1. Cells of yeasts adhered in corn grains and the storage perspective for use as probiotic

    Directory of Open Access Journals (Sweden)

    Antonio Sampaio Baptista

    2005-03-01

    Full Text Available Yeasts were applied to corn grains, containing 16 or 20% moisture, at concentrations of 1 and 2%. The cellular viability was assayed at 0, 15, 30, 90 and 110 days. The cellular viability did not differ statistically among the treatments up to 30 days of storage, with the median viability of 89.10%. The average viability found at 90 days (72.20% was lower than in the first three storage periods. After 110 days, the average viability was 61.14%. In conclusion, since yeast cells were viable up to 110 days in storage on corn grains, these can be used as a vehicle for the application of yeast as a probiotic.O comportamento da viabilidade celular de leveduras é essencial quando se almeja utilizá-la como um probiótico e o veículo que assegure esta propriedade até o momento do consumo é particularmente. Sobre grãos de milho, contendo 16 ou 20% de umidade, foram aplicadas leveduras desidratadas vivas, nas concentrações de 1 e de 2%. A viabilidade celular foi avaliada nos períodos de 0, 15, 30, 90 e 110 dias de armazenamento. A viabilidade celular nas leveduras não diferiu estatisticamente entre os tratamentos aos 30 dias em armazenamento, com a viabilidade média de 89,10%. A viabilidade média encontrada aos 90 dias de 72,20% foi menor do que aquelas nos três primeiros períodos de armazenamento. Após 110 dias de armazenamento foi de 61,14%. Conclue-se que leveduras apresentam viabilidade celular até 110 dias em armazenamento sobre milho e que estes podem ser utilizados como um veículo para a aplicação da levedura como um probiótico.

  2. Single cell oils of the cold-adapted oleaginous yeast Rhodotorula glacialis DBVPG 4785

    Directory of Open Access Journals (Sweden)

    De Lucia Marzia

    2010-09-01

    Full Text Available Abstract Background The production of microbial lipids has attracted considerable interest during the past decade since they can be successfully used to produce biodiesel by catalyzed transesterification with short chain alcohols. Certain yeast species, including several psychrophilic isolates, are oleaginous and accumulate lipids from 20 to 70% of biomass under appropriate cultivation conditions. Among them, Rhodotorula glacialis is a psychrophilic basidiomycetous species capable to accumulate intracellular lipids. Results Rhodotorula glacialis DBVPG 4785 is an oleaginous psychrophilic yeast isolated from a glacial environment. Despite its origin, the strain abundantly grew and accumulated lipids between -3 to 20°C. The temperature did not influence the yield coefficients of both biomass and lipids production, but had positive effect on the growth rate and thus on volumetric productivity of lipid. In glucose-based media, cellular multiplication occurred first, while the lipogenic phase followed whenever the culture was limited by a nutrient other than glucose. The extent of the carbon excess had positive effects on triacylglycerols production, that was maximum with 120 g L-1 glucose, in terms of lipid concentration (19 g L-1, lipid/biomass (68% and lipid/glucose yields (16%. Both glucose concentration and growth temperature influenced the composition of fatty acids, whose unsaturation degree decreased when the temperature or glucose excess increased. Conclusions This study is the first proposed biotechnological application for Rhodotorula glacialis species, whose oleaginous biomass accumulates high amounts of lipids within a wide range of temperatures through appropriate cultivation C:N ratio. Although R. glacialis DBVPG 4785 is a cold adapted yeast, lipid production occurs over a broad range of temperatures and it can be considered an interesting microorganism for the production of single cell oils.

  3. Deficiencies in mitochondrial DNA compromise the survival of yeast cells at critically high temperatures.

    Science.gov (United States)

    Zubko, Elena I; Zubko, Mikhajlo K

    2014-01-01

    To address possible roles of mitochondrial genes in adaptation of eukaryotic cells to critical temperatures, we compared thermotolerance of mitochondrial rho mutants and wild type cells of six rho positive yeast species: Candida glabrata, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces paradoxus and Saccharomyces pastorianus. All rho mutants manifested compromised thermotolerance as a common phenotype. Analysis of viabilities at critical temperatures (32-45 °C) showed the reduction of maximum permissive temperatures (MPTs) in all rho mutants in comparison to their wild type counterparts. Degrees of the compromised thermotolerance depended on maximum permissive temperatures for wild type cells: the highest levels of MPT reductions for rho mutants took place in species and strains with highest MPTs for wild types. Short term exposures of S. cerevisiae cells (up to 3.5h) at non-permissive temperatures (45 °C and 50 °C) also lead to more rapid cell death of rho mutants as compared to wild type cells. We conclude that: (1) compromised thermotolerance could be a generic phenotypic property of rho mutants; (2) the enhanced thermotolerance of cells possessing mitochondrial genomes could be one of selective advantages in adaptation to environmental factors, in particular to enhanced temperatures. Copyright © 2013 Elsevier GmbH. All rights reserved.

  4. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    Directory of Open Access Journals (Sweden)

    Sho W Suzuki

    2011-02-01

    Full Text Available Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA. We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  5. Ammonium is toxic for aging yeast cells, inducing death and shortening of the chronological lifespan.

    Directory of Open Access Journals (Sweden)

    Júlia Santos

    Full Text Available Here we show that in aging Saccharomyces cerevisiae (budding yeast cells, NH(4 (+ induces cell death associated with shortening of chronological life span. This effect is positively correlated with the concentration of NH(4 (+ added to the culture medium and is particularly evident when cells are starved for auxotrophy-complementing amino acids. NH(4 (+-induced cell death is accompanied by an initial small increase of apoptotic cells followed by extensive necrosis. Autophagy is inhibited by NH(4 (+, but this does not cause a decrease in cell viability. We propose that the toxic effects of NH(4 (+ are mediated by activation of PKA and TOR and inhibition of Sch9p. Our data show that NH(4 (+ 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.

  6. Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must.

    Directory of Open Access Journals (Sweden)

    Catherine Tesnière

    Full Text Available We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

  7. An assessment of increased levels of yeast single cell protein (SCP ...

    African Journals Online (AJOL)

    An investigation was carried out to ascertain the effect of higher levels of yeast SCP on growth and nutrient utilization of O. niloticus fingerlings. This was a follow up of an earlier work in which 10-50% substitution of fish meal was carried out with yeast SCP. In the present work the yeast SCP levels were increased in a ...

  8. A rapid method for the differentiation of yeast cells grown under carbon and nitrogen-limited conditions by means of partial least squares discriminant analysis employing infrared micro-spectroscopic data of entire yeast cells

    OpenAIRE

    Kuligowski, Julia; Quintás, Guillermo; Herwig, Christoph; Lendl, Bernhard

    2012-01-01

    This paper shows the ease of application and usefulness of mid-IR measurements for the investigation of orthogonal cell states on the example of the analysis of Pichia pastoris cells. A rapid method for the discrimination of entire yeast cells grown under carbon and nitrogen-limited conditions based on the direct acquisition of mid-IR spectra and partial least squares discriminant analysis (PLS-DA) is described. The obtained PLS-DA model was extensively validated employing two different valid...

  9. Dynamical Adaptation in Terrorist Cells/Networks

    DEFF Research Database (Denmark)

    Hussain, Dil Muhammad Akbar; Ahmed, Zaki

    2010-01-01

    Typical terrorist cells/networks have dynamical structure as they evolve or adapt to changes which may occur due to capturing or killing of a member of the cell/network. Analytical measures in graph theory like degree centrality, betweenness and closeness centralities are very common and have long...... and followers etc. In this research we analyze and predict the most likely role a particular node can adapt once a member of the network is either killed or caught. The adaptation is based on computing Bayes posteriori probability of each node and the level of the said node in the network structure....

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

    Directory of Open Access Journals (Sweden)

    Jakub Orzechowski Westholm

    Full Text Available 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.

  11. [Optimization of synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid].

    Science.gov (United States)

    Wang, Dong; Wang, Bei-Bei; Liu, Yi; Shi, Ming-Yu; Xiao, Dong-Guang; Huang, Lu-Qi; Dai, Zhu-Bo; Zhang, Xue-Li

    2014-07-01

    To optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid. Using the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA. Increasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose. Production of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.

  12. A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells

    Science.gov (United States)

    Kasbawati; Kalondeng, A.; Aris, N.; Erawaty, N.; Azis, M. I.

    2018-03-01

    Metabolic regulation plays an important role in the metabolic engineering of a cellular process. It is conducted to improve the productivity of a microbial process by identifying the important regulatory nodes of a metabolic pathway such as fermentation pathway. Regulation of enzymes involved in a particular pathway can be held to improve the productivity of the system. In the central metabolism of yeast cell, some enzymes are known as regulating enzymes that can be inhibited to increase the production of ethanol. In this research we study the kinetic modelling of the enzymes in the central pathway of yeast metabolism by taking into consideration the enzyme inhibition effects to the ethanol production. The existence of positive steady state solution and the stability of the system are also analysed to study the property and dynamical behaviour of the system. One stable steady state of the system is produced if some conditions are fulfilled. The conditions concern to the restriction of the maximum reactions of the enzymes in the pyruvate and acetaldehyde branch points. There exists a certain time of fermentation reaction at which a maximum and a minimum ethanol productions are attained after regulating the system. Optimal ethanol concentration is also produced for a certain initial concentration of inhibitor.

  13. Adhesion of yeast cells to different porous supports, stability of cell-carrier systems and formation of volatile by-products.

    Science.gov (United States)

    Kregiel, Dorota; Berlowska, Joanna; Ambroziak, Wojciech

    2012-12-01

    The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular α-amylase and glucoamylase, were used in this study. Different media, including cell starvation, were applied for immobilization of yeast strains as well as different phases of cell growth. Immobilization of selected yeasts on a hydroxylapatite carrier was rather weak. However, when incubation of starved yeast cells was conducted in the minimal medium supplemented by calcium carbonate, the scale of immobilization after 24 h was higher, especially for the D. occidentalis strain. Adhesion to hydroxylapatite carriers in wort broth was of reversible character and better results of adhesion were observed in the case of another ceramic carrier-chamotte. The number of immobilized cells was about 10(6)-10(7) per tablet and cell adhesion was stable during the whole fermentation process. The comparison of the volatile products that were formed during fermentation did not show any significant qualitative and quantitative differences between the free and the immobilized cells. This is the first time when a cheap, porous chamotte surface has been applied to yeast adhesion and fermentation processes.

  14. LC3-Associated Phagocytosis Is Required for Dendritic Cell Inflammatory Cytokine Response to Gut Commensal Yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dimitra Lamprinaki

    2017-10-01

    Full Text Available The human fungal microbiota known as mycobiota is increasingly recognized as a critical factor in human gut health and disease. Non-pathogenic commensal yeasts such as Saccharomyces cerevisiae promote homeostasis in the gut, whereas dysbiosis of the gut mycobiota is associated with inflammation. Glycan-binding receptors (lectins are key host factors in host–mycobiota interaction in the gut. They are expressed on immune cells such as dendritic cells (DCs and recognize fungal polysaccharides. This interaction is imperative to mount appropriate immune responses for immune homeostasis in the gut as well as clearance of fungal pathogens. Recent studies demonstrate that microtubule-associated protein light-chain 3 (LC3-associated phagocytosis (LAP is involved in lectin–fungi interactions. Yet, the biological impact of LAP on the lectin function remains largely elusive. In this report, we demonstrate that in mouse LAP is linked to dendritic cell-associated lectin 2 (Dectin-2, a C-type lectin specific to fungal α-mannan polysaccharide. We found that mouse Dectin-2 recognizes commensal yeast S. cerevisiae and Kazachstania unispora. Mouse bone marrow-derived DCs (BMDCs produced inflammatory cytokines TNFα and IL-1β in response to the yeasts in a Dectin-2 and spleen tyrosine kinase (Syk-dependent manner. We found that S. cerevisiae and K. unispora induced LAP in mouse BMDCs upon internalization. Furthermore, LC3 was activated by stimulation of BMDCs with the yeasts in a Dectin-2 and Syk-dependent manner. To address the biological impact of LAP on Dectin-2 yeast interaction, we established a knock-in mouse strain (Atg16L1E230, thereafter called E230, which BMDCs exhibit autophagy-active and LAP-negative phenotypes. When stimulated with yeasts, E230 BMDCs produced significantly less amounts of TNFα and IL-1β. Taken together, we revealed a novel link between Dectin-2 and LAP that enables host immune cells to respond to mycobiota.

  15. Zygocin, a secreted antifungal toxin of the yeast Zygosaccharomyces bailii, and its effect on sensitive fungal cells.

    Science.gov (United States)

    Weiler, Frank; Schmitt, Manfred J

    2003-03-01

    Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.

  16. Yeast Cells Exposed to Exogenous Palmitoleic Acid Either Adapt to Stress and Survive or Commit to Regulated Liponecrosis and Die

    Directory of Open Access Journals (Sweden)

    Karamat Mohammad

    2018-01-01

    Full Text Available A disturbed homeostasis of cellular lipids and the resulting lipotoxicity are considered to be key contributors to many human pathologies, including obesity, metabolic syndrome, type 2 diabetes, cardiovascular diseases, and cancer. The yeast Saccharomyces cerevisiae has been successfully used for uncovering molecular mechanisms through which impaired lipid metabolism causes lipotoxicity and elicits different forms of regulated cell death. Here, we discuss mechanisms of the “liponecrotic” mode of regulated cell death in S. cerevisiae. This mode of regulated cell death can be initiated in response to a brief treatment of yeast with exogenous palmitoleic acid. Such treatment prompts the incorporation of exogenously added palmitoleic acid into phospholipids and neutral lipids. This orchestrates a global remodeling of lipid metabolism and transfer in the endoplasmic reticulum, mitochondria, lipid droplets, and the plasma membrane. Certain features of such remodeling play essential roles either in committing yeast to liponecrosis or in executing this mode of regulated cell death. We also outline four processes through which yeast cells actively resist liponecrosis by adapting to the cellular stress imposed by palmitoleic acid and maintaining viability. These prosurvival cellular processes are confined in the endoplasmic reticulum, lipid droplets, peroxisomes, autophagosomes, vacuoles, and the cytosol.

  17. A stochastic model correctly predicts changes in budding yeast cell cycle dynamics upon periodic expression of CLN2.

    Directory of Open Access Journals (Sweden)

    Cihan Oguz

    Full Text Available In this study, we focus on a recent stochastic budding yeast cell cycle model. First, we estimate the model parameters using extensive data sets: phenotypes of 110 genetic strains, single cell statistics of wild type and cln3 strains. Optimization of stochastic model parameters is achieved by an automated algorithm we recently used for a deterministic cell cycle model. Next, in order to test the predictive ability of the stochastic model, we focus on a recent experimental study in which forced periodic expression of CLN2 cyclin (driven by MET3 promoter in cln3 background has been used to synchronize budding yeast cell colonies. We demonstrate that the model correctly predicts the experimentally observed synchronization levels and cell cycle statistics of mother and daughter cells under various experimental conditions (numerical data that is not enforced in parameter optimization, in addition to correctly predicting the qualitative changes in size control due to forced CLN2 expression. Our model also generates a novel prediction: under frequent CLN2 expression pulses, G1 phase duration is bimodal among small-born cells. These cells originate from daughters with extended budded periods due to size control during the budded period. This novel prediction and the experimental trends captured by the model illustrate the interplay between cell cycle dynamics, synchronization of cell colonies, and size control in budding yeast.

  18. Non-uniform electric field-induced yeast cell electrokinetic behavior

    Directory of Open Access Journals (Sweden)

    Flavio Humberto Fernández Morales

    2008-09-01

    Full Text Available Common dielectrophoresis (c-DEP, i.e. neutral matter motion induced by non-uniform electric fields has become a basic pheno-menon of biochips intended for medical, biological and chemical assays, especially when they imply bioparticle handling. This paper deals with modelling and experimental verification of a castellated, c-DEP-based, microelectrode array intended to handle biological objects. The proposed microsystem was developed employing platinum electrodes patterned by lift-off, silicon micro-machining and photoresin patterning techniques. Saccharomyces cerevisiae were used as test bioparticles for experimental verifi-cation. Yeast cells were repelled toward electrode bays and toward interelectrodic gaps tor frequencies around 20 MHz where there is minimum electric field strength, corresponding to a negative dielectrophoretic phenomenon. Yeast cell agglomerations were observed around electrode edges for frequencies of around 2 MHz where there is maximum electric field strength, thereby verifying the positive dielectrophoretic phenomenon. Bioparticles were separated from the electrode edges when the working fre-quency was reduced and they were dragged towards the electrode centre, remaining there while the frequency was low enough. Such atypical pattern may be explained due to the occurrence of positive dielectrophoresis overlap with electrohydrodynamic effects (i.e. the viscous drag force acting on the particles was greater than the dielectrophoretic force at frequencies where positi-ve dielectrophoresis should occur. The experiments illustrated microsystem convenience in microhandling biological objects, the-reby providing these microarrays’ possible use with other cells. Liquid motion resulting from electrohydrodynamic effects must also be taken into account when designing bioparticle micromanipulators, and could be used as a mechanism for cleaning electrode surfaces.

  19. Single cell analysis of yeast replicative aging using a new generation of microfluidic device.

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    Full Text Available A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.

  20. Dynamic Metabolic Footprinting Reveals the Key Components of Metabolic Network in Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Chumnanpuen, Pramote; Hansen, Michael Adsetts Edberg; Smedsgaard, Jørn

    2014-01-01

    relies on analysis at a single time point. Using direct infusion-mass spectrometry (DI-MS), we could observe the dynamic metabolic footprinting in yeast S. cerevisiae BY4709 (wild type) cultured on 3 different C-sources (glucose, glycerol, and ethanol) and sampled along 10 time points with 5 biological...... ionization (ESI) modes were performed to obtain the complete information about the metabolite content. Using sparse principal component analysis (Sparse PCA), we further identified those pairs of metabolites that significantly contribute to the separation. From the list of significant metabolite pairs, we...

  1. The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast.

    Directory of Open Access Journals (Sweden)

    Magdalena Mos

    Full Text Available The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1 is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase.

  2. The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast

    Science.gov (United States)

    Mos, Magdalena; Esparza-Franco, Manuel A.; Godfrey, Emma L.; Richardson, Kathryn; Davey, John; Ladds, Graham

    2013-01-01

    The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase. PMID:23843946

  3. Contractile network models for adherent cells.

    Science.gov (United States)

    Guthardt Torres, P; Bischofs, I B; Schwarz, U S

    2012-01-01

    Cells sense the geometry and stiffness of their adhesive environment by active contractility. For strong adhesion to flat substrates, two-dimensional contractile network models can be used to understand how force is distributed throughout the cell. Here we compare the shape and force distribution for different variants of such network models. In contrast to Hookean networks, cable networks reflect the asymmetric response of biopolymers to tension versus compression. For passive networks, contractility is modeled by a reduced resting length of the mechanical links. In actively contracting networks, a constant force couple is introduced into each link in order to model contraction by molecular motors. If combined with fixed adhesion sites, all network models lead to invaginated cell shapes, but only actively contracting cable networks lead to the circular arc morphology typical for strongly adhering cells. In this case, shape and force distribution are determined by local rather than global determinants and thus are suited to endow the cell with a robust sense of its environment. We also discuss nonlinear and adaptive linker mechanics as well as the relation to tissue shape. © 2012 American Physical Society

  4. Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Michael A. [Oxiage Cosmeceutical Research Institute, Virginia (United States)

    2013-05-15

    Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD{sub 50}) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative

  5. Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

    International Nuclear Information System (INIS)

    Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung; Nili, Michael A.

    2013-01-01

    Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD 50 ) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative stress by

  6. Use of INAA to study the determination of Se, Th, Zn, Co and Fe levels of yeast cells

    International Nuclear Information System (INIS)

    Czauderna, M.; Turska, M.; Sitowska, B.

    1996-01-01

    Differences in the effects of seleno-cystine (CySe) 2 , glutathione (GSH), Se(IV) [as SeO 2 ] and Se(VI) [as (NH 4 ) 2 SeO 4 ] on Th(IV) [as Th(CO 3 ) 2 ] uptake by the cells, Saccharomyces cerevisiae, have been studied. The Th, Se, Zn, Co and Fe levels of the yeast cells were measured by instrumental neutron activation analysis. Results obtained show that the addition of Th alone to the culture medium resulting in the Th content of the cells and the Th level of the yeast slightly decreased during the incubation. The addition of Th in combination with GSH produced a higher decrease of the Th content in comparison with the single Th dosage. During the initial 48 h of the incubation the presence of Th and Se(VI) in the medium produced a decrease of the Th level of the cells in comparison with the addition of Th alone. (CySe) 2 or SeO 2 does not produce a regular change of the Th level of the cells. Th uptake by the yeast influenced the retention of Se in the cells. In fact, the Se levels of the cells were always significantly higher when the yeast was incubated in the medium containing Th and SeO 2 or Se(VI). The enhance in the Se level of the cells rises increasing concentrations of SeO 2 in the culture medium. Th decreased the Se content of the yeast when the cells were incubated in the medium containing (CySe) 2 and Th. GSH supply in combination with Th and SeO 2 produced a very significant enhancement of the Se abundance in the cells in comparison with the single addition of SeO 2 . Se-compounds and/or Th dosages affected the Zn, Co and Fe contents of the cells. The Fe level of the yeast is below the quantitative detection limit of Fe when the cells were incubated in the medium containing Th. (Author)

  7. Physicochemical characterization of the yeast cells and the waste lignocellulosic particles in the immobilization process for ethanol production

    DEFF Research Database (Denmark)

    Agudelo-Escobar, Lina María; Mussatto, Solange I.; Peñuela, Mariana

    2017-01-01

    Ethanol is one of the leading alternative fuels. Efforts have increased the development of technologies for producing ethanol efficiently and economically. The continuous fermentation using yeast cells immobilized in low‐cost materials is presented as an excellent alternative. We used four...

  8. Methionine restriction activates the retrograde response and confers both stress tolerance and lifespan extension to yeast, mouse and human cells.

    Directory of Open Access Journals (Sweden)

    Jay E Johnson

    Full Text Available A methionine-restricted diet robustly improves healthspan in key model organisms. For example, methionine restriction reduces age-related pathologies and extends lifespan up to 45% in rodents. However, the mechanisms underlying these benefits remain largely unknown. We tested whether the yeast chronological aging assay could model the benefits of methionine restriction, and found that this intervention extends lifespan when enforced by either dietary or genetic approaches, and furthermore, that the observed lifespan extension is due primarily to reduced acid accumulation. In addition, methionine restriction-induced lifespan extension requires the activity of the retrograde response, which regulates nuclear gene expression in response to changes in mitochondrial function. Consistent with an involvement of stress-responsive retrograde signaling, we also found that methionine-restricted yeast are more stress tolerant than control cells. Prompted by these findings in yeast, we tested the effects of genetic methionine restriction on the stress tolerance and replicative lifespans of cultured mouse and human fibroblasts. We found that such methionine-restricted mammalian cells are resistant to numerous cytotoxic stresses, and are substantially longer-lived than control cells. In addition, similar to yeast, the extended lifespan of methionine-restricted mammalian cells is associated with NFκB-mediated retrograde signaling. Overall, our data suggest that improved stress tolerance and extension of replicative lifespan may contribute to the improved healthspan observed in methionine-restricted rodents, and also support the possibility that manipulation of the pathways engaged by methionine restriction may improve healthspan in humans.

  9. Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers

    DEFF Research Database (Denmark)

    Haaning, J; Oxvig, C; Overgaard, Michael Toft

    1997-01-01

    by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature...

  10. VLSI Cells Placement Using the Neural Networks

    International Nuclear Information System (INIS)

    Azizi, Hacene; Zouaoui, Lamri; Mokhnache, Salah

    2008-01-01

    The artificial neural networks have been studied for several years. Their effectiveness makes it possible to expect high performances. The privileged fields of these techniques remain the recognition and classification. Various applications of optimization are also studied under the angle of the artificial neural networks. They make it possible to apply distributed heuristic algorithms. In this article, a solution to placement problem of the various cells at the time of the realization of an integrated circuit is proposed by using the KOHONEN network

  11. Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Perou, C.M.; Pryor, R.J.; Kaplan, J. [Univ. of Utah, Salt Lake City, UT (United States); Justice, M.J. [Oak Ridge National Labortory, TN (United States)

    1996-06-11

    Chediak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melansosome morphology and function. We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation. The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418. When YAC-complemented beige cells were fused to human Chediak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred. These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans. 16 refs., 5 figs.

  12. A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells.

    Science.gov (United States)

    Tomaszewska, Ludwika; Rakicka, Magdalena; Rymowicz, Waldemar; Rywińska, Anita

    2014-09-01

    Citric acid and erythritol biosynthesis from pure and crude glycerol by three acetate-negative mutants of Yarrowia lipolytica yeast was investigated in batch cultures in a wide pH range (3.0-6.5). Citric acid biosynthesis was the most effective at pH 5.0-5.5 in the case of Wratislavia 1.31 and Wratislavia AWG7. With a decreasing pH value, the direction of biosynthesis changed into erythritol synthesis accompanied by low production of citric acid. Pathways of glycerol conversion into erythritol and citric acid were investigated in Wratislavia K1 cells. Enzymatic activity was compared in cultures run at pH 3.0 and 4.5, that is, under conditions promoting the production of erythritol and citric acid, respectively. The effect of pH value (3.0 and 4.5) and NaCl presence on the extracellular production and intracellular accumulation of citric acid and erythritol was compared as well. Low pH and NaCl resulted in diminished activity of glycerol kinase, whereas such conditions stimulated the activity of glycerol-3-phosphate dehydrogenase. The presence of NaCl strongly influenced enzymes activity - the effective erythritol production was correlated with a high activity of transketolase and erythrose reductase. Therefore, presented results confirmed that transketolase and erythrose reductase are involved in the overproduction of erythritol in the cells of Y. lipolytica yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  13. Effects of adding yeast cell walls and Yucca schidigera extract to diets of layer chicks.

    Science.gov (United States)

    Gurbuz, E; Balevi, T; Kurtoglu, V; Oznurlu, Y

    2011-10-01

    This research was conducted to determine the impact of diet supplementation with yeast cell walls and Yucca schidigera extract on the growth performance, antibody titres, and intestinal tissue histology of layer chicks. White, 1-d-old, Hy-Line hybrid chicks (n = 840) were divided into 4 main groups, each comprising 7 replicates of 30 chicks (n = 210): (1) control; (2) 1000 mg/kg yeast cell walls (YCW) added; (3) 1000 mg/kg Yucca schidigera extract (YE) added; and (4) 500 mg/kg YE + 500 mg/kg YCW added. The trial lasted 60 d. Daily weight gain of the chicks was positively affected between d 45-60 in the YE and YCW + YE groups compared with the control group. Overall, feed consumption did not differ between the control and YCW, YE, YCW + YE groups during the 60 d study period. Feed efficiency was better in the YE and YCW + YE groups than in the control group between d 1-60. During the 60 d evaluation period, live weight gain, and final live weight were higher in YE and YCW + YE groups than in the control group. Antibody titres against infectious bronchitis and infectious bursal disease did not differ among the 4 treatments, but those for Newcastle disease were higher in the YE + YCW groups than in the control, YCW and YE groups on d 45. There were differences in intestinal histomorphometry between the 4 treatments. The height of the jejunal and ileal villi was greater in the YE and YCW + YE groups than in the control and YCW groups. It can be concluded that YCW and YE supplementation for layer chicks is beneficial for growth performance and intestinal histology during the 1-60 d growing period.

  14. Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

    Science.gov (United States)

    Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

    2017-03-01

    Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

  15. Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

    DEFF Research Database (Denmark)

    Spåhr, H; Samuelsen, C O; Baraznenok, V

    2001-01-01

    . cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10...... essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated....

  16. Toxicology of the aqueous extract from the flowers of Butea monosperma Lam. and it's metabolomics in yeast cells.

    Science.gov (United States)

    Khan, Washim; Gupta, Shreesh; Ahmad, Sayeed

    2017-10-01

    Due to lack of scientific evidence for the safety of Butea monosperma (Fabaceae), our study aimed to carry out its toxicological profile and to identify its metabolic pattern in yeast cell. The effect of aqueous extract of B. monosperma flower on glucose uptake in yeast cell was evaluated through optimizing pH, temperature, incubation time, substrate concentration and kinetic parameters. Further, the metabolic pattern of extract as such and in yeast cell were analyzed by gas chromatography-mass spectrometry. Mice were administered aqueous extract up to 6000 and 4000 mg/kg for acute oral and intraperitoneal toxicity, respectively, while up to 4500 mg/kg for sub-acute oral toxicity (30 days). Elongation in the lag and log phase was observed in yeast cells supplemented with extract as compared to control. A maximum of 184.9% glucose uptake was observed whereas kinetic parameters (K m and V max ) were 1.38 and 41.91 mol/s, respectively. Out of 75 metabolites found in the extract, 14 and 18 metabolites were utilized by yeast cell after 15 and 30 min of incubation, respectively. The LD 50 of extract administered through intraperitoneal route was estimated to be 3500 mg/kg. The extract did not elicit any significant difference (P ≥ 0.05) in weight gain, food consumption, water intake, hematological, biochemical parameters and histological changes as compared to the normal control. Results ascertained the safety of B. monosperma flower extract which can be explored as potential candidates for the development of anti-diabetic phytopharmaceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. A consensus yeast metabolic network obtained from a community approach to systems biology.

    NARCIS (Netherlands)

    Herrgard, M.J.; Swainston, N.; Dobson, P.; Dunn, W.B.; Arga, K.Y.; Arvas, M.; Bluthgen, N.; Borger, S.; Costenoble, E.R.; Heinemann, M.; Hucka, M.; Li, P.; Liebermeister, W.; Mo, M.L.; Oliveira, A.P.; Petranovic, D.; Pettifer, S.; Simeonides, E.; Smallbone, K.; Spasi, I.; Weichart, D.; Brent, R.; Broomhead, D.S.; Westerhoff, H.V.; Kirdar, B.; Penttila, M.; Klipp, E.; Paton, N.; Palsson, B.O.; Sauer, U.; Oliver, S.G.; Mendes, P.; Nielsen, J.; Kell, D.B.

    2008-01-01

    Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and

  18. A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology

    NARCIS (Netherlands)

    Herrgård, Markus J.; Swainston, Neil; Dobson, Paul; Dunn, Warwick B.; Arga, K. Yalçin; Arvas, Mikko; Blüthgen, Nils; Borger, Simon; Costenoble, Roeland; Heinemann, Matthias; Hucka, Michael; Novère, Nicolas Le; Li, Peter; Liebermeister, Wolfram; Mo, Monica L.; Oliveira, Ana Paula; Petranovic, Dina; Pettifer, Stephen; Simeonidis, Evangelos; Smallbone, Kieran; Spasić, Irena; Weichart, Dieter; Brent, Roger; Broomhead, David S.; Westerhoff, Hans V.; Kırdar, Betül; Penttilä, Merja; Klipp, Edda; Palsson, Bernhard Ø.; Sauer, Uwe; Oliver, Stephen G.; Mendes, Pedro; Nielsen, Jens; Kell, Douglas B.

    2008-01-01

    Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and

  19. A Nonlinear Mixed Effects Approach for Modeling the Cell-To-Cell Variability of Mig1 Dynamics in Yeast.

    Directory of Open Access Journals (Sweden)

    Joachim Almquist

    Full Text Available The last decade has seen a rapid development of experimental techniques that allow data collection from individual cells. These techniques have enabled the discovery and characterization of variability within a population of genetically identical cells. Nonlinear mixed effects (NLME modeling is an established framework for studying variability between individuals in a population, frequently used in pharmacokinetics and pharmacodynamics, but its potential for studies of cell-to-cell variability in molecular cell biology is yet to be exploited. Here we take advantage of this novel application of NLME modeling to study cell-to-cell variability in the dynamic behavior of the yeast transcription repressor Mig1. In particular, we investigate a recently discovered phenomenon where Mig1 during a short and transient period exits the nucleus when cells experience a shift from high to intermediate levels of extracellular glucose. A phenomenological model based on ordinary differential equations describing the transient dynamics of nuclear Mig1 is introduced, and according to the NLME methodology the parameters of this model are in turn modeled by a multivariate probability distribution. Using time-lapse microscopy data from nearly 200 cells, we estimate this parameter distribution according to the approach of maximizing the population likelihood. Based on the estimated distribution, parameter values for individual cells are furthermore characterized and the resulting Mig1 dynamics are compared to the single cell times-series data. The proposed NLME framework is also compared to the intuitive but limited standard two-stage (STS approach. We demonstrate that the latter may overestimate variabilities by up to almost five fold. Finally, Monte Carlo simulations of the inferred population model are used to predict the distribution of key characteristics of the Mig1 transient response. We find that with decreasing levels of post-shift glucose, the transient

  20. Mechanical Cell-Cell Communication in Fibrous Networks: The Importance of Network Geometry.

    Science.gov (United States)

    Humphries, D L; Grogan, J A; Gaffney, E A

    2017-03-01

    Cells contracting in extracellular matrix (ECM) can transmit stress over long distances, communicating their position and orientation to cells many tens of micrometres away. Such phenomena are not observed when cells are seeded on substrates with linear elastic properties, such as polyacrylamide (PA) gel. The ability for fibrous substrates to support far reaching stress and strain fields has implications for many physiological processes, while the mechanical properties of ECM are central to several pathological processes, including tumour invasion and fibrosis. Theoretical models have investigated the properties of ECM in a variety of network geometries. However, the effects of network architecture on mechanical cell-cell communication have received little attention. This work investigates the effects of geometry on network mechanics, and thus the ability for cells to communicate mechanically through different networks. Cell-derived displacement fields are quantified for various network geometries while controlling for network topology, cross-link density and micromechanical properties. We find that the heterogeneity of response, fibre alignment, and substrate displacement fields are sensitive to network choice. Further, we show that certain geometries support mechanical communication over longer distances than others. As such, we predict that the choice of network geometry is important in fundamental modelling of cell-cell interactions in fibrous substrates, as well as in experimental settings, where mechanical signalling at the cellular scale plays an important role. This work thus informs the construction of theoretical models for substrate mechanics and experimental explorations of mechanical cell-cell communication.

  1. Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

    Science.gov (United States)

    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.

  2. Tomato Phospholipid Hydroperoxide Glutathione Peroxidase Inhibits Cell Death Induced by Bax and Oxidative Stresses in Yeast and Plants1

    Science.gov (United States)

    Chen, Shaorong; Vaghchhipawala, Zarir; Li, Wei; Asard, Han; Dickman, Martin B.

    2004-01-01

    Using a conditional life or death screen in yeast, we have isolated a tomato (Lycopersicon esculentum) gene encoding a phospholipid hydroperoxide glutathione peroxidase (LePHGPx). The protein displayed reduced glutathione-dependent phospholipid hydroperoxide peroxidase activity, but differs from counterpart mammalian enzymes that instead contain an active seleno-Cys. LePHGPx functioned as a cytoprotector in yeast (Saccharomyces cerevisiae), preventing Bax, hydrogen peroxide, and heat stress induced cell death, while also delaying yeast senescence. When tobacco (Nicotiana tabacum) leaves were exposed to lethal levels of salt and heat stress, features associated with mammalian apoptosis were observed. Importantly, transient expression of LePHGPx protected tobacco leaves from salt and heat stress and suppressed the apoptotic-like features. As has been reported, conditional expression of Bax was lethal in tobacco, resulting in tissue collapse and membrane permeability to Evans blue. When LePHGPx was coexpressed with Bax, little cell death and no vital staining were observed. Moreover, stable expression of LePHGPx in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea. Taken together, our data indicated that LePHGPx can protect plant tissue from a variety of stresses. Moreover, functional screens in yeast are a viable tool for the identification of plant genes that regulate cell death. PMID:15235116

  3. Single Cell Oil Producing Yeasts Lipomyces starkeyi and Rhodosporidium toruloides: Selection of Extraction Strategies and Biodiesel Property Prediction

    Directory of Open Access Journals (Sweden)

    Nemailla Bonturi

    2015-05-01

    Full Text Available Single cell oils (SCOs are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi. Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi. Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

  4. Consequences of calorie restriction and calorie excess for the physiological parameters of the yeast Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Maslanka, Roman; Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata

    2017-12-01

    Glucose plays an important role in cell metabolism and has an impact on cellular physiology. Changes in glucose availability may strongly influence growth rate of the cell size, cell metabolism and the rate of generation of cellular by-products, such as reactive oxygen species. The positive effect of low glucose concentration conditions-calorie restriction is observed in a wide range of species, including the Saccharomyces cerevisiae yeast, yet little is known about the effect of high glucose concentrations-calorie excess. Such analysis seems to be particularly important due to recently common problem of diabetes and obesity. The effect of glucose on morphological and physiological parameters of the yeast cell was conducted using genetic alteration (disruption of genes involved in glucose signalling) and calorie restriction and calorie excess conditions. The results show a significant relationship among extracellular glucose concentration, cell size and reactive oxygen species generation in yeast cells. Furthermore, the results obtained through the use of mutant strains with disorders in glucose signalling pathways suggest that the intracellular level of glucose is more important than its extracellular concentration. These data also suggest that the calorie excess as a factor, which has a significant impact on cell physiology, requires further comprehensive analyses. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Effect of yeast TEL1 gene expression in A-T cells on chromosome aberration induced by radiation

    International Nuclear Information System (INIS)

    Cao Jianping; Zheng Siying; Zhu Wei; Luo Jialin; Zhou Xianfeng; Wang Mingming

    2004-01-01

    Objective: To analyze the effect of yeast TEL1 gene expression in human A-T (ataxia-telangiectasia) cells on chromosome aberration induced by radiation. Methods: Yeast TEL1 gene (TEL1rep), TEL1 fragment (deltaTELrep), pRep5 vector were stably transfected into A-T cells and pRep5 vector was stably transfected into GM639 cells. respectively. The stably transfected A-T and GM639 cells, namely, AT-TEL1, AT-deltaTEL1, AT-rep, GM-rep, were grown selectively in Dulbecco's minimal essential medium (DMEM) containing 100 μg/ml hygromycin, while non-transfected A-T and GM639 cells were grown in DMEM containing 15%220% FBS. 60 Co γ-rays were used to irradiate the above cells at exponential phase. The irradiation dose was 0, 1, 3 Gy, respectively. A hundred metaphases were analyzed for microscopically detectable chromosome type and chromatid type aberrations. Results: The chromosome numbers of all transfected, and non-transfected A-T, GM639 fobroblasts were aneuploid. Both spontaneous and radiation-induced chromosome aberration frequencies of A-T cells were higher than those of GM639 control cells. There is no significant difference in the spontaneous chromosome aberration between TEL1 gene stably transfected A-T cells (AT-TEL1) and non-transfected A-T cells (P>0.05). Chromosome aberration frequencies induced by radiation in AT-TEL1 cells were significantly lower than those of A-T cells (P<0.01). However, chromosome aberration frequencies induced by radiation in AT-deltaTEL1 and AT-rep cells were higher than those of A-T cells. Conclusion: The expression of yeast TEL1 gene in human A-T cells can significantly decrease the chromosome aberration frequencies induced by radiation. (authors)

  6. Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity

    International Nuclear Information System (INIS)

    Moore, C.W.

    1982-01-01

    Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 0 C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +- 0.07 and 5.7 +- 0.6 single-strand breaks per 10 8 daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 0 C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +- 0.3 (at 23 0 C) or 0.21 +- 0.05 (at 37 0 C) per 10 8 daltons in parental cells, but remained at 2.1 +- 0.06 (at 23 0 C) or 2.3 +- 0.07 (at 37 0 C) per 10 8 daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 0 C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 0 C, DNAs in both strains were completely rejoined

  7. Modification of aflatoxin B1 and ochratoxin A toxicokinetics in rats administered a yeast cell wall preparation

    OpenAIRE

    2010-01-01

    Abstract The cell wall of Saccharomyces cerevisiae can bind mycotoxins in vitro but there is scarce information on whether this property decreases the absorption of mycotoxins in vivo. The effect of a yeast cell wall preparation (YCW) on toxicokinetics and balance excretion (urine and faeces) of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was tested in rats after oral administration of each toxin. The 3H-labelled mycotoxins were used at low doses. Co-administration of YCW with AF...

  8. Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells.

    Science.gov (United States)

    Nielsen, Jens Christian; Senne de Oliveira Lino, Felipe; Rasmussen, Thomas Gundelund; Thykær, Jette; Workman, Christopher T; Basso, Thiago Olitta

    2017-11-01

    The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO 2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates and glucose uptake rates, while commercial AFA had no effect in concentrations relevant for defoaming purposes. Industrial AFA were further tested in laboratory scale simulations of the Brazilian ethanol production process and proved to decrease cell viability compared to the control, and the effects were intensified with increasing AFA concentrations and exposure time. Transcriptome analysis showed that AFA treatments induced additional stress responses in yeast cells compared to the control, shown by an up-regulation of stress-specific genes and a down-regulation of lipid biosynthesis, especially ergosterol. By documenting the detrimental effects associated with chemical AFA, we highlight the importance of developing innocuous systems for foam control in industrial fermentation processes.

  9. The measurement temperature: an important factor relating physicochemical and adhesive properties of yeast cells to biomaterials.

    Science.gov (United States)

    Gallardo-Moreno, A M; González-Martín, M L; Pérez-Giraldo, C; Bruque, J M; Gómez-García, A C

    2004-03-15

    Flow chambers applied to the study of the initial adhesion process of Candida parapsilosis are rarely found in the literature. The ability of these microorganisms to proliferate and form biofilms in environments at temperatures around 22 or 37 degrees C is reflected in the contamination of laboratory instruments and material or in human implant infections, respectively. The initial interaction between yeasts and substrata is mediated by physicochemical forces, which in turn originate from the physicochemical surface properties of both interacting phases. In this context, this work aims to relate the initial rates of adhesion rates to glass and silicone of Candida parapsilosis, strains 294 and 289, grown at 22 and 37 degrees C with the theoretical predictions of the adhesion process, expressed by the interaction free energies and calculated through the physicochemical parameters, which are also measured at 22 and 37 degrees C. The results indicate that physicochemical parameters of yeasts are changed not only by the culture temperature but also by the measurement temperature; only when the measurement temperature is equal to the growth temperature a coherent relation between in vitro adhesion data and interaction free energies can be established. In this sense, the adhesion to glass is mediated by long-range forces or, what amounts to the same thing, by Lifshitz-van der Waals interaction free energy. On the other hand, the adhesion to silicone rubber seems to be moderated by acid-base interaction free energy, which involves the presence of short-range forces. Based on these results, it can be assumed that the substratum surface properties are directly related to the kind of force acting on the initial microbial adhesion process, while cell surface properties dictate the changes in the strength of the force between different samples.

  10. The Bioeffects Resulting from Prokaryotic Cells and Yeast Being Exposed to an 18 GHz Electromagnetic Field.

    Directory of Open Access Journals (Sweden)

    The Hong Phong Nguyen

    Full Text Available The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMFwere studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure, independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM and confocal laser scanning microscopy (CLSM. Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid may affect the extent of uptake of the large nanospheres (46 nm. Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T.

  11. Performance study of sugar-yeast-ethanol bio-hybrid fuel cells

    Science.gov (United States)

    Jahnke, Justin P.; Mackie, David M.; Benyamin, Marcus; Ganguli, Rahul; Sumner, James J.

    2015-05-01

    Renewable alternatives to fossil hydrocarbons for energy generation are of general interest for a variety of political, economic, environmental, and practical reasons. In particular, energy from biomass has many advantages, including safety, sustainability, and the ability to be scavenged from native ecosystems or from waste streams. Microbial fuel cells (MFCs) can take advantage of microorganism metabolism to efficiently use sugar and other biomolecules as fuel, but are limited by low power densities. In contrast, direct alcohol fuel cells (DAFCs) take advantage of proton exchange membranes (PEMs) to generate electricity from alcohols at much higher power densities. Here, we investigate a novel bio-hybrid fuel cell design prepared using commercial off-the-shelf DAFCs. In the bio-hybrid fuel cells, biomass such as sugar is fermented by yeast to ethanol, which can be used to fuel a DAFC. A separation membrane between the fermentation and the DAFC is used to purify the fermentate while avoiding any parasitic power losses. However, shifting the DAFCs from pure alcohol-water solutions to filtered fermented media introduces complications related to how the starting materials, fermentation byproducts, and DAFC waste products affect both the fermentation and the long-term DAFC performance. This study examines the impact of separation membrane pore size, fermentation/fuel cell byproducts, alcohol and salt concentrations, and load resistance on fuel cell performance. Under optimized conditions, the performance obtained is comparable to that of a similar DAFC run with a pure alcohol-water mixture. Additionally, the modified DAFC can provide useable amounts of power for weeks.

  12. Fabrication of microstamps and patterned cell network

    International Nuclear Information System (INIS)

    Seong, Nak Seon; Pak, James Jung Ho; Choi, Ju Hee; Ahn, Dong June; Hwang, Seong Min; Lee, Kyung J.

    2002-01-01

    Elastomeric stamps with micrometer-sized grids are fabricated for building biological cell networks by design. Polymerized polydimethyl-siloxane (PDMS) stamps are cast in a variety of different molds prepared by micro-electro mechanical systems (MEMS) technology. Micro square-grid patterns of 3-aminopropyl triethoxy silane (APS) are successfully imprinted on glass plates, and patterned networks of cardiac cells are obtained as designed. The resulting cellular networks clearly demonstrate that cell attachment and growth are greatly favored on APS-treated thin tracks. Here, we report the technical details related to the fabrication of microstamps, to the stamping procedure, and to the culture method. The potential applications of patterned cellular networks are also discussed

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Effect of mercury and organomercurials on cellular glucose utilization: a study using resting mercury-resistant yeast cells.

    Science.gov (United States)

    Ghosh, S K; Chaudhuri, J; Gachhui, R; Mandal, A; Ghosh, S

    2007-02-01

    Mercury compounds are highly toxic to all types of living cells. Isolated yeast strains of Rhodotorula rubra showed high and low resistance pattern towards mercury and organomercurial compounds. To investigate the basis of differential sensitivity of these two types of strains, glucose utilization was measured in the presence of mercury compounds. Glucose utilization process remained unaffected in resting cells of highly Hg(2+)-resistant strain in the presence of HgCl(2) but not in the presence of phenylmercuric acetate and thimerosal. However, HgCl(2) significantly affected glucose utilization in the case of low-resistant cells. The Hg-retaining ability of the cell wall of highly Hg(2+)-resistant yeast strain was greater than that of the weakly Hg(2+)-resistant strain. The spheroplast-bound Hg(2+) was also significantly less in the highly Hg(2+)-resistant strain than in the weakly Hg(2+)-resistant strain. Glucose uptake machinery was not affected in the presence of toxic metal ions in the case of high-resistant strains. But in the case of low Hg(2+)-resistant strain, glucose transport system may be affected either by inactivation of sensor proteins containing -SH group associated with glucose uptake. Cell wall of mercury-resistant yeast cells may play an important role in heavy metal bioremediation process.

  15. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    Science.gov (United States)

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  16. The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 → S transition

    International Nuclear Information System (INIS)

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

    2011-01-01

    Highlights: → TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. → TCP4 expression in yeast retards cell division by blocking G1 → S transition. → 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 → 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 → S arrest is discussed.

  17. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar: Increased Abundance and Expression of a Calreticulin-Like Protein.

    Directory of Open Access Journals (Sweden)

    Giulia Micallef

    Full Text Available In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  18. Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels

    Science.gov (United States)

    Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie HD

    2015-01-01

    Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production. DOI: http://dx.doi.org/10.7554/eLife.05896.001 PMID:25647728

  19. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    Science.gov (United States)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  20. L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches.

    Science.gov (United States)

    Karkovska, Maria; Smutok, Oleh; Stasyuk, Nataliya; Gonchar, Mykhailo

    2015-11-01

    In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics. The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel. The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells. The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials.

    Science.gov (United States)

    Zhao, Chun-Hai; Cui, Wei; Liu, Xiao-Yan; Chi, Zhen-Ming; Madzak, Catherine

    2010-11-01

    Yarrowia lipolytica ACA-DC 50109 has been reported to be an oleaginous yeast and significant quantities of lipids were accumulated inside the yeast cells. In this study, the INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. The activity of the inulinase with 6 × His tag secreted by the transformant Z31 obtained was found to be 41.7U mL(-1) after cell growth for 78 h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L(-1) within 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g L(-1) within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g L(-1) within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C(16:0), C(18:1) and C(18:2), especially C(18:1) (58.5%). Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Extension of Yeast Chronological Lifespan by Methylamine

    NARCIS (Netherlands)

    Kumar, Sanjeev; Lefevre, Sophie D.; Veenhuis, Marten; van der Klei, Ida J.

    2012-01-01

    Background: Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast

  3. Bacterial Networks in Cells and Communities.

    Science.gov (United States)

    Sourjik, Victor; Vorholt, Julia A

    2015-11-20

    Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Optimization and model reduction in the high dimensional parameter space of a budding yeast cell cycle model

    Science.gov (United States)

    2013-01-01

    Background Parameter estimation from experimental data is critical for mathematical modeling of protein regulatory networks. For realistic networks with dozens of species and reactions, parameter estimation is an especially challenging task. In this study, we present an approach for parameter estimation that is effective in fitting a model of the budding yeast cell cycle (comprising 26 nonlinear ordinary differential equations containing 126 rate constants) to the experimentally observed phenotypes (viable or inviable) of 119 genetic strains carrying mutations of cell cycle genes. Results Starting from an initial guess of the parameter values, which correctly captures the phenotypes of only 72 genetic strains, our parameter estimation algorithm quickly improves the success rate of the model to 105–111 of the 119 strains. This success rate is comparable to the best values achieved by a skilled modeler manually choosing parameters over many weeks. The algorithm combines two search and optimization strategies. First, we use Latin hypercube sampling to explore a region surrounding the initial guess. From these samples, we choose ∼20 different sets of parameter values that correctly capture wild type viability. These sets form the starting generation of differential evolution that selects new parameter values that perform better in terms of their success rate in capturing phenotypes. In addition to producing highly successful combinations of parameter values, we analyze the results to determine the parameters that are most critical for matching experimental outcomes and the most competitive strains whose correct outcome with a given parameter vector forces numerous other strains to have incorrect outcomes. These “most critical parameters” and “most competitive strains” provide biological insights into the model. Conversely, the “least critical parameters” and “least competitive strains” suggest ways to reduce the computational complexity of the

  5. Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

    Science.gov (United States)

    Hauptmann, Peter; Lehle, Ludwig

    2008-07-04

    N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

  6. Cell mass and cell cycle dynamics of an asynchronous budding yeast population

    DEFF Research Database (Denmark)

    Lencastre Fernandes, Rita; Carlquist, Magnus; Lundin, Luisa

    2013-01-01

    in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation...... and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation.......Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development...

  7. Signaling pathways and posttranslational modifications of tau in Alzheimer’s disease: the humanization of yeast cells

    Directory of Open Access Journals (Sweden)

    Jürgen J. Heinisch

    2016-03-01

    Full Text Available 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 neurodegenerative diseases collectively called tauopathies and which includes Alzheimer’s disease (AD as its most common representative. In the course of the disease, tau changes its phosphorylation state and becomes hyperphosphorylated, 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 heterologous gene expression. Here, we present an overview on common modifications 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.

  8. Effect of Shock Waves Generated by Pulsed Electric Discharges in Water on Yeast Cells and Virus Particles

    Science.gov (United States)

    Girdyuk, A. E.; Gorshkov, A. N.; Egorov, V. V.; Kolikov, V. A.; Snetov, V. N.; Shneerson, G. A.

    2018-02-01

    The aim of this study is to determine the optimal parameters of the electric pulses and shock waves generated by them for the soft destruction of the virus and yeast envelopes with no changes in the structure of antigenic surface albumin and in the cell morphology in order to use them to produce antivirus vaccines and in biotechnology. The pulse electric discharges in water have been studied for different values of amplitude, pulse duration and the rate of the rise in the current. A mathematical model has been developed to estimate the optimal parameters of pulsed electric charges and shock waves for the complete destruction of the yeast cell envelopes and virus particles at a minimum of pulses.

  9. Immunogenic evaluation of Paracoccidioides brasiliensis radioattenuated yeast cells in murine model

    International Nuclear Information System (INIS)

    Martins, Estefania M.N.; Andrade, Antero S.R.; Fernandes, Viviane Cristina; Morais, Elis Araujo; Goes, Alfredo M.; Resende, Maria Aparecida de

    2011-01-01

    Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a chronic systemic disease prevalent in Latin American, which is characterized by the formation of granulomatous lesions. To date, there is no effective vaccine to PCM or to any systemic mycosis. In an attempt to induce an efficient response to such agent in an animal model, gamma radiation attenuated P. brasiliensis yeast cells (LevRad) were developed at the Radiobiology Laboratory from CDTN/CNEN. A gamma radiation dose was defined in which the pathogen loses its ability to multiply, while retaining its viability, metabolic activity and antigenic profile. The prophylactic potential of LevRad was assessed after its intravenous administration in male Balb/C mice, challenged 45 days after immunization with intratracheal administration of 3x105 cells of a highly virulent non-radiated P.brasiliensis isolate. At 30 and 90 days post challenge (dpc), lungs, spleen and liver were collected to analyse CFU (colony forming units) recovery, histology, cell proliferation, cytokine (IFN-gamma, IL-4, IL-10, TNF-alpha and TGF-beta) and iNOS production. The sera were used to evaluate the immunization efficacy, and to assess IgG isotypes (IgG1, IgG2a, IgG2b, IgG3) and total IgG levels. The present data show that there was no significant decrease in the CFU counts of the lungs of immunized animals 30 dpc. Nevertheless, no CFU or histopathological alterations were visualized at the organs of immunized animals at 90 dpc. During the same period, IgG2a, IgG2b, IFN-alpha and iNOS levels raised while IL-10, TNF-alpha, TGF-beta and IL-4 maintained low levels, suggesting the prevalence of Th1 response profile. Our results confirmed the protective (author)

  10. Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

    Directory of Open Access Journals (Sweden)

    Tolliday Nicola J

    2006-06-01

    Full Text Available Abstract Background All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae, in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD of budding yeast Myo1. Results We show that the MLD is a small region in the centre of the tail of Myo1 and that it is both necessary and sufficient for localisation of Myo1 to the yeast bud neck, the pre-determined site of cell division. Hydrodynamic measurements of the MLD, purified from bacteria or yeast, show that it is likely to exist as a trimer. We also examine the importance of a small region of low coiled coil forming probability within the MLD, which we call the hinge region. Removal of the hinge region prevents contraction of the CR. Using fluorescence recovery after photobleaching (FRAP, we show that GFP-tagged MLD is slightly more dynamic than the GFP-tagged full length molecule but less dynamic than the GFP-tagged Myo1 construct lacking the hinge region. Conclusion Our results define the intrinsic determinant for the localization of budding yeast myosin II and show

  11. Coevolution Trumps Pleiotropy: Carbon Assimilation Traits Are Independent of Metabolic Network Structure in Budding Yeast

    Science.gov (United States)

    Opulente, Dana A.; Morales, Christopher M.; Carey, Lucas B.; Rest, Joshua S.

    2013-01-01

    Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). However, the extent to which network pleiotropy versus environmental coevolution shapes shared responses has not been addressed. To test these alternatives, we took advantage of the fact that the genus Saccharomyces has variation in habitat usage and diversity in the carbon sources that a given strain can metabolize. We examined patterns of gain and loss in carbon utilization traits across 488 strains of Saccharomyces to investigate whether the structure of metabolic pathways or selection pressure from common environments may have caused carbon utilization traits to be gained and lost together. While most carbon sources were gained and lost independently of each other, we found four clusters that exhibit non-random patterns of gain and loss across strains. Contrary to the network pleiotropy hypothesis, we did not find that these patterns are explained by the structure of metabolic pathways or shared enzymes. Consistent with the hypothesis that common environments shape suites of phenotypes, we found that the environment a strain was isolated from partially predicts the carbon sources it can assimilate. PMID:23326606

  12. METHOD FOR THE PRODUCTION OF HETEROLOGOUS POLYPEPTIDES IN TRANSFORMED YEAST CELLS

    DEFF Research Database (Denmark)

    2000-01-01

    The invention describes industrial fermentation of a $i(Saccharomyces) yeast species for production of a heterologous product encoded by a plasmid or DNA contained in said $i(Saccharomyces) yeast species with method utilizes the substrate more efficiently and without fermentative metabolism...... resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained. The $i(Saccharomyces) yeast species is preferably a Crabtree negative $i(Saccharomyces species) in particular $i(Saccharomyces kluyveri)....

  13. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  14. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

    2013-01-01

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions

  15. Yeast Cells Lacking the CIT1-encoded Mitochondrial Citrate Synthase Are Hypersusceptible to Heat- or Aging-induced Apoptosis

    OpenAIRE

    Lee, Yong Joo; Hoe, Kwang Lae; Maeng, Pil Jae

    2007-01-01

    In Saccharomyces cerevisiae, the initial reaction of the tricarboxylic acid cycle is catalyzed by the mitochondrial citrate synthase Cit1. The function of Cit1 has previously been studied mainly in terms of acetate utilization and metabolon construction. Here, we report the relationship between the function of Cit1 and apoptosis. Yeast cells with cit1 deletion showed a temperature-sensitive growth phenotype, and they displayed a rapid loss in viability associated with typical apoptotic hallma...

  16. Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

    Czech Academy of Sciences Publication Activity Database

    Procházka, Václav; Cifra, Michal; Kulha, Pavel; Ižák, Tibor; Rezek, Bohuslav; Kromka, Alexander

    2017-01-01

    Roč. 395, Feb (2017), s. 214-219 ISSN 0169-4332 R&D Projects: GA ČR(CZ) GBP108/12/G108 Institutional support: RVO:68378271 ; RVO:67985882 Keywords : nanocrystalline diamond * yeast cells * field-effect transistor * transfer characteristics pH sensitivity Subject RIV: BO - Bio physics OBOR OECD: Bio physics Impact factor: 3.387, year: 2016

  17. Forces in yeast flocculation

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

    2015-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  18. Endometritis therapy in sows by intra uterine instillation of yeast cell wall solution

    Directory of Open Access Journals (Sweden)

    Lazarević M.

    2012-01-01

    Full Text Available On the basis of our investigations it was possible to conclude that intrauterine treatment of sows with puerperal uterine infections with sterile YCW (Yeast Cell Wall resulted in significant clinical improvement. The percent of recidivism was the lowest (10% in groups of sows treated with 10 and 20 g of YCW. The degree of bacterial CFU (Colony Forming Units reduction in samples of sows uterine flushings following instillation of YCW (5, 10 and 20 g was wery high and ranged from 1361 to 1444 times, while in sows treated with Lotagen 2% solution (100 mL this parametar was only 32. At the moment of weaning, piglets from sows treated with 10 and 20 g of YCW were heavier when compared to the control and Lotagen group and their DBWG (Daily Body Weight Gain was higher when compared to the Lotagen and control group. Treatment of sows by IU instillation of YCW did not influence the number of piglets in the next breeding cycle.

  19. Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

    Science.gov (United States)

    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.

  20. Use of yeast cell walls and Yucca schidigera extract in layer hens’ diets

    Directory of Open Access Journals (Sweden)

    Yasemin Oznurlu

    2011-04-01

    Full Text Available This research was conducted to determine the impact of diet supplementation with yeast cell walls (YCW and Yucca schidigera extract (YE on performance, egg weight, specific gravity, body weight, and intestinal tissue hist­ology in layer hens. White, 48-week-old, Hy-line hybrid hens (n=320 were divided into four main groups, each comprising eight groups of 10 hens: (1 control, (2 500 mg/kg YCW added, (3 500 mg/kg YE added and (4 250 mg/kg YE plus 2500 mg/kg YCW added. While the egg production and feed intake of the hens was significantly affected, overall feed efficiency, damaged-egg ratio, dirty-egg ratio, egg weight and specific gravity did not differ between the control group and the YCW, YE or YCW+YE groups. Final body weight was higher in the YCW, YE and YCW+YE groups than in the control group. There were differences in the width, muscle layer thickness and height/crypt depth ratio of the duodenal villus and the width of the ileal villus among the four groups. It can be concluded that YCW and YCW+YE supplementation for layer hens are beneficial for egg production.

  1. Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

    Directory of Open Access Journals (Sweden)

    Miklos Halmai

    Full Text Available Proliferating cell nuclear antigen (PCNA plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

  2. The TORC2-Dependent Signaling Network in the Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Roelants, Françoise M; Leskoske, Kristin L; Martinez Marshall, Maria Nieves; Locke, Melissa N; Thorner, Jeremy

    2017-09-05

    To grow, eukaryotic cells must expand by inserting glycerolipids, sphingolipids, sterols, and proteins into their plasma membrane, and maintain the proper levels and bilayer distribution. A fungal cell must coordinate growth with enlargement of its cell wall. In Saccharomyces cerevisiae, a plasma membrane-localized protein kinase complex, Target of Rapamicin (TOR) complex-2 (TORC2) (mammalian ortholog is mTORC2), serves as a sensor and masterregulator of these plasma membrane- and cell wall-associated events by directly phosphorylating and thereby stimulating the activity of two types of effector protein kinases: Ypk1 (mammalian ortholog is SGK1), along with a paralog (Ypk2); and, Pkc1 (mammalian ortholog is PKN2/PRK2). Ypk1 is a central regulator of pathways and processes required for plasma membrane lipid and protein homeostasis, and requires phosphorylation on its T-loop by eisosome-associated protein kinase Pkh1 (mammalian ortholog is PDK1) and a paralog (Pkh2). For cell survival under various stresses, Ypk1 function requires TORC2-mediated phosphorylation at multiple sites near its C terminus. Pkc1 controls diverse processes, especially cell wall synthesis and integrity. Pkc1 is also regulated by Pkh1- and TORC2-dependent phosphorylation, but, in addition, by interaction with Rho1-GTP and lipids phosphatidylserine (PtdSer) and diacylglycerol (DAG). We also describe here what is currently known about the downstream substrates modulated by Ypk1-mediated and Pkc1-mediated phosphorylation.

  3. A predictive model of the oxygen and heme regulatory network in yeast.

    Directory of Open Access Journals (Sweden)

    Anshul Kundaje

    2008-11-01

    Full Text Available Deciphering gene regulatory mechanisms through the analysis of high-throughput expression data is a challenging computational problem. Previous computational studies have used large expression datasets in order to resolve fine patterns of coexpression, producing clusters or modules of potentially coregulated genes. These methods typically examine promoter sequence information, such as DNA motifs or transcription factor occupancy data, in a separate step after clustering. We needed an alternative and more integrative approach to study the oxygen regulatory network in Saccharomyces cerevisiae using a small dataset of perturbation experiments. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes, and only a handful of oxygen regulators have been identified in previous studies. We used a new machine learning algorithm called MEDUSA to uncover detailed information about the oxygen regulatory network using genome-wide expression changes in response to perturbations in the levels of oxygen, heme, Hap1, and Co2+. MEDUSA integrates mRNA expression, promoter sequence, and ChIP-chip occupancy data to learn a model that accurately predicts the differential expression of target genes in held-out data. We used a novel margin-based score to extract significant condition-specific regulators and assemble a global map of the oxygen sensing and regulatory network. This network includes both known oxygen and heme regulators, such as Hap1, Mga2, Hap4, and Upc2, as well as many new candidate regulators. MEDUSA also identified many DNA motifs that are consistent with previous experimentally identified transcription factor binding sites. Because MEDUSA's regulatory program associates regulators to target genes through their promoter sequences, we directly tested the predicted regulators for OLE1, a gene specifically induced under hypoxia, by experimental analysis of the activity of its promoter. In each case, deletion of

  4. Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering.

    Science.gov (United States)

    Nakanishi, Akihito; Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2012-10-23

    To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

  5. Evaluation of the Efficiency of Different Disruption Methods on Yeast Cell Wall Preparation for β-Glucan Isolation

    Directory of Open Access Journals (Sweden)

    Anna Bzducha-Wróbel

    2014-12-01

    Full Text Available 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.

  6. Stochastic simulation for the inference of transcriptional control network of yeast cyclins genes

    Czech Academy of Sciences Publication Activity Database

    Vohradský, Jiří

    2012-01-01

    Roč. 40, č. 15 (2012), s. 7096-7103 ISSN 0305-1048 R&D Projects: GA ČR GAP302/11/0229 Institutional support: RVO:61388971 Keywords : Cell cycle * gene expression * protein complexes Subject RIV: CE - Biochemistry Impact factor: 8.278, year: 2012

  7. Measurement variation determines the gene network topology reconstructed from experimental data: a case study of the yeast cyclin network

    Czech Academy of Sciences Publication Activity Database

    To, C. Ch.; Vohradský, Jiří

    2010-01-01

    Roč. 24, č. 9 (2010), s. 3468-3478 ISSN 0892-6638 R&D Projects: GA ČR GA310/07/1009 Institutional research plan: CEZ:AV0Z50200510 Keywords : numerical modeling * genetic programming * cell-cycle Subject RIV: EE - Microbiology, Virology Impact factor: 6.515, year: 2010

  8. Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification.

    Science.gov (United States)

    Srimhan, Purimprat; Kongnum, Khanitta; Taweerodjanakarn, Siriporn; Hongpattarakere, Tipparat

    2011-03-07

    Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. CellNet: network biology applied to stem cell engineering.

    Science.gov (United States)

    Cahan, Patrick; Li, Hu; Morris, Samantha A; Lummertz da Rocha, Edroaldo; Daley, George Q; Collins, James J

    2014-08-14

    Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. The time-profile of cell growth in fission yeast: model selection criteria favoring bilinear models over exponential ones

    Directory of Open Access Journals (Sweden)

    Sveiczer Akos

    2006-03-01

    Full Text Available Abstract Background There is considerable controversy concerning the exact growth profile of size parameters during the cell cycle. Linear, exponential and bilinear models are commonly considered, and the same model may not apply for all species. Selection of the most adequate model to describe a given data-set requires the use of quantitative model selection criteria, such as the partial (sequential F-test, the Akaike information criterion and the Schwarz Bayesian information criterion, which are suitable for comparing differently parameterized models in terms of the quality and robustness of the fit but have not yet been used in cell growth-profile studies. Results Length increase data from representative individual fission yeast (Schizosaccharomyces pombe cells measured on time-lapse films have been reanalyzed using these model selection criteria. To fit the data, an extended version of a recently introduced linearized biexponential (LinBiExp model was developed, which makes possible a smooth, continuously differentiable transition between two linear segments and, hence, allows fully parametrized bilinear fittings. Despite relatively small differences, essentially all the quantitative selection criteria considered here indicated that the bilinear model was somewhat more adequate than the exponential model for fitting these fission yeast data. Conclusion A general quantitative framework was introduced to judge the adequacy of bilinear versus exponential models in the description of growth time-profiles. For single cell growth, because of the relatively limited data-range, the statistical evidence is not strong enough to favor one model clearly over the other and to settle the bilinear versus exponential dispute. Nevertheless, for the present individual cell growth data for fission yeast, the bilinear model seems more adequate according to all metrics, especially in the case of wee1Δ cells.

  11. Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis.

    Science.gov (United States)

    Gervais, Thomas R; Carta, Giorgio; Gainer, John L

    2003-01-01

    A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents. The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions. The effect of reactant partitioning on the initial rate of the reactions is also investigated. The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads. A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads.

  12. Different cell fates from cell-cell interactions: core architectures of two-cell bistable networks.

    Science.gov (United States)

    Rouault, Hervé; Hakim, Vincent

    2012-02-08

    The acquisition of different fates by cells that are initially in the same state is central to development. Here, we investigate the possible structures of bistable genetic networks that can allow two identical cells to acquire different fates through cell-cell interactions. Cell-autonomous bistable networks have been previously sampled using an evolutionary algorithm. We extend this evolutionary procedure to take into account interactions between cells. We obtain a variety of simple bistable networks that we classify into major subtypes. Some have long been proposed in the context of lateral inhibition through the Notch-Delta pathway, some have been more recently considered and others appear to be new and based on mechanisms not previously considered. The results highlight the role of posttranscriptional interactions and particularly of protein complexation and sequestration, which can replace cooperativity in transcriptional interactions. Some bistable networks are entirely based on posttranscriptional interactions and the simplest of these is found to lead, upon a single parameter change, to oscillations in the two cells with opposite phases. We provide qualitative explanations as well as mathematical analyses of the dynamical behaviors of various created networks. The results should help to identify and understand genetic structures implicated in cell-cell interactions and differentiation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... that the minimum number of genes from each species that need to be compared to produce a reliable phylogeny is about 20. Yeast has also become an attractive model to study speciation in eukaryotes, especially to understand molecular mechanisms behind the establishment of reproductive isolation. Comparison...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

  14. Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

    Directory of Open Access Journals (Sweden)

    Tania Bose

    Full Text Available Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the

  15. Sporothrix schenckii yeasts induce ERK pathway activation and secretion of IL-6 and TNF-α in rat mast cells, but no degranulation.

    Science.gov (United States)

    Romo-Lozano, Yolanda; Hernández-Hernández, Francisca; Salinas, Eva

    2014-11-01

    Sporothrix schenckii is a dimorphic fungus that causes sporotrichosis, a subcutaneous mycosis found throughout the world in humans and other mammals. After contact with conidia, transition to the yeast stage is required for establishment of infection. Mast cells are one of the first components of the immune system to make contact with invading pathogens. They release potent mediators that are decisive in initiating and directing the course of immune and inflammatory responses in the host. It remains unknown whether or not yeast cells of S. schenckii activate mast cells. Our aim in this study was to evaluate the in vitro response of mast cells to S. schenckii yeasts cells. Mast cells became activated after interaction with the yeasts, although exocytosis of preformed mediators was not stimulated. Sporothrix schenckii yeasts induced the release of early response cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 and activation of the extracellular signal-regulated kinase (ERK) signaling pathway in mast cells. As TNF-α and IL-6 are considered crucial mediators in the defense of the host against fungal disease, the release of both mediators from mast cells may contribute to the overall response of the host immune system during S. schenckii infection. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Immunologic proof of DNS irradiation damages and their repair in stationary yeast cells

    International Nuclear Information System (INIS)

    Waller, H.

    1980-08-01

    In rabbits an antiserum was produced by injecting UV-irradiated denaturated calf-thymus DNS; after inhibiting unspecific bindings, a specific serological reaction with UV-induced irradiation damages could be taken as present in this antiserum. By the ammonium sulphate precipitation as immunologic method of detection, after UV-irradiation the genesis of damages at certain sites in the DNS of different yeast lineages and their repair was observed. The elemination of UV-induced DNS damages was observed after an incubation in a nutrien medium, after photo-reactivation and after combining both therapeutic treatments. The following results were obtained: the detected DNS damage (number of induced dimeres/yeast genomes) had the same degree in the four yeast lineages. Apart from the excision-negative mutante 2094 for all yeast lineages a repair efficiency of 60% could be detected. All yeast lineages presented themselves as photographically to be reactivated; however, in all cases a DNS damage of 40 to 50% remained. The examinations for the specificity of antiserum against roentgenologically irradiated DNS led to the conclusion that the antibody population of the serum consisted mainly of immunoglobulines against unchanged DNS areas. A specific immunological reaction of only about 10% could be achieved. (orig./MG) [de

  17. Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

    Science.gov (United States)

    Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

    2015-04-01

    The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

  18. In Vitro Binding Potentials of Bentonite, Yeast Cell Wall and Lactic Acid Bacteria for Aflatoxin B1 and Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Damoon Ghofrani Tabari

    2018-02-01

    Full Text Available Background: This study intended to assess individual and combined adsorption potentials of three adsorbents (processed bentonite as an inorganic adsorbent, and cell walls of Saccharomyces cerevisiae and of the GG strain of Lactobacillus rhamnosus as organic adsorbents for aflatoxin B1 and ochratoxin A under in vitro conditions. Methods: This study was conducted in Ferdowsi University of Mashhad, Mashhad, Iran in 2106. A pre-test with four treatments of bacteria and yeast (live or dead with five replications was designed and conducted to study the effects of these biotic and abiotic organic adsorbents on toxin adsorption efficiency. The experiment in the main study had seven treatments including two toxins (2 ppm and three adsorbents with five replications using the completely randomized design. Toxin quantities were measured by an HPLC instrument. Results: The various types of dead organic adsorbents were more capable of adsorbing toxins compared to the live ones. The processed bentonite was considerably more efficient in adsorbing aflatoxin B1 (93.51 compared to the other treatment groups (P<0.05. No significant differences were observed between the effects of yeast and bacterial cell walls in adsorbing aflatoxin B1. However, the bacterial cell walls (61.71 had higher adsorption efficiencies in adsorbing ochratoxin A than the processed bentonite and yeast cell walls (P<0.05. Conclusion: Processed bentonite (or montmorillonite and bacterial cell walls are able to adsorb considerable quantities of aflatoxin B1and ochratoxin A, and can be used in multiple mycotoxin contaminations as an effective strategy for preventing or reducing the harmful effects of these toxins.

  19. Evolution of transcriptional networks in yeast: alternative teams of transcriptional factors for different species

    Directory of Open Access Journals (Sweden)

    Adriana Muñoz

    2016-11-01

    Full Text Available Abstract Background The diversity in eukaryotic life reflects a diversity in regulatory pathways. Nocedal and Johnson argue that the rewiring of gene regulatory networks is a major force for the diversity of life, that changes in regulation can create new species. Results We have created a method (based on our new “ping-pong algorithm for detecting more complicated rewirings, where several transcription factors can substitute for one or more transcription factors in the regulation of a family of co-regulated genes. An example is illustrative. A rewiring has been reported by Hogues et al. that RAP1 in Saccharomyces cerevisiae substitutes for TBF1/CBF1 in Candida albicans for ribosomal RP genes. There one transcription factor substitutes for another on some collection of genes. Such a substitution is referred to as a “rewiring”. We agree with this finding of rewiring as far as it goes but the situation is more complicated. Many transcription factors can regulate a gene and our algorithm finds that in this example a “team” (or collection of three transcription factors including RAP1 substitutes for TBF1 for 19 genes. The switch occurs for a branch of the phylogenetic tree containing 10 species (including Saccharomyces cerevisiae, while the remaining 13 species (Candida albicans are regulated by TBF1. Conclusions To gain insight into more general evolutionary mechanisms, we have created a mathematical algorithm that finds such general switching events and we prove that it converges. Of course any such computational discovery should be validated in the biological tests. For each branch of the phylogenetic tree and each gene module, our algorithm finds a sub-group of co-regulated genes and a team of transcription factors that substitutes for another team of transcription factors. In most cases the signal will be small but in some cases we find a strong signal of switching. We report our findings for 23 Ascomycota fungi species.

  20. Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

    Directory of Open Access Journals (Sweden)

    Felipe O Bendezú

    Full Text Available The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

  1. Towards systematic discovery of signaling networks in budding yeast filamentous growth stress response using interventional phosphorylation data.

    Science.gov (United States)

    Zhang, Yan; Kweon, Hye Kyong; Shively, Christian; Kumar, Anuj; Andrews, Philip C

    2013-01-01

    Reversible phosphorylation is one of the major mechanisms of signal transduction, and signaling networks are critical regulators of cell growth and development. However, few of these networks have been delineated completely. Towards this end, quantitative phosphoproteomics is emerging as a useful tool enabling large-scale determination of relative phosphorylation levels. However, phosphoproteomics differs from classical proteomics by a more extensive sampling limitation due to the limited number of detectable sites per protein. Here, we propose a comprehensive quantitative analysis pipeline customized for phosphoproteome data from interventional experiments for identifying key proteins in specific pathways, discovering the protein-protein interactions and inferring the signaling network. We also made an effort to partially compensate for the missing value problem, a chronic issue for proteomics studies. The dataset used for this study was generated using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) technique with interventional experiments (kinase-dead mutations). The major components of the pipeline include phosphopeptide meta-analysis, correlation network analysis and causal relationship discovery. We have successfully applied our pipeline to interventional experiments identifying phosphorylation events underlying the transition to a filamentous growth form in Saccharomyces cerevisiae. We identified 5 high-confidence proteins from meta-analysis, and 19 hub proteins from correlation analysis (Pbi2p and Hsp42p were identified by both analyses). All these proteins are involved in stress responses. Nine of them have direct or indirect evidence of involvement in filamentous growth. In addition, we tested four of our predicted proteins, Nth1p, Pbi2p, Pdr12p and Rcn2p, by interventional phenotypic experiments and all of them present differential invasive growth, providing prospective validation of our approach. This comprehensive pipeline presents a

  2. Towards systematic discovery of signaling networks in budding yeast filamentous growth stress response using interventional phosphorylation data.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available Reversible phosphorylation is one of the major mechanisms of signal transduction, and signaling networks are critical regulators of cell growth and development. However, few of these networks have been delineated completely. Towards this end, quantitative phosphoproteomics is emerging as a useful tool enabling large-scale determination of relative phosphorylation levels. However, phosphoproteomics differs from classical proteomics by a more extensive sampling limitation due to the limited number of detectable sites per protein. Here, we propose a comprehensive quantitative analysis pipeline customized for phosphoproteome data from interventional experiments for identifying key proteins in specific pathways, discovering the protein-protein interactions and inferring the signaling network. We also made an effort to partially compensate for the missing value problem, a chronic issue for proteomics studies. The dataset used for this study was generated using SILAC (Stable Isotope Labeling with Amino acids in Cell culture technique with interventional experiments (kinase-dead mutations. The major components of the pipeline include phosphopeptide meta-analysis, correlation network analysis and causal relationship discovery. We have successfully applied our pipeline to interventional experiments identifying phosphorylation events underlying the transition to a filamentous growth form in Saccharomyces cerevisiae. We identified 5 high-confidence proteins from meta-analysis, and 19 hub proteins from correlation analysis (Pbi2p and Hsp42p were identified by both analyses. All these proteins are involved in stress responses. Nine of them have direct or indirect evidence of involvement in filamentous growth. In addition, we tested four of our predicted proteins, Nth1p, Pbi2p, Pdr12p and Rcn2p, by interventional phenotypic experiments and all of them present differential invasive growth, providing prospective validation of our approach. This comprehensive

  3. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    Science.gov (United States)

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

  4. Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950.

    Science.gov (United States)

    Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna

    2015-01-01

    Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g) were found in yeast cells after 24-hour culture conducted in control (YPD) medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours) the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium) of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

  5. Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950

    Directory of Open Access Journals (Sweden)

    Marek Kieliszek

    2015-01-01

    Full Text Available Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g were found in yeast cells after 24-hour culture conducted in control (YPD medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

  6. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  7. Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2.

    Science.gov (United States)

    Shkil, Halyna; Stoica, Leonard; Dmytruk, Kostyantyn; Smutok, Oleh; Gonchar, Mykhailo; Sibirny, Andriy; Schuhmann, Wolfgang

    2009-09-01

    In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.

  8. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, T.; Fukuda, H. [Kobe University, Kobe (Japan). Graduate School of Science and Technology, Division of Molecular Science; Takahashi, S.; Ueda, M.; Tanaka, A. [Kyoto University, Kyoto (Japan). Dept. of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering; Kaieda, M.; Kondo, A. [Kobe University, Kobe (Japan). Faculty of Engineering, Dept. of Chemical Science and Engineering

    2001-07-01

    Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rPRoROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization. (orig.)

  9. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

    Full Text Available The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L and silver nitrate (30 mg/L for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

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

    Czech Academy of Sciences Publication Activity Database

    Borovikova, D.; Herynková, Pavla; Rapoport, A.; Sychrová, Hana

    2014-01-01

    Roč. 350, č. 1 (2014), s. 28-33 ISSN 0378-1097 R&D Projects: GA MŠk(CZ) LD13037; GA ČR(CZ) GAP503/10/0307 Institutional support: RVO:67985823 Keywords : desiccation * yeast * potassium uptake * stress survival Subject RIV: EE - Microbiology, Virology Impact factor: 2.121, year: 2014

  11. Yeast Cells-Derived Hollow Core/Shell Heteroatom-Doped Carbon Microparticles for Sustainable Electrocatalysis

    Czech Academy of Sciences Publication Activity Database

    Huang, X.; Zou, X.; Meng, Y.; Mikmeková, Eliška; Chen, H.; Voiry, D.; Goswami, A.; Chhowalla, M.; Asefa, T.

    2015-01-01

    Roč. 7, č. 3 (2015), s. 1978-1986 ISSN 1944-8244 R&D Projects: GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : yeast * heteroatom-doped carbon * oxygen reduction * ORR * hydrazine electrooxidation Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 7.145, year: 2015

  12. A comparison of the radiosensitivity of stationary, exponential and G1 phase wild type and repair deficient yeast cultures: supporting evidence for stationary phase yeast cells being in G0

    International Nuclear Information System (INIS)

    Tippins, R.S.; Parry, J.M.

    1982-01-01

    The main points to emerge from this comparison of the radiosensitivity of stationary, exponential and G 1 phase yeast cultures were: (1) In wild type yeast cultures, G 1 cells were the most sensitive to the lethal effects of X-rays, exponential phase cells were the most resistant and stationary phase cells were intermediate in sensitivity. (2) With the excision-repair-defective strain D61-3 (rad 3) stationary phase cells were more resistant than exponential cells with G 1 cells again being most sensitive. (3) The rad 50 gene present in JD50 had a marked effect on the X-ray inactivation response of this strain. In the presence of the defective rad 50 allele, exponential phase cells were as sensitive as G 1 phase cells, with stationary phase cells being more resistant than either. (4) There were marked differences in sensitivity between stationary phase and G 1 phase cells. These differences, along with other physiological differences reported by other workers, lead the authors to suggest that stationary phase cells can be better described as being in G 0 phase, i.e. a stage which is outside the normal mitotic cell cycle of an exponential culture. (author)

  13. Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption.

    Science.gov (United States)

    Müller, F M; Werner, K E; Kasai, M; Francesconi, A; Chanock, S J; Walsh, T J

    1998-06-01

    Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (10(8) and 10(7) CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10(8) CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P extraction and PC extraction. For 10(7) CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P fungi than for yeasts by the HSCD extraction procedure (P extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

  14. Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability.

    Science.gov (United States)

    Wang, Jin-jing; Xu, Wei-na; Li, Xin'er; Li, Jia; Li, Qi

    2014-06-01

    The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.

  15. Evaluation of the protection induced by the immunization with radioiodinated yeast cells of Paracoccidioides brasiliensis in animal model

    International Nuclear Information System (INIS)

    Martins, Estefania Mara do Nascimento

    2007-01-01

    Paracoccidioides brasiliensis is fungus agent of paracoccidioidomycosis (PCM), a chronic systemic disease prevalent in Latin American. To date, there is no effective vaccine. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory were developed yeast cells of P. brasiliensis attenuated by gamma radiation, which lose the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins, the oxidative metabolism and the expression of the antigens present in the native yeast. The aim of the present work was to evaluate the protection elicited by the immunization with this cells in animal model. The virulence attenuated was evaluated in BALB/c and Nude-Nude mice. The protector effect was evaluated in BALB/c mice groups immunized once or twice. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. The mice were sacrificed 30 and 90 days after challenge. The removed organs were used for colony forming units (CFU's) recover, histopathological analysis and cytokine determination. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. To evaluate the type of elicited immune response the cytokines IFN - γ, TNF - α, IL - 10 and IL - 5 were determined by real time PCR. The radio attenuated yeast loses its virulence since fails in producing infection in BALB/c and Nude-Nude mice. No CFU's were recovered neither histological changes observed in the mice infected with the radio attenuated cells. The mice infected with the not irradiated P. brasiliensis showed a high level of antibody production while the infection with the radio attenuated yeast did not significantly change the antibody level. The mice infected with the radio attenuated yeast presented an increase in the IFN - γ and TNF - α production and an inhibition of the IL-10 synthesis, indicating a

  16. A vibrating membrane bioreactor operated at supra- and sub-critical flux: Influence of extracellular polymeric substances from yeast cells

    DEFF Research Database (Denmark)

    Beier, Søren Prip; Jonsson, Gunnar Eigil

    2007-01-01

    A vibrating membrane bioreactor, in which the fouling problems are reduced by vibrating a hollow fiber membrane module, has been tested in constant flux microfiltration above (supra-critical) and below (sub-critical) an experimentally determined critical flux. Suspensions of bakers yeast cells were...... is continually washed out during supra-critical flux operation whereas the washing out at sub-critical flux operation is not observed. This might be due to locally different hydrodynamic conditions at the membrane surface and pore entrances at supra- and sub-critical flux respectively....

  17. Adhesion of yeast cells to different porous supports, stability of cell-carrier systems and formation of volatile by-products

    OpenAIRE

    Kregiel, Dorota; Berlowska, Joanna; Ambroziak, Wojciech

    2012-01-01

    The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular α-amylase and glucoamylase, were used in this study. Diffe...

  18. The Reticular Cell Network : A Robust Backbone for Immune Responses

    NARCIS (Netherlands)

    Textor, Johannes; Mandl, Judith N; de Boer, Rob J

    2016-01-01

    Lymph nodes are meeting points for circulating immune cells. A network of reticular cells that ensheathe a mesh of collagen fibers crisscrosses the tissue in each lymph node. This reticular cell network distributes key molecules and provides a structure for immune cells to move around on. During

  19. Effects of yeast extract and methyl jasmonate on the enhancement of solasodine biosynthesis in cell cultures of Solanum hainanense Hance

    Directory of Open Access Journals (Sweden)

    NGUYEN HOANG LOC

    2014-04-01

    Full Text Available In this work, the effects of the elicitors methyl jasmonate (MeJA and yeast extract (YE on the growth and solasodine production of Solanum hainanense cells were investigated. The results showed that various concentrations of MeJA (50-250 µM and YE (1-4 g/L have different eliciting influences. The increase of solasodine content induced by the elicitation of 3 g/L of YE and 50 µM of MeJA at the beginning of cell culture was about 1.9- and 1.3-fold, respectively, as compared with that of the non-elicitated cells. In general, YE (biotic elicitor was more effective in enhancing solasodine production than MeJA (abiotic elicitor.

  20. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  1. Use of Saccharomyces cerevisiae expressing beta-galactosidase to screen for antimycotic agents directed against yeast cell wall biosynthesis and possible application to pathogenic fungi.

    OpenAIRE

    Zaworski, P G; Gill, G S

    1990-01-01

    Saccharomyces cerevisiae expressing beta-galactosidase was used to develop a screen for compounds active against formation of the yeast cell wall. The screen detailed here is based on the release of beta-galactosidase from cells which had received an osmotic shock.

  2. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan); Ohta, Miho [Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033 (Japan); Sakaki, Toshiyuki, E-mail: tsakaki@pu-toyama.ac.jp [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)

    2013-05-03

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.

  3. A new approach on Brewer's spent grains treatment and potential use as lignocellulosic yeast cells carriers.

    Science.gov (United States)

    Pires, Eduardo J; Ruiz, Héctor A; Teixeira, José A; Vicente, António A

    2012-06-13

    The major objective of this work is to improve the pretreatments of brewer's spent grains (BSG) aiming at their use as a source for lignocellulosic yeast carriers (LCYC) production. Therefore, several pretreatments of BSG have been designed aiming at obtaining various yeast carriers, differing on their physicochemical composition. Cellulose, hemicellulose, lignin, fat, protein, and ash content were determined for crude BSG and the LCYCs. The long chain fatty acids profile for the crude BSG was also analyzed. Chemical treatments successfully produced several different LCYC based on BSG. The highest cellulose content in LCYC was achieved upon application of caustic (NaOH) treatment during 40 min. Either caustic or combined acid-caustic treatments predominately generated hydrophobic, negatively charged LCYC. The feasibility of using BSG for LCYC production is strengthened by the fact that added-value byproduct can be extracted before the chemical treatments are applied.

  4. Continuous production of pectinase by immobilized yeast cells on spent grains

    OpenAIRE

    Almeida, Catarina; Brányik, Tomáš; Ferreira, Pedro Moradas; Teixeira, J. A.

    2003-01-01

    A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors’ productivity, a cellulosic carrier obtained from barley spent grain...

  5. Glucose-induced MDR pump resynthesis in respiring yeast cells depends on nutrient level

    Czech Academy of Sciences Publication Activity Database

    Maláč, J.; Sigler, Karel; Gášková, D.

    -, č. 337 (2005), s. 138-141 ISSN 0006-291X R&D Projects: GA ČR GD204/03/H066; GA ČR GP202/04/P110 Grant - others:GA FRVŠ FRVS 555/2005/G3 Institutional research plan: CEZ:AV0Z50200510 Keywords : MDR pump s * yeast * fluorimetric assay Subject RIV: EE - Microbiology, Virology Impact factor: 3.000, year: 2005

  6. Synthetic Genetic Arrays: Automation of Yeast Genetics.

    Science.gov (United States)

    Kuzmin, Elena; Costanzo, Michael; Andrews, Brenda; Boone, Charles

    2016-04-01

    Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype. © 2016 Cold Spring Harbor Laboratory Press.

  7. Aging Yeast Cells Undergo a Sharp Entry into Senescence Unrelated to the Loss of Mitochondrial Membrane Potential

    Directory of Open Access Journals (Sweden)

    Steffen Fehrmann

    2013-12-01

    Full Text Available In budding yeast, a mother cell can produce a finite number of daughter cells before it stops dividing and dies. Such entry into senescence is thought to result from a progressive decline in physiological function, including a loss of mitochondrial membrane potential (ΔΨ. Here, we developed a microfluidic device to monitor the dynamics of cell division and ΔΨ in real time at single-cell resolution. We show that cells do not enter senescence gradually but rather undergo an abrupt transition to a slowly dividing state. Moreover, we demonstrate that the decline in ΔΨ, which is observed only in a fraction of cells, is not responsible for entry into senescence. Rather, the loss of ΔΨ is an age-independent and heritable process that leads to clonal senescence and is therefore incompatible with daughter cell rejuvenation. These results emphasize the importance of quantitative single-cell measurements to decipher the causes of cellular aging.

  8. Interference Management with Successive Cancellation for Dense Small Cell Networks

    DEFF Research Database (Denmark)

    Lopez, Victor Fernandez; Pedersen, Klaus I.; Steiner, Jens

    2016-01-01

    Network-Assisted Interference Cancellation and Suppression (NAICS) receivers have appeared as a promising way to curb inter-cell interference in future dense network deployments. This investigation compares the performance of a NAICS receiver with successive interference cancellation capabilities...

  9. Overexpression of O-methyltransferase leads to improved vanillin production in baker's yeast only when complemented with model-guided network engineering.

    Science.gov (United States)

    Brochado, Ana Rita; Patil, Kiran R

    2013-02-01

    Overproduction of a desired metabolite is often achieved via manipulation of the pathway directly leading to the product or through engineering of distant nodes within the metabolic network. Empirical examples illustrating the combined effect of these local and global strategies have been so far limited in eukaryotic systems. In this study, we compared the effects of overexpressing a key gene in de novo vanillin biosynthesis (coding for O-methyltransferase, hsOMT) in two yeast strains, with and without model-guided global network modifications. Overexpression of hsOMT resulted in increased vanillin production only in the strain with model-guided modifications, exemplifying advantage of using a global strategy prior to local pathway manipulation. Copyright © 2012 Wiley Periodicals, Inc.

  10. Simulation of developing human neuronal cell networks.

    Science.gov (United States)

    Lenk, Kerstin; Priwitzer, Barbara; Ylä-Outinen, Laura; Tietz, Lukas H B; Narkilahti, Susanna; Hyttinen, Jari A K

    2016-08-30

    Microelectrode array (MEA) is a widely used technique to study for example the functional properties of neuronal networks derived from human embryonic stem cells (hESC-NN). With hESC-NN, we can investigate the earliest developmental stages of neuronal network formation in the human brain. In this paper, we propose an in silico model of maturating hESC-NNs based on a phenomenological model called INEX. We focus on simulations of the development of bursts in hESC-NNs, which are the main feature of neuronal activation patterns. The model was developed with data from developing hESC-NN recordings on MEAs which showed increase in the neuronal activity during the investigated six measurement time points in the experimental and simulated data. Our simulations suggest that the maturation process of hESC-NN, resulting in the formation of bursts, can be explained by the development of synapses. Moreover, spike and burst rate both decreased at the last measurement time point suggesting a pruning of synapses as the weak ones are removed. To conclude, our model reflects the assumption that the interaction between excitatory and inhibitory neurons during the maturation of a neuronal network and the spontaneous emergence of bursts are due to increased connectivity caused by the forming of new synapses.

  11. Influence of N-glycans on Expression of Cell Wall Remodeling Related Genes in Paracoccidioides brasiliensis Yeast Cells

    Science.gov (United States)

    Almeida, Fausto; Antoniêto, Amanda Cristina Campos; Pessoni, André Moreira; Monteiro, Valdirene Neves; Alegre-Maller, Ana Claudia Paiva; Pigosso, Laurine Lacerda; Pereira, Maristela; Soares, Célia Maria de Almeida; Roque-Barreira, Maria Cristina

    2016-01-01

    Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as β-1,3-glucanosyltransferase (PbGEL3), 1,3-β-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-β-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and β-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM. PMID:27226767

  12. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...

  13. Cell outage compensation in LTE networks: Algorithms and performance assessment

    NARCIS (Netherlands)

    Amirijoo, M.; Jorguseski, L.; Litjens, R.; Schmelz, L.C.

    2011-01-01

    Cell outage compensation is a self-healing function and as such part of the Self-Organising Networks concept for mobile wireless networks. It aims at mitigating the degradation of coverage, capacity and service quality caused by a cell or site level outage. Upon detection of such an outage, cell

  14. VDAC regulates AAC-mediated apoptosis and cytochromecrelease in yeast.

    Science.gov (United States)

    Trindade, Dário; Pereira, Clara; Chaves, Susana R; Manon, Stéphen; Côrte-Real, Manuela; Sousa, Maria J

    2016-08-25

    Mitochondrial outer membrane permeabilization is a key event in apoptosis processes leading to the release of lethal factors. We have previously shown that absence of the ADP/ATP carrier (AAC) proteins (yeast orthologues of mammalian ANT proteins) increased the resistance of yeast cells to acetic acid, preventing MOMP and the release of cytochrome c from mitochondria during acetic acid - induced apoptosis. On the other hand, deletion of POR1 (yeast voltage-dependent anion channel - VDAC) increased the sensitivity of yeast cells to acetic acid. In the present work, we aimed to further characterize the role of yeast VDAC in acetic acid - induced apoptosis and assess if it functionally interacts with AAC proteins. We found that the sensitivity to acetic acid resulting from POR1 deletion is completely abrogated by the absence of AAC proteins, and propose that Por1p acts as a negative regulator of acetic acid - induced cell death by a mechanism dependent of AAC proteins, by acting on AAC - dependent cytochrome c release. Moreover, we show that Por1p has a role in mitochondrial fusion that, contrary to its role in apoptosis, is not affected by the absence of AAC, and demonstrate that mitochondrial network fragmentation is not sufficient to induce release of cytochrome c or sensitivity to acetic acid - induced apoptosis. This work enhances our understanding on cytochrome c release during cell death, which may be relevant in pathological scenarios where MOMP is compromised.

  15. Downsides and benefits of unicellularity in budding yeast

    Science.gov (United States)

    Balazsi, Gabor; Chen, Lin; Kuzdzal-Fick, Jennie

    Yeast cells that do not separate after cell division form clumps. Clumping was shown to aid utilization of certain sugars, but its effects in stressful conditions are unknown. Generally speaking, what are the costs and benefits of unicellularity versus clumping multicellularity in normal and stressful conditions? To address this question, we evolved clumping yeast towards unicellularity by continuously propagating only those cells that remain suspended in liquid culture after settling. Whole-genome sequencing indicated that mutations in the AMN1 (antagonist of mitotic exit network) gene underlie the changes from clumping to unicellular phenotypes in these evolved yeast cells. Simple models predict that clumping should hinder growth in normal conditions while being protective in stress. Accordingly, we find experimentally that yeast clumps are more resistant to freeze/thaw, hydrogen peroxide, and ethanol stressors than their unicellular counterparts. On the other hand, unicellularity seems to be advantageous in normal conditions. Overall, these results reveal the downsides and benefits of unicellularity in different environmental conditions and uncover its genetic bases in yeast. This research was supported by the NIH Director's New Innovator Award Program (1DP2 OD006481-01), by NSF/IOS 1021675 and the Laufer Center for Physical & Quantitative Biology.

  16. Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

    Science.gov (United States)

    Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

    2013-01-01

    Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593

  17. Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

    Science.gov (United States)

    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 (Pkefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.

  18. Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Kazuyoshi, E-mail: kazum@nips.ac.jp [National Institute for Physiological Sciences, Okazaki, Aichi 444-8585 (Japan); Esaki, Masatoshi; Ogura, Teru [Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811 (Japan); Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo [Ecotopia Science Institute, Nagoya University, Nagoya, Aichi 464-8603 (Japan)

    2014-11-15

    Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ∼3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology. - Highlights: • High voltage TEM and STEM tomography were compared to visualize whole yeast cells. • 1-MeV STEM-BF tomography had significant improvements in image contrast and SNR. • 1-MeV STEM tomography showed less specimen shrinkage than the TEM tomography. • KMnO{sub 4} post-treatment permitted segmenting the major cellular components.

  19. Effects of N-acetyl-L-cysteine on gene expression of antioxidant enzymes in yeast cells after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu; Park, Ji Young; Ryu, Tae Ho; Roh, Chang Hyun [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2012-04-15

    Ionizing radiation induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage. When exposed to ionizing radiation, cells activates ROS scavenging detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. SOD scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast has two catalase and three GPx proteins. The biochemical function of GPx is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. N-acetylL-cysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity and alkaline phosphatase. In this study, the role of NAC as an antioxidant and a radioprotector was examined on cell survival, transcriptional level, and protein level. through observing viability of cells, analyzing the gene expression of antioxidant enzyme, measuring the SOD activity and intracellular GSH levels in yeast W303-1A strain The cell viability of haploid S. cerevisiae W303-1A strain was reduced significantly at the low dose (10∼30 Gy). The half-lethal dose of the strain was about 20 Gy. The CFU assay result confirmed that NAC could not rescue the cells from radiation-induced death. When irradiated with 100 Gy, an increase in the transcriptional expression was observed in the antioxicant genes. The expression of these genes decreased by treatment of NAC in irradiated cells. NAC decline SOD activity and intracellular GSH levels. The present study shows that NAC can directly scavenge

  20. Aerobic decolorization and degradation of azo dyes by suspended growing cells and immobilized cells of a newly isolated yeast Magnusiomyces ingens LH-F1.

    Science.gov (United States)

    Tan, Liang; Li, Hua; Ning, Shuxiang; Xu, Bingwen

    2014-04-01

    Aerobic decolorization and degradation of azo dyes by both of suspended growing cells and immobilized cells of a newly isolated yeast strain LH-F1 were investigated in this study. A yeast strain LH-F1 capable of aerobically decolorizing various azo dyes (20mg/L) was identified as Magnusiomyces ingens basing on 26S rDNA analysis. Meanwhile, effects of different parameters on decolorization of Acid Red B by both of suspended growing cells and immobilized cells of strain LH-F1 were investigated. Furthermore, possible degradation pathway of the dye was proposed through analyzing metabolic intermediates using UV-Vis and HPLC-MS methods. As far as it is known, it is the first systematic research on a M. ingens strain which is capable of efficiently decolorizing azo dyes under aerobic condition. Additionally, this work would also provide a potentially useful microbial strain LH-F1 for treatment of industrial wastewaters containing azo dyes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A rapid method for the differentiation of yeast cells grown under carbon and nitrogen-limited conditions by means of partial least squares discriminant analysis employing infrared micro-spectroscopic data of entire yeast cells

    Science.gov (United States)

    Kuligowski, Julia; Quintás, Guillermo; Herwig, Christoph; Lendl, Bernhard

    2012-01-01

    This paper shows the ease of application and usefulness of mid-IR measurements for the investigation of orthogonal cell states on the example of the analysis of Pichia pastoris cells. A rapid method for the discrimination of entire yeast cells grown under carbon and nitrogen-limited conditions based on the direct acquisition of mid-IR spectra and partial least squares discriminant analysis (PLS-DA) is described. The obtained PLS-DA model was extensively validated employing two different validation strategies: (i) statistical validation employing a method based on permutation testing and (ii) external validation splitting the available data into two independent sub-sets. The Variable Importance in Projection scores of the PLS-DA model provided deeper insight into the differences between the two investigated states. Hence, we demonstrate the feasibility of a method which uses IR spectra from intact cells that may be employed in a second step as an in-line tool in process development and process control along Quality by Design principles. PMID:22967595

  2. Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid

    Science.gov (United States)

    Silva, Alexandra; Sampaio-Marques, Belém; Fernandes, Ângela; Carreto, Laura; Rodrigues, Fernando; Holcik, Martin; Santos, Manuel A. S.; Ludovico, Paula

    2013-01-01

    Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress. PMID:23967187

  3. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sofie eVan Rossom

    2012-07-01

    Full Text Available DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine-nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2 to 6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human HEK293T cells. Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  4. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

    DEFF Research Database (Denmark)

    Knudsen, Jenny Dahl; Hägglöf, Cecilia; Weber, Nora

    2016-01-01

    -expressing SADH. CONCLUSIONS: Overall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i......, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over...

  5. Production of N2O in soil during decomposition of dead yeast cells with different spatial distributions

    DEFF Research Database (Denmark)

    Ambus, P.

    1996-01-01

    Production and sources of N2O were determined in soil columns amended with autoclaved yeast cells either mixed into or added as 0.5 cm(3) lumps to the soil in combination with no or 200 mu g NO3--Ng(-1). At four occasions over a two-week study period, subsets of cores were measured for N2O...... production during 4-hour incubations under atmospheres of ambient air, 10 Pa of C2H2, and N-2, respectively. Denitrification enzyme activity (DEA) was assessed in subsamples of cores that had been incubated continuously under air. Autoclaved yeast provided a C-source readily available for denitrifying...

  6. Transcriptional, proteomic, and metabolic responses to lithium in galactose-grown yeast cells

    DEFF Research Database (Denmark)

    Bro, Christoffer; Regenberg, Birgitte; Lagniel, G.

    2003-01-01

    , and nucleotide metabolism were down-regulated at the transcriptional level, whereas genes responsive to different stresses as well as genes from energy reserve metabolism and monosaccharide metabolism were up-regulated. Compared with the proteomic data, 26% of the down-regulated and 48% of the up......Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition...... of lithium. These results were related to physiological studies where both secreted and intracellular metabolites were determined. Microarray analysis showed that 664 open reading frames were down-regulated and 725 up-regulated in response to addition of lithium. Genes involved in transcription, translation...

  7. The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae.

    Science.gov (United States)

    Pereira, Luciana Filgueira; Bassi, Ana Paula Guarnieri; Avansini, Simoni Helena; Neto, Adauto Gomes Barbosa; Brasileiro, Bereneuza Tavares Ramos Valente; Ceccato-Antonini, Sandra Regina; de Morais, Marcos Antonio

    2012-03-01

    The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.

  8. Overexpression of O‐methyltransferase leads to improved vanillin production in baker's yeast only when complemented with model‐guided network engineering

    DEFF Research Database (Denmark)

    Brochado, Ana Rita; Patil, Kiran R.

    2013-01-01

    Overproduction of a desired metabolite is often achieved via manipulation of the pathway directly leading to the product or through engineering of distant nodes within the metabolic network. Empirical examples illustrating the combined effect of these local and global strategies have been so far ...... vanillin production only in the strain with model‐guided modifications, exemplifying advantage of using a global strategy prior to local pathway manipulation. Biotechnol. Bioeng. 2013; 110: 656–659. © 2012 Wiley Periodicals, Inc.......Overproduction of a desired metabolite is often achieved via manipulation of the pathway directly leading to the product or through engineering of distant nodes within the metabolic network. Empirical examples illustrating the combined effect of these local and global strategies have been so far...... limited in eukaryotic systems. In this study, we compared the effects of overexpressing a key gene in de novo vanillin biosynthesis (coding for O‐methyltransferase, hsOMT) in two yeast strains, with and without model‐guided global network modifications. Overexpression of hsOMT resulted in increased...

  9. Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

    Science.gov (United States)

    Kistowska, Magdalena; Fenini, Gabriele; Jankovic, Dragana; Feldmeyer, Laurence; Kerl, Katrin; Bosshard, Philipp; Contassot, Emmanuel; French, Lars E

    2014-12-01

    Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1β secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1β when exposed to Malassezia spp. Moreover, we demonstrate that IL-1β secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast Papiliotrema laurentii AM113 for Biodiesel Making.

    Science.gov (United States)

    Wang, Guangyuan; Liu, Lin; Liang, Wenxing

    2018-01-01

    Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C 14:0 (0.9%), C 16:0 (10.8%), C 16:1 (9.7%), C 18:0 (6.5%), C 18:1 (60.3%), and C 18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.

  11. Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

    Directory of Open Access Journals (Sweden)

    Hinnebusch Alan G

    2011-01-01

    Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

  12. Effects of the strain background and autolysis process on the composition and biophysical properties of the cell wall from two different industrial yeasts.

    Science.gov (United States)

    Schiavone, Marion; Sieczkowski, Nathalie; Castex, Mathieu; Dague, Etienne; Marie François, Jean

    2015-03-01

    The Saccharomyces cerevisiae cell surface is endowed with some relevant technological properties, notably antimicrobial and biosorption activities. For these purposes, yeasts are usually processed and packaged in an 'autolysed/dried' formula, which may have some impacts on cell surface properties. In this report, we showed using a combination of biochemical, biophysical and molecular methods that the composition of the cell wall of two wine yeast strains was not altered by the autolysis process. In contrast, this process altered the nanomechanical properties as shown by a 2- to 4-fold increased surface roughness and to a higher adhesion to the atomic force microscope tips of the autolysed cells as compared to live yeast cells. Besides, we found that the two strains harboured differences in biomechanical properties that could be due in part to higher levels of mannan in one of them, and to the fact that the surface of this mannan-enriched strain is decorated with highly adhesive patches forming nanodomains. The presence of these nanodomains could be correlated with the upregulation of flocculin encoding FLO11 as well as to higher expression of few other genes encoding cell wall mannoproteins in this mannan-enriched strain as compared to the other strain. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  13. Evaluation of Relative Yeast Cell Surface Hydrophobicity Measured by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Lisa Colling

    2005-01-01

    Full Text Available Objective: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties.

  14. Continuous production of pectinase by immobilized yeast cells on spent grains.

    Science.gov (United States)

    Almeida, Catarina; Brányik, Tomás; Moradas-Ferreira, Pedro; Teixeira, José

    2003-01-01

    A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors' productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source--a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (P(V)=0.98 U ml(-1) h(-1)) was achieved in the PBR for D=0.40 h(-1), a glucose concentration on the inlet of S(in)=20 g l(-1), and a biomass load in the support of X(i)=0.225 g g(-1). The results demonstrate the attractiveness of the packed bed system for pectinase production.

  15. Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

    NARCIS (Netherlands)

    Amantonico, Andrea; Oh, Joo Yeon; Sobek, Jens; Heinemann, Matthias; Zenobi, Renato

    2008-01-01

    Getting a look-in: An optimized MALDI-MS procedure has been developed to detect endogenous primary metabolites directly in the cell extract. A detection limit corresponding to metabolites from less than a single cell has been attained, opening the door to single-cell metabolomics by mass

  16. A CDK-independent metabolic oscillator orchestrates the budding yeast cell cycle

    NARCIS (Netherlands)

    Papagiannakis, A.; Niebel, B.; Wit, E.; Heinemann, M.

    2017-01-01

    Eukaryotic cell division is known to be controlled by the cyclin/ CDK machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell cycle

  17. Interaction Between Yeasts and Zinc

    Science.gov (United States)

    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

  18. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    Science.gov (United States)

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  19. Complementation of a yeast cell cycle mutant by an alfalfa cDNA encoding a protein kinase homologous to p34cdc2.

    Science.gov (United States)

    Hirt, H; Páy, A; Györgyey, J; Bakó, L; Németh, K; Bögre, L; Schweyen, R J; Heberle-Bors, E; Dudits, D

    1991-03-01

    The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.

  20. Immunization with Paracoccidioides brasiliensis radioattenuated yeast cells induces Th1 immune response in Balb/C mice

    Energy Technology Data Exchange (ETDEWEB)

    Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)], e-mail: estefaniabio@yahoo.com.br, e-mail: antero@cdtn.br; Resende, Maria Aparecida de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: maresend@mono.icb.ufmg.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia], e-mail: goes@mono.icb.ufmg.br, e-mail: brsgarbi@mono.icb.ufmg.br

    2009-07-01

    Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. To date, there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated by gamma irradiation. We defined an absorbed dose in which the pathogen loses the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins and the oxidative metabolism. The immunization with these cells was able to confer protection in BALB/c mice. The aim of the present work was evaluate the immune response pathway activated in mice immunized with P. brasiliensis radioattenuated yeast cells. The protector effect was evaluated in BALB/c mice groups immunized once or twice, respectively. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. These groups were called G1A, G1B and G1C in the group immunized once and G2A, G2B and G2C in the group immunized twice. Recovery of CFUs and cytokines determination (IFN - {gamma}, IL - 10 and IL IV 4) were performed three months post challenge. Quantitative RT-PCR was the method of choice used to quantify the expression of cytokines. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. A significant reduction in CFUs recovery was verified 90 days post challenge in mice submitted to one immunization: 73.0%, 96.0% and 76.3% for sub-groups G1A, G1B and G1C, respectively. In the group submitted to two immunizations, a remarkable increase in the protection was obtained. No CFUs was recovered from sub-groups G2B and G2C and very few CFUs (reduction of 98.6%) were recovered from the lungs of sub group G2A. In mice submitted to one immunization, Th1 and Th2 cytokines were simultaneously produced. In the group submitted to two immunizations, levels of IL-10 and IL-4 were very low, while IFN-{gamma} production was maintained indicating that a Th1 pattern was

  1. Immunization with Paracoccidioides brasiliensis radioattenuated yeast cells induces Th1 immune response in Balb/C mice

    International Nuclear Information System (INIS)

    Martins, Estefania M.N.; Andrade, Antero S.R.; Resende, Maria Aparecida de; Reis, Bernardo S.; Goes, Alfredo M.

    2009-01-01

    Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. To date, there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated by gamma irradiation. We defined an absorbed dose in which the pathogen loses the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins and the oxidative metabolism. The immunization with these cells was able to confer protection in BALB/c mice. The aim of the present work was evaluate the immune response pathway activated in mice immunized with P. brasiliensis radioattenuated yeast cells. The protector effect was evaluated in BALB/c mice groups immunized once or twice, respectively. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. These groups were called G1A, G1B and G1C in the group immunized once and G2A, G2B and G2C in the group immunized twice. Recovery of CFUs and cytokines determination (IFN - γ, IL - 10 and IL IV 4) were performed three months post challenge. Quantitative RT-PCR was the method of choice used to quantify the expression of cytokines. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. A significant reduction in CFUs recovery was verified 90 days post challenge in mice submitted to one immunization: 73.0%, 96.0% and 76.3% for sub-groups G1A, G1B and G1C, respectively. In the group submitted to two immunizations, a remarkable increase in the protection was obtained. No CFUs was recovered from sub-groups G2B and G2C and very few CFUs (reduction of 98.6%) were recovered from the lungs of sub group G2A. In mice submitted to one immunization, Th1 and Th2 cytokines were simultaneously produced. In the group submitted to two immunizations, levels of IL-10 and IL-4 were very low, while IFN-γ production was maintained indicating that a Th1 pattern was dominant. For

  2. CHANGES IN LIPID CONTENT OF WINE YEASTS DURING FERMENTATION BY IMMOBILIZED CELLS

    Directory of Open Access Journals (Sweden)

    Fedor Malik

    2010-05-01

    Full Text Available Comparison of the lipid composition of immobilised and non-immobilised cells of the wine cell strain Saccharomyces cerevisiae 6C subjected to ethanol stress indicates that the whole impact of the ethanol stress on the fatty acids composition is less influenced with immobilised cells as with non- immobilised ones. The ethanol stress raised in immobilised and free cells occurrence of palmitoleic acid to the detriment of palmitic acid. The character of changes in lipid composition during immobilisation probably has an impact upon slightly increased stress resistance. The immobilised cells are as well resistive against passive membrane fluidisation by ethanol. doi:10.5219/56

  3. The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: potential and limitations.

    Science.gov (United States)

    Cottier, Stéphanie; Mönig, Timon; Wang, Zheming; Svoboda, Jiří; Boland, Wilhelm; Kaiser, Markus; Kombrink, Erich

    2011-01-01

    Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

  4. The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

    Directory of Open Access Journals (Sweden)

    Stéphanie eCottier

    2011-12-01

    Full Text Available Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx. In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA binding domain (encoded in the yeast strain, and the bioactive molecule part binding to its potential protein target fused to a DNA activating domain (encoded on a cDNA expression vector. During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discussed the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

  5. Current Techniques for Studying Oligomer Formations of G-Protein-Coupled Receptors Using Mammalian and Yeast Cells.

    Science.gov (United States)

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2016-05-27

    G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques.

  6. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    Science.gov (United States)

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  7. Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

    Science.gov (United States)

    Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

    2017-08-01

    In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

  8. The structure of cell wall alpha-glucan from fission yeast

    NARCIS (Netherlands)

    Grün, Christian H.; Hochstenbach, Frans; Humbel, Bruno M.; Verkleij, Arie J.; Sietsma, J. Hans; Klis, Frans M.; Kamerling, Johannis P.; Vliegenthart, Johannes F. G.

    2005-01-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the

  9. The structure of cell wall alpha-glucan from fission yeast.

    NARCIS (Netherlands)

    Grün, C.H.; Hochstenbach, F.; Humbel, B.M.; Verkleij, A.J.; Sietsma, J.H.; Klis, F.M.; Kamerling, J.P.; Vliegenthart, J.F.G.

    2005-01-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1rarr3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the

  10. To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

    Science.gov (United States)

    Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

    2014-01-01

    Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

  11. Lager Yeast Comes of Age

    Science.gov (United States)

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  12. A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens.

    Science.gov (United States)

    Arnold, S F; Robinson, M K; Notides, A C; Guillette, L J; McLachlan, J A

    1996-01-01

    Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol. To test this hypothesis, the yeast estrogen screen (YES) was created by expressing human estrogen receptor (hER) and two estrogen response elements (ERE) linked to the lacZ gene. The beta-galactosidase activity of the YES system was significantly increased after treatment with 17 beta-estradiol or the xenoestrogens diethylstilbestrol (DES), o,p'-DDT, and OP but not with vehicle, antiestrogen ICI 164,384, dexamethasone, or testosterone. To determine whether serum proteins affected the bioavailability of natural estrogens compared to xenoestrogens, albumin, sex hormone binding globulin (SHBG), or charcoal-stripped serum were added to the YES system and beta-galactosidase activity assayed. Albumin and SHBG decreased beta-galactosidase activity in the presence of estradiol to a greater extent than DES, o,p'-DDT, and OP. Human and alligator charcoal-stripped serum were also effective at selectively reducing beta-galactosidase activity in the presence of estradiol compared to xenoestrogens. Human serum was more effective than alligator serum in reducing beta-galactosidase activity in the presence of xenoestrogens, indicating that serum may serve as a biomarker for sensitivity to xenoestrogens. Selective binding of 17 beta-estradiol by proteins in serum indicates that certain xenoestrogens may exert greater estrogenicity than originally predicted. The estrogenic potency of a compound involves its binding affinity, bioavailability in serum, and persistence in the environment. Our data

  13. Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development.

    Science.gov (United States)

    Becker, Emmanuelle; Liu, Yuchen; Lardenois, Aurélie; Walther, Thomas; Horecka, Joe; Stuparevic, Igor; Law, Michael J; Lavigne, Régis; Evrard, Bertrand; Demougin, Philippe; Riffle, Michael; Strich, Randy; Davis, Ronald W; Pineau, Charles; Primig, Michael

    2015-04-24

    Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/α strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis. Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development

  14. Therapeutic action of taurine on the postirradiation recovery of the yeast cells

    International Nuclear Information System (INIS)

    Benevolenskij, V.N.; Yartsev, E.I.; Novosteltseva, S.D.; Yakovlev, V.G.

    1975-01-01

    It has been shown on X-irradiated Saccharomyces ellipsoides cells that taurine-potassium phosphate applied after the exposure has a therapeutic action, that is, it intensifies the natural process of intracellular dark repair

  15. Live-cell imaging of budding yeast telomerase RNA and TERRA.

    Science.gov (United States)

    Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

    2017-02-01

    In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure.

    Science.gov (United States)

    Grand, Ralph S; Pichugina, Tatyana; Gehlen, Lutz R; Jones, M Beatrix; Tsai, Peter; Allison, Jane R; Martienssen, Robert; O'Sullivan, Justin M

    2014-11-10

    Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S. pombe chromosomes. Known features of genome organization (e.g. the clustering of telomeres and retrotransposon long terminal repeats (LTRs)) were observed throughout the cell cycle. There were clear correlations between transcript levels and chromosomal interactions between genes, consistent with a role for interactions in transcriptional regulation at specific stages of the cell cycle. In silico reconstructions of the chromosome organization within the S. pombe nuclei were made by polymer modeling. These models suggest that groups of genes with high and low, or differentially regulated transcript levels have preferred positions within the S. pombe nucleus. We conclude that the S. pombe nucleus is spatially divided into functional sub-nuclear domains that correlate with gene activity. The observation that chromosomal interactions are maintained even when chromosomes are fully condensed in M phase implicates genome organization in epigenetic inheritance and bookmarking. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Directory of Open Access Journals (Sweden)

    Flávia A Resende

    Full Text Available Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA and the MCF-7 proliferation assay (E-screen, since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  18. Involvement of GDH3-encoded NADP+-dependent glutamate dehydrogenase in yeast cell resistance to stress-induced apoptosis in stationary phase cells.

    Science.gov (United States)

    Lee, Yong Joo; Kim, Kyung Jin; Kang, Hong Yong; Kim, Hye-Rim; Maeng, Pil Jae

    2012-12-28

    Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP(+)-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.

  19. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and γ-rays

    International Nuclear Information System (INIS)

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-01-01

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (γ)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and γ-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and γ-rays). Similarly, for X- and γ-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and γ-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-a-vis their energy levels

  20. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

    Science.gov (United States)

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

  1. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    Directory of Open Access Journals (Sweden)

    Mari Narusaka

    Full Text Available Housaku Monogatari (HM is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.

  2. The comparative effect of prebiotic, growth promoter antibiotic, probiotic, yeast cell wall and acid fire on broiler chickens performance

    Directory of Open Access Journals (Sweden)

    A Zakeri

    2010-05-01

    Full Text Available In this study 600 Cobb 500 broiler chickens were divided in six similar groups (A, B, C, D, E&F with 100 chickens in each group (with four replicates of 25 chickens in each group. 100 g/ton growth promoter antibiotic in experimental group (B, 100 g/ton Prebiotic in experimental group (C, 800 g/ton Acid fires in experimental group (D, 1 kg/ton MOS in experimental group (E and 4 kg/ton Yeast cell wall in experimental group (F were added to the basic diets however the control group (A chickens were fed only with the basic diet. Every week 100 chickens from each group were selected randomly and productive parameters such as weight gain, FCR, EEF, mortality, feed intake (FI were calculated. On days 9, 17 and 25 of growth (1 day before and 7, 14 days after first Newcastle B1 vaccination, from each groups, each time 20 chickens were chosen randomly and serum antibody titres of these chickens were measured against Newcastle vaccine by HI test. The results obtained from statistical analysis indicated that using natural growth promoting substances not only increases humoral immunity but it also leads to improvement of productive parameters (p

  3. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    International Nuclear Information System (INIS)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

    2016-01-01

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  4. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig, E-mail: blackstc@ninds.nih.gov

    2016-11-15

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  5. The resistance of the yeast Saccharomyces cerevisiae to the biocide polyhexamethylene biguanide: involvement of cell wall integrity pathway and emerging role for YAP1

    Directory of Open Access Journals (Sweden)

    de Morais Marcos A

    2011-08-01

    Full Text Available Abstract Background Polyhexamethylene biguanide (PHMB is an antiseptic polymer that is mainly used for cleaning hospitals and pools and combating Acantamoeba infection. Its fungicide activity was recently shown by its lethal effect on yeasts that contaminate the industrial ethanol process, and on the PE-2 strain of Saccharomyces cerevisiae, one of the main fermenting yeasts in Brazil. This pointed to the need to know the molecular mechanism that lay behind the cell resistance to this compound. In this study, we examined the factors involved in PHMB-cell interaction and the mechanisms that respond to the damage caused by this interaction. To achieve this, two research strategies were employed: the expression of some genes by RT-qPCR and the analysis of mutant strains. Results Cell Wall integrity (CWI genes were induced in the PHMB-resistant Saccharomyces cerevisiae strain JP-1, although they are poorly expressed in the PHMB-sensitive Saccharomyces cerevisiae PE2 strain. This suggested that PHMB damages the glucan structure on the yeast cell wall. It was also confirmed by the observed sensitivity of the yeast deletion strains, Δslg1, Δrom2, Δmkk2, Δslt2, Δknr4, Δswi4 and Δswi4, which showed that the protein kinase C (PKC regulatory mechanism is involved in the response and resistance to PHMB. The sensitivity of the Δhog1 mutant was also observed. Furthermore, the cytotoxicity assay and gene expression analysis showed that the part played by YAP1 and CTT1 genes in cell resistance to PHMB is unrelated to oxidative stress response. Thus, we suggested that Yap1p can play a role in cell wall maintenance by controlling the expression of the CWI genes. Conclusion The PHMB treatment of the yeast cells activates the PKC1/Slt2 (CWI pathway. In addition, it is suggested that HOG1 and YAP1 can play a role in the regulation of CWI genes.

  6. Evolutionarily conserved sites in yeast tropomyosin function in cell polarity, transport and contractile ring formation

    Directory of Open Access Journals (Sweden)

    Susanne Cranz-Mileva

    2015-08-01

    Full Text Available Tropomyosin is a coiled-coil protein that binds and regulates actin filaments. The tropomyosin gene in Schizosaccharomyces pombe, cdc8, is required for formation of actin cables, contractile rings, and polar localization of actin patches. The roles of conserved residues were investigated in gene replacement mutants. The work validates an evolution-based approach to identify tropomyosin functions in living cells and sites of potential interactions with other proteins. A cdc8 mutant with near-normal actin affinity affects patch polarization and vacuole fusion, possibly by affecting Myo52p, a class V myosin, function. The presence of labile residual cell attachments suggests a delay in completion of cell division and redistribution of cell patches following cytokinesis. Another mutant with a mild phenotype is synthetic negative with GFP-fimbrin, inferring involvement of the mutated tropomyosin sites in interaction between the two proteins. Proteins that assemble in the contractile ring region before actin do so in a mutant cdc8 strain that cannot assemble condensed actin rings, yet some cells can divide. Of general significance, LifeAct-GFP negatively affects the actin cytoskeleton, indicating caution in its use as a biomarker for actin filaments.

  7. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Wang, Z.; Wu, X.; Friedberg, E.C.

    1993-01-01

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg 2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  8. Nano-topography Enhances Communication in Neural Cells Networks.

    Science.gov (United States)

    Onesto, V; Cancedda, L; Coluccio, M L; Nanni, M; Pesce, M; Malara, N; Cesarelli, M; Di Fabrizio, E; Amato, F; Gentile, F

    2017-08-29

    Neural cells are the smallest building blocks of the central and peripheral nervous systems. Information in neural networks and cell-substrate interactions have been heretofore studied separately. Understanding whether surface nano-topography can direct nerve cells assembly into computational efficient networks may provide new tools and criteria for tissue engineering and regenerative medicine. In this work, we used information theory approaches and functional multi calcium imaging (fMCI) techniques to examine how information flows in neural networks cultured on surfaces with controlled topography. We found that substrate roughness S a affects networks topology. In the low nano-meter range, S a  = 0-30 nm, information increases with S a . Moreover, we found that energy density of a network of cells correlates to the topology of that network. This reinforces the view that information, energy and surface nano-topography are tightly inter-connected and should not be neglected when studying cell-cell interaction in neural tissue repair and regeneration.

  9. Nano-topography Enhances Communication in Neural Cells Networks

    KAUST Repository

    Onesto, V.

    2017-08-23

    Neural cells are the smallest building blocks of the central and peripheral nervous systems. Information in neural networks and cell-substrate interactions have been heretofore studied separately. Understanding whether surface nano-topography can direct nerve cells assembly into computational efficient networks may provide new tools and criteria for tissue engineering and regenerative medicine. In this work, we used information theory approaches and functional multi calcium imaging (fMCI) techniques to examine how information flows in neural networks cultured on surfaces with controlled topography. We found that substrate roughness Sa affects networks topology. In the low nano-meter range, S-a = 0-30 nm, information increases with Sa. Moreover, we found that energy density of a network of cells correlates to the topology of that network. This reinforces the view that information, energy and surface nano-topography are tightly inter-connected and should not be neglected when studying cell-cell interaction in neural tissue repair and regeneration.

  10. A simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies.

    Science.gov (United States)

    Rivas, E-M; Gil de Prado, E; Wrent, P; de Silóniz, M-I; Barreiro, P; Correa, E C; Conejero, F; Murciano, A; Peinado, J M

    2014-12-01

    We propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks. Mathematical models used to predict microbial growth are based on liquid cultures data. Models describing growth on solid surfaces, highlighting the differences with liquids cultures, are scarce. In this work, we have demonstrated that a single Gompertz equation describes accurately the increase of the yeast colonies, up to the point where they reach their maximum size. The model can be used to quantify the differences in growth kinetics between solid and liquid media. Moreover, as all its parameters have biological meaning, it could be used to build secondary models predicting yeast growth on solid surfaces under several environmental conditions. © 2014 The Society for Applied Microbiology.

  11. Yeast Cell Factory-Platform for the Screening and the Industrial Production of Flavonoids and other Phenolic Compounds

    DEFF Research Database (Denmark)

    Lehka, Beata Joanna

    is a precursor for other commercially relevant flavonoids we developed a platform for the production of a library of flavonoid derivatives and screened them for antibacterial properties. Seven different yeast strains producing flavonoids (naringenin, kaempferol, dihydrokaempferol, apigenin and afzelechin...

  12. Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients

    NARCIS (Netherlands)

    Laheij, A.M.G.A.; de Soet, J.J.; von dem Borne, P.A.; Kuijper, E.J.; Kraneveld, E.A.; van Loveren, C.; Raber-Durlacher, J.E.

    2012-01-01

    BACKGROUND: Oral mucositis is a serious and debilitating side effect of conditioning regimens for hematopoietic stem cell transplant (HSCT). Through HSCT, the homeostasis in the oral cavity is disrupted. The contribution of the oral microflora to mucositis remains to be clarified. The aim of our

  13. Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients

    NARCIS (Netherlands)

    Laheij, Alexa M. G. A.; de Soet, Johannes J.; von dem Borne, Peter A.; Kuijper, Ed J.; Kraneveld, Eefje A.; van Loveren, Cor; Raber-Durlacher, Judith E.

    2012-01-01

    Oral mucositis is a serious and debilitating side effect of conditioning regimens for hematopoietic stem cell transplant (HSCT). Through HSCT, the homeostasis in the oral cavity is disrupted. The contribution of the oral microflora to mucositis remains to be clarified. The aim of our study was to

  14. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

    Science.gov (United States)

    Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

    1991-03-01

    It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

  15. Stable inheritable postradiation increase in the death-rate of yeast cells

    International Nuclear Information System (INIS)

    Bychkovskaya, I.B.; Stanzhevskaya, T.I.

    1980-01-01

    It was demonstrated by the use of serial equal dilution of irradiated and control cultures, which extended the logarithmic phase of growth, that irradiation of 0.5-2.5 krad, as well as the effect of a preirradiated medium, induce in Saccharomyces ellipsoideus Megri an inheritable stable increase in the death-rate of cells which was previously demonstrated on individually cultured protozoa

  16. Industrial systems biology and its impact on synthetic biology of yeast cell factories

    DEFF Research Database (Denmark)

    Fletcher, Eugene; Krivoruchko, Anastasia; Nielsen, Jens

    2016-01-01

    , microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex...

  17. Energy Efficiency Challenges of 5G Small Cell Networks.

    Science.gov (United States)

    Ge, Xiaohu; Yang, Jing; Gharavi, Hamid; Sun, Yang

    2017-05-01

    The deployment of a large number of small cells poses new challenges to energy efficiency, which has often been ignored in fifth generation (5G) cellular networks. While massive multiple-input multiple outputs (MIMO) will reduce the transmission power at the expense of higher computational cost, the question remains as to which computation or transmission power is more important in the energy efficiency of 5G small cell networks. Thus, the main objective in this paper is to investigate the computation power based on the Landauer principle. Simulation results reveal that more than 50% of the energy is consumed by the computation power at 5G small cell base stations (BSs). Moreover, the computation power of 5G small cell BS can approach 800 watt when the massive MIMO (e.g., 128 antennas) is deployed to transmit high volume traffic. This clearly indicates that computation power optimization can play a major role in the energy efficiency of small cell networks.

  18. Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells.

    Science.gov (United States)

    Zhao, Chun-Hai; Chi, Zhe; Zhang, Fang; Guo, Feng-Jun; Li, Mei; Song, Wei-Bo; Chi, Zhen-Ming

    2011-05-01

    In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C(16:0), C(18:1) and C(18:2), especially C(18:1) (50.6%). Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Functional Networks Involved in Cell Wall Biosynthesis and the Isoprenoid Pathway in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Kuranda, Klaudia

    2006-01-01

    Ce travail porte sur la réponse de la levure à des composés chimiques, qui de manière similaire aux antifongiques, altèrent la structure pariétale et/ou la biosynthèse de l'ergostérol. Ces deux voies sont essentielles pour la survie des champignons, la paroi les protège de leur environnement, alors que l'ergostérol garantit un fonctionnement correct de la membrane plasmique. Pour comprendre ces mécanismes et leurs interactions, nous avons choisi un organisme modèle, la levure Saccharomyces ce...

  20. Why do hubs in the yeast protein interaction network tend to be essential: reexamining the connection between the network topology and essentiality.

    Directory of Open Access Journals (Sweden)

    Elena Zotenko

    2008-08-01

    Full Text Available The centrality-lethality rule, which notes that high-degree nodes in a protein interaction network tend to correspond to proteins that are essential, suggests that the topological prominence of a protein in a protein interaction network may be a good predictor of its biological importance. Even though the correlation between degree and essentiality was confirmed by many independent studies, the reason for this correlation remains illusive. Several hypotheses about putative connections between essentiality of hubs and the topology of protein-protein interaction networks have been proposed, but as we demonstrate, these explanations are not supported by the properties of protein interaction networks. To identify the main topological determinant of essentiality and to provide a biological explanation for the connection between the network topology and essentiality, we performed a rigorous analysis of six variants of the genomewide protein interaction network for Saccharomyces cerevisiae obtained using different techniques. We demonstrated that the majority of hubs are essential due to their involvement in Essential Complex Biological Modules, a group of densely connected proteins with shared biological function that are enriched in essential proteins. Moreover, we rejected two previously proposed explanations for the centrality-lethality rule, one relating the essentiality of hubs to their role in the overall network connectivity and another relying on the recently published essential protein interactions model.

  1. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  2. Microbial colonization of irradiated pathogenic yeast to catheter surfaces: Relationship between adherence, cell surface hydrophobicity, biofilm formation and antifungal susceptibility. A scanning electron microscope analysis.

    Science.gov (United States)

    Farrag, Hala Abdallah; A-Karam El-Din, Alzahraa; Mohamed El-Sayed, Zeinab Galal; Abdel-Latifissa, Soheir; Kamal, Mona Mohamed

    2015-06-01

    Technological advances such as long-term indwelling catheters have created milieu in which infections are a major complication. Thus it is essential to be able to recognize, diagnose, and treat infections occurring in immunocompromised patients. Adherence assay and quantitation of biofilms was performed by a spectrophotometric method, hydrophobicity was evaluated by adhesion to p-xylene. The minimum inhibitory concentration (MIC) of Nystatin was carried out by a well dilution method. Out of 100 bladder cancer patients, 23 pathogenic yeast isolates were identified. The samples were taken from urinary catheters and urine collected from their attached drainage bags. Pathogenic yeast identified were species of Candida, Cryptococcus, Saccharomyces, Blastoschizomyces, Trichosporn, Hansenula, Prototheca and Rhodotorula. With the exception of Rhodotorula minuta, the yeast were sensitive to the antimycotic agent (Nystatin) used before and after in vitro gamma irradiation at 24.41 Gy as measured by a disc diffusion method. All tested yeast strains were slime producers and showed positive adherence reactions. There were considerable differences in adherence measurements after irradiation. An increase in adherence measurement values (using a spectrophotometric method) after irradiation were detected in four strains whereas eight other strains showed a reduction in their adherence reaction. The cell surface hydrophobicity (CSH) was evaluated by adhesion to p-xylene. Candida tropicalis showed a hydrophobic reaction with an increase in the cell surface hydrophobicity after irradiation. Scanning electron microscopy of irradiated C. tropicalis showed marked abnormalities in cell shape and size with significant reduction in adherence ability at the MIC level of Nystatin (4 μg/ml). More basic research at the level of pathogenesis and catheter substance is needed to design novel strategies to prevent fungal adherence and to inhibit biofilm formation.

  3. Systems biology of yeast: enabling technology for development of cell factories for production of advanced biofuels.

    Science.gov (United States)

    de Jong, Bouke; Siewers, Verena; Nielsen, Jens

    2012-08-01

    Transportation fuels will gradually shift from oil based fuels towards alternative fuel resources like biofuels. Current bioethanol and biodiesel can, however, not cover the increasing demand for biofuels and there is therefore a need for advanced biofuels with superior fuel properties. Novel cell factories will provide a production platform for advanced biofuels. However, deep cellular understanding is required for improvement of current biofuel cell factories. Fast screening and analysis (-omics) methods and metabolome-wide mathematical models are promising techniques. An integrated systems approach of these techniques drives diversity and quantity of several new biofuel compounds. This review will cover the recent technological developments that support improvement of the advanced biofuels 1-butanol, biodiesels and jetfuels. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Correction to: Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells

    OpenAIRE

    Nielsen, Jens Christian; Lino, Felipe Senne de Oliveira; Rasmussen, Thomas Gundelund; Thykær, Jette; Workman, Christopher T.; Basso, Thiago Olitta

    2017-01-01

    The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have...

  5. Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Maršíková, J.; Wilkinson, D.; Hlaváček, Otakar; Gilfillan, G.D.; Mizeranschi, A.; Hughes, T.; Begany, Markéta; Rešetárová, Stanislava; Váchová, Libuše; Palková, Z.

    2017-01-01

    Roč. 18, OCT 23 (2017), s. 814 ISSN 1471-2164 R&D Projects: GA MŠk(CZ) 7F14083; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Saccharomyces cerevisiae * Colony biofilms * Cell differentiation Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.729, year: 2016

  6. Metabolic network analysis on Phaffia rhodozyma yeast using C-13-labeled glucose and gas chromatography-mass spectrometry

    DEFF Research Database (Denmark)

    Cannizzaro, C.; Christensen, B.; Nielsen, Jens

    2004-01-01

    labeling patterns, as determined by GC-MS, were in accordance with a metabolic network consisting of the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (similar to65% for wildtype strain), which reflected...... to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower....

  7. Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

    Directory of Open Access Journals (Sweden)

    Cory H.T. Boone

    2017-08-01

    Full Text Available The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC50 dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by ICP-MS. Measurement of the relative ATP and ADP concentrations indicates an initial 3-fold depletion of ATP suggesting an alteration in the ATP:ADP ratio. At the 6 h time point the lidocaine exposed population contained ATP concentrations roughly 85% that of the negative control suggesting the surviving population adapted its metabolic pathways to, at least partially restore cellular bioenergetics. Metabolite analysis indicates an increase of intermediates in the pentose phosphate pathway, the preparatory phase of glycolysis, and NADPH. Oxidative stress produced by lidocaine exposure targets aconitase decreasing its activity with an observed decrease in isocitrate and an increase citrate. Similarly, increases in α-ketoglutarate, malate, and oxaloacetate imply activation of anaplerotic reactions. Antioxidant molecule glutathione and its precursor amino acids, cysteine and glutamate were greatly increased at later time points. Phosphatidylserine externalization suggestive of early phase apoptosis was also observed. Genetic studies using metacaspase null strains showed resistance to lidocaine induced cell death. These data suggest lidocaine induces perpetual mitochondrial depolarization, ROS/RNS generation along with increased

  8. Determination of Concentration of Living Immobilized Yeast Cells by Fluorescence Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Podrazký, Ondřej; Kuncová, Gabriela

    2005-01-01

    Roč. 107, č. 1 (2005), s. 126-134 ISSN 0925-4005. [European Conference on Optical Chemical Sensors and Biosensors EUROPT(R)ODE /7./. Madrid, 04.04.2004-07.04.2004] R&D Projects: GA ČR GA104/01/0461; GA MŠk(CZ) OC 840.10 Institutional research plan: CEZ:AV0Z40720504 Keywords : immobilization of cells * 2-D fluorescence spectroscopy * sol–gel Subject RIV: CE - Biochemistry Impact factor: 2.646, year: 2005

  9. Capillary network formation from dispersed endothelial cells: Influence of cell traction, cell adhesion, and extracellular matrix rigidity

    Science.gov (United States)

    Ramos, João R. D.; Travasso, Rui; Carvalho, João

    2018-01-01

    The formation of a functional vascular network depends on biological, chemical, and physical processes being extremely well coordinated. Among them, the mechanical properties of the extracellular matrix and cell adhesion are fundamental to achieve a functional network of endothelial cells, able to fully cover a required domain. By the use of a Cellular Potts Model and Finite Element Method it is shown that there exists a range of values of endothelial traction forces, cell-cell adhesion, and matrix rigidities where the network can spontaneously be formed, and its properties are characterized. We obtain the analytical relation that the minimum traction force required for cell network formation must obey. This minimum value for the traction force is approximately independent on the considered cell number and cell-cell adhesion. We quantify how these two parameters influence the morphology of the resulting networks (size and number of meshes).

  10. Workshop: Theory an Applications of Coupled Cell Networks

    Science.gov (United States)

    2006-03-22

    Economia and Centro de Matematica , Universidade do Porto) Application of coupled cell systems have been made to a wide range of problems in the physical and...Departamento de Matematica Pura da Faculdade de Ciencias do Porto) As pointed by [1], in the class of coupled cell networks that permits self-coupling

  11. Cell proliferation along vascular islands during microvascular network growth

    Directory of Open Access Journals (Sweden)

    Kelly-Goss Molly R

    2012-06-01

    Full Text Available Abstract Background Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth. Results Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels. Conclusions These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth.

  12. Nuclear Transport of Yeast Proteasomes

    Directory of Open Access Journals (Sweden)

    Cordula Enenkel

    2014-10-01

    Full Text Available Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin αβ. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin β, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells.

  13. Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning

    KAUST Repository

    Feizi, Alborz

    2016-09-24

    Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm2. This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP\\'s performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

  14. Selenium speciation and isotope composition in 77Se-enriched yeast using gradient elution HPLC separation and ICP-dynamic reaction cell-MS

    DEFF Research Database (Denmark)

    Larsen, Erik Huusfeldt; Sloth, Jens Jørgen; Hansen, M.

    2003-01-01

    A batch of Se-77-labelled and enriched yeast was characterised with regard to isotopic composition and content of selenium species for later use in a human absorption study based on the method of enriched stable isotopes. The abundance of the six stable selenium isotopes was determined by ICP- MS...... equipped with a dynamic reaction cell (DRC). The results showed that the Se-77 isotope was enriched to 98.5 atom-%, whereas the remaining selenium was present as the other five isotopes at low abundance. The low-molecular Se-77 containing species, which were biosynthesised by the yeast during fermentation...... using the enriched Se-77-selenite as substrate, were released by enzymatic hydrolysis using (I), a beta-glucosidase followed by a protease mixture, and (II), a commercial protease preparation. For selenium speciation the chromatographic selectivity of the cation exchange HPLC system was adjusted...

  15. yeast transformation of Mucor circinelloides Tieghe

    African Journals Online (AJOL)

    GRACE

    2006-05-02

    May 2, 2006 ... enhanced growth by 32.95, 65.07 and 63.82%, respectively, over control mean growth. Proliferating yeast cells induced from .... Table 2. A nested model analysis of variance of growth data of induced yeast cells of M. ..... Pullman B (ed) Frontiers in Physicochemical Biology. New York: Academic Press. p.

  16. Aerobic decolorization and degradation of Acid Orange G (AOG) by suspended growing cells and immobilized cells of a yeast strain Candida tropicalis TL-F1.

    Science.gov (United States)

    Tan, Liang; Li, Hua; Ning, Shuxiang; Hao, Jia

    2014-10-01

    In this study, aerobic decolorization and degradation of azo dye Acid Orange G (AOG) by both suspended growing cells and immobilized cells of a yeast strain Candida tropicalis TL-F1 were studied. The effects of different parameters on decolorization of AOG by both growing suspended and immobilized strain TL-F1 were investigated. Furthermore, a possible decolorization mechanism of AOG was proposed through analyzing metabolic intermediates using UV-vis and high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. Strain TL-F1 could decolorize AOG in both liquid and solid mediums through degradation. The optimal conditions for decolorization with suspended growing cells of strain TL-F1 were as follows: 6-10 g/L sucrose, 5-7 g/L urea, ≥6 % (v/v) inoculation size, ≥160 rpm, 35-40 °C, and pH 5.0-6.0; and those for immobilized cells, the conditions were as follows: 4-6 g/L glucose, 0.2-0.4 g/L urea, 6-10 g/L (wet cell pellets) inoculation size, ≥160 rpm, 35-40 °C, and pH 5.0-7.0. Results of UV-vis scanning spectra suggested that AOG was decolorized through biodegradation, and the possible pathway was proposed through the results of HPLC-MS analysis and related literature. This is a systematic research on aerobic decolorization and degradation of AOG by both suspended and immobilized cells of a C. tropicalis strain.

  17. Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

    1984-01-01

    Cell-free extracts from wild-type yeast (RAD + ) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4-1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD 37 (about 10 4 PD per haploid genome). (Auth.)

  18. Comparison of Yeast Cell Protein Solubilization Procedures for Two-dimensional Electrophoresis

    DEFF Research Database (Denmark)

    Harder, A; Wildgruber, R; Nawrocki, A

    1999-01-01

    with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea...... sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii)....... molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins...

  19. Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells

    DEFF Research Database (Denmark)

    Nielsen, Jens Christian; Senne de Oliveira Lino, Felipe; Rasmussen, Thomas Gundelund

    2017-01-01

    The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2...... generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial...... ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates...

  20. Correction to: Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells

    DEFF Research Database (Denmark)

    Nielsen, Jens Christian Frøslev; Senne de Oliveira Lino, Felipe; Rasmussen, Thomas Gundelund

    2018-01-01

    The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2...... generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial...... ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates...

  1. Changes of respiration and of specific growth rate during cell cycle of yeast cells of different genealogical age.

    Science.gov (United States)

    Vraná, D

    1988-01-01

    When investigating changes of respiratory activity during the cell cycle of mother and daughter Candida cells significant oscillations of specific rate of oxygen consumption were detected; specific growth rate also varied. The oscillations were less pronounced when the inoculum was obtained from the chemostat at the high dilution rates of 0.25 and 0.35/h.

  2. Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

    Science.gov (United States)

    Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal

    2015-12-01

    Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  3. Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.

    Science.gov (United States)

    Bögre, L; Jonak, C; Mink, M; Meskiene, I; Traas, J; Ha, D T; Swoboda, I; Plank, C; Wagner, E; Heberle-Bors, E; Hirt, H

    1996-03-01

    We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.

  4. The Use of HIS6 Gene as a Selectable Marker for Yeast Vector

    Directory of Open Access Journals (Sweden)

    IMADEARTIKA

    2009-03-01

    Full Text Available The yeast Saccharomyces cerevisiae HIS6 gene has been shown to be functional as a selectable marker for selecting and maintaining a yeast vector in yeast S. cerevisiae host cells. The yeast HIS6 gene encodes an enzyme involved in the yeast histidine biosynthesis. The yeast HIS6 gene was cloned into a yeast expression vector. The resultant recombinant plasmid was introduced into yeast host cells defective in endogenous HIS6 gene. The functionality of the HIS6 gene as a selectable marker was tested by growing transformed cells on selective minimum medium lacking histidine supplementation.

  5. The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway.

    Science.gov (United States)

    Heeren, Gino; Jarolim, Stefanie; Laun, Peter; Rinnerthaler, Mark; Stolze, Klaus; Perrone, Gabriel G; Kohlwein, Sepp D; Nohl, Hans; Dawes, Ian W; Breitenbach, Michael

    2004-11-01

    We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.

  6. Replacement of Fishmeal by Single Cell Protein Derived from Yeast Grown on Date (Phoenix dactylifera) Industry Waste in the Diet of Nile Tilapia (Oreochromis niloticus) Fingerlings

    KAUST Repository

    Al-Hafedh, Yousef S.

    2013-10-02

    Isonitrogenous and isocaloric diets (32% protein, 4.3 Kcal/g) were formulated to replace fishmeal by single cell protein (SCP) from two yeasts, Saccharomyces cerevisiae and Candida utilis, grown on date (Phoenix dactylifera) processing waste in diets for two size groups (avg 15.39 g and 25.14 g) of juvenile Nile tilapia (Oreochromis niloticus). A control diet (T1) with fishmeal and six experimental diets (S1, S2, and S3 with S. cerevisiae, and C1, C2, and C3 with C. utilis) each containing 11.6%, 23.2%, and 34.2% yeast as SCP were prepared to replace 25%, 50%, and 75% of fishmeal, respectively. Tilapia fed on the control and experimental diets (S1, S2, C1, C2) with 25% and 50% replacement of fishmeal showed better growth and feed utilization. Fish fed on diets S3 and C3 (75% fishmeal replacement) had significantly (p < 0.05) poorer growth suggesting that yeast SCP can replace up to 50% of fishmeal in juvenile tilapia diets. © 2013 Copyright Taylor and Francis Group, LLC.

  7. Primary Cilia, Signaling Networks and Cell Migration

    DEFF Research Database (Denmark)

    Veland, Iben Rønn

    Primary cilia are microtubule-based, sensory organelles that emerge from the centrosomal mother centriole to project from the surface of most quiescent cells in the human body. Ciliary entry is a tightly controlled process, involving diffusion barriers and gating complexes that maintain a unique...... and cytoskeletal organization. Further, cell migration and polarization in are impaired in Invs MEFs. In two-dimensional cell migration, the centrosome is positioned between the nucleus and the leading edge with the primary cilium directed towards the direction of migration. PDGFRα is activated in the primary......, which leads to uncontrolled cell movements. Together, the results obtained from my PhD studies reflect the high level of complexity within signaling systems regulated by the primary cilium that control cellular processes during embryonic development and in tissue homeostasis. As such, this dissertation...

  8. Social structures in Russia : cells and networks

    OpenAIRE

    Yefimov, Vladimir

    2001-01-01

    Russian companies heirs of Soviet enterprises are not Western-style companies, a significant difference is that they represent the basic structures of social life in the USSR : cells. The Soviet cellular system itself has deep roots in the history of Russia. The principal social structure of pre-revolutionary Russia was the rural community. In the late 1950s, Soviet society began to move away from the classic model. Cells gradually lose their exclusive role in the functioning of society. New ...

  9. Sociobiology of the budding yeast

    Indian Academy of Sciences (India)

    2014-03-15

    Mar 15, 2014 ... media (figure 2). On solid substrates exposed to air, cells that do not produce flocculins will develop nonadhesive colonies, such as seen for the ..... Programmed cell death. Escherichia coli, protozoa, bacteria, slime moulds. Yeast apoptosis (Madeo et al. 1997; Honigberg 2011). Communication via.

  10. Yeast ABC transporters in lipid trafficking.

    Science.gov (United States)

    Prasad, Rajendra; Khandelwal, Nitesh Kumar; Banerjee, Atanu

    2016-08-01

    Throughout its evolution, the ATP-binding cassette (ABC) transporter superfamily has experienced a rapid expansion in its substrate repertoire and functions. Of the diverse functions that these pumps offer, their drug transport properties have attracted considerable attention primarily owing to their clinical significance. Despite this fact, emerging evidence suggests that physiological substrates of transporters also affect the overall functioning of an organism. Lipids, as substrates of ABC transporters, constitute one feature found in all representative groups of the living kingdom. Due to the importance of lipid species in the cellular physiology of an organism, their proper distribution within cells is crucial. This fact is well exemplified by the vast number of medical conditions that have been caused as a result of perturbations in ABC transporter-mediated lipid transport in higher organisms. In yeasts, apart from providing transport functions, ABC transporters also coordinate regulatory networks with lipids. This review focuses on yeast ABC transporters involved in the transport of lipids and briefly discusses the integration of their regulatory network with that of the lipid species. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer.

    Science.gov (United States)

    Bom, I J; Klis, F M; de Nobel, H; Brul, S

    2001-12-01

    Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.

  12. Hemicellulosic Ethanol Production by Immobilized Wild Brazilian Yeast Scheffersomyces shehatae UFMG-HM 52.2: Effects of Cell Concentration and Stirring Rate.

    Science.gov (United States)

    Antunes, F A F; Santos, J C; Chandel, A K; Milessi, T S S; Peres, G F D; da Silva, S S

    2016-02-01

    The use of sugarcane bagasse hemicellulosic hydrolysates presents an interesting alternative to second generation (2G) ethanol production. Techniques to enhance the fermentation process, e.g., the use of immobilized cells, is one of the key factors for efficient production. Here, the effect of two important parameters (cell concentration in immobilized system and stirring rate) on the 2G ethanol production using the wild Brazilian yeast S. shehatae UFMG-HM 52.2 immobilized in calcium alginate matrix are presented. A 2(2) full factorial design of experiments was carried out to evaluate the effect of cell concentrations in sodium alginate solution for immobilized bead production (3.0, 6.0, and 9.0 g/L) and stirring rate (150, 200, and 250 rpm) for 2G ethanol production. Statistical analysis showed that the use of both variables at low levels enhanced ethanol yield (YP/S). Under these process conditions, YP/S of 0.31 g/g and ethanol productivity (Qp) of 0.12 g/L h were achieved. Results showed the potential of this immobilized yeast in 2G ethanol production from C5 sugars and demonstrate the importance of adequate cell concentration in immobilized systems, a finding that stands to increase bioprocesses yields and productivity.

  13. The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

    Science.gov (United States)

    Tinoco-Nunes, Bruno; Telleria, Erich Loza; da Silva-Neves, Monique; Marques, Christiane; Azevedo-Brito, Daisy Aline; Pitaluga, André Nóbrega; Traub-Csekö, Yara Maria

    2016-04-20

    Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5. The expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania. These studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi. We demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.

  14. Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

    Science.gov (United States)

    Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

    2013-01-01

    The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133

  15. Construction of a computable cell proliferation network focused on non-diseased lung cells

    Directory of Open Access Journals (Sweden)

    Veljkovic Emilija

    2011-07-01

    Full Text Available Abstract Background Critical to advancing the systems-level evaluation of complex biological processes is the development of comprehensive networks and computational methods to apply to the analysis of systems biology data (transcriptomics, proteomics/phosphoproteomics, metabolomics, etc.. Ideally, these networks will be specifically designed to capture the normal, non-diseased biology of the tissue or cell types under investigation, and can be used with experimentally generated systems biology data to assess the biological impact of perturbations like xenobiotics and other cellular stresses. Lung cell proliferation is a key biological process to capture in such a network model, given the pivotal role that proliferation plays in lung diseases including cancer, chronic obstructive pulmonary disease (COPD, and fibrosis. Unfortunately, no such network has been available prior to this work. Results To further a systems-level assessment of the biological impact of perturbations on non-diseased mammalian lung cells, we constructed a lung-focused network for cell proliferation. The network encompasses diverse biological areas that lead to the regulation of normal lung cell proliferation (Cell Cycle, Growth Factors, Cell Interaction, Intra- and Extracellular Signaling, and Epigenetics, and contains a total of 848 nodes (biological entities and 1597 edges (relationships between biological entities. The network was verified using four published gene expression profiling data sets associated with measured cell proliferation endpoints in lung and lung-related cell types. Predicted changes in the activity of core machinery involved in cell cycle regulation (RB1, CDKN1A, and MYC/MYCN are statistically supported across multiple data sets, underscoring the general applicability of this approach for a network-wide biological impact assessment using systems biology data. Conclusions To the best of our knowledge, this lung-focused Cell Proliferation Network

  16. Cell-specific synaptic plasticity induced by network oscillations.

    Science.gov (United States)

    Zarnadze, Shota; Bäuerle, Peter; Santos-Torres, Julio; Böhm, Claudia; Schmitz, Dietmar; Geiger, Jörg Rp; Dugladze, Tamar; Gloveli, Tengis

    2016-05-24

    Gamma rhythms are known to contribute to the process of memory encoding. However, little is known about the underlying mechanisms at the molecular, cellular and network levels. Using local field potential recording in awake behaving mice and concomitant field potential and whole-cell recordings in slice preparations we found that gamma rhythms lead to activity-dependent modification of hippocampal networks, including alterations in sharp wave-ripple complexes. Network plasticity, expressed as long-lasting increases in sharp wave-associated synaptic currents, exhibits enhanced excitatory synaptic strength in pyramidal cells that is induced postsynaptically and depends on metabotropic glutamate receptor-5 activation. In sharp contrast, alteration of inhibitory synaptic strength is independent of postsynaptic activation and less pronounced. Further, we found a cell type-specific, directionally biased synaptic plasticity of two major types of GABAergic cells, parvalbumin- and cholecystokinin-expressing interneurons. Thus, we propose that gamma frequency oscillations represent a network state that introduces long-lasting synaptic plasticity in a cell-specific manner.

  17. NKT Cell Networks in the Regulation of Tumor Immunity

    Science.gov (United States)

    Robertson, Faith C.; Berzofsky, Jay A.; Terabe, Masaki

    2014-01-01

    CD1d-restricted natural killer T (NKT) cells lie at the interface between the innate and adaptive immune systems and are important mediators of immune responses and tumor immunosurveillance. These NKT cells uniquely recognize lipid antigens, and their rapid yet specific reactions influence both innate and adaptive immunity. In tumor immunity, two NKT subsets (type I and type II) have contrasting roles in which they not only cross-regulate one another, but also impact innate immune cell populations, including natural killer, dendritic, and myeloid lineage cells, as well as adaptive populations, especially CD8+ and CD4+ T cells. The extent to which NKT cells promote or suppress surrounding cells affects the host’s ability to prevent neoplasia and is consequently of great interest for therapeutic development. Data have shown the potential for therapeutic use of NKT cell agonists and synergy with immune response modifiers in both pre-clinical studies and preliminary clinical studies. However, there is room to improve treatment efficacy by further elucidating the biological mechanisms underlying NKT cell networks. Here, we discuss the progress made in understanding NKT cell networks, their consequent role in the regulation of tumor immunity, and the potential to exploit that knowledge in a clinical setting. PMID:25389427

  18. NKT cell networks in the regulation of tumor immunity.

    Science.gov (United States)

    Robertson, Faith C; Berzofsky, Jay A; Terabe, Masaki

    2014-01-01

    CD1d-restricted natural killer T (NKT) cells lie at the interface between the innate and adaptive immune systems and are important mediators of immune responses and tumor immunosurveillance. These NKT cells uniquely recognize lipid antigens, and their rapid yet specific reactions influence both innate and adaptive immunity. In tumor immunity, two NKT subsets (type I and type II) have contrasting roles in which they not only cross-regulate one another, but also impact innate immune cell populations, including natural killer, dendritic, and myeloid lineage cells, as well as adaptive populations, especially CD8(+) and CD4(+) T cells. The extent to which NKT cells promote or suppress surrounding cells affects the host's ability to prevent neoplasia and is consequently of great interest for therapeutic development. Data have shown the potential for therapeutic use of NKT cell agonists and synergy with immune response modifiers in both pre-clinical studies and preliminary clinical studies. However, there is room to improve treatment efficacy by further elucidating the biological mechanisms underlying NKT cell networks. Here, we discuss the progress made in understanding NKT cell networks, their consequent role in the regulation of tumor immunity, and the potential to exploit that knowledge in a clinical setting.