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Sample records for budding yeast cell

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

    International Nuclear Information System (INIS)

    Asami, Koji; Sekine, Katsuhisa

    2007-01-01

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

  2. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    DNA synthesis is one of the landmark events in the cell cycle: G 1 cells have one copy of the genome, S phase cells are actively engaged in DNA synthesis, and G 2 cells have twice as much nuclear DNA as G 1 cells. Cellular DNA content can be measured by staining with a fluorescent dye followed by a flow-cytometric readout. This method provides a quantitative measurement of cell cycle position on a cell-by-cell basis at high speed. Using flow cytometry, tens of thousands of single-cell measurements can be generated in a few seconds. This protocol details staining of cells of the budding yeast Saccharomyces cerevisiae for flow cytometry using Sytox Green dye in a method that can be scaled widely-from one sample to many thousands and operating on inputs ranging from 1 million to more than 100 million cells. Flow cytometry is preferred over light microscopy or Coulter analyses for the analysis of the cell cycle as DNA content and cell cycle position are being directly measured. © 2017 Cold Spring Harbor Laboratory Press.

  3. 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.

  4. Study of budding yeast colony formation and its characterizations by using circular granular cell

    Science.gov (United States)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  5. 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

  6. 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.

  7. Experimental evolution in budding yeast

    Science.gov (United States)

    Murray, Andrew

    2012-02-01

    I will discuss our progress in analyzing evolution in the budding yeast, Saccharomyces cerevisiae. We take two basic approaches. The first is to try and examine quantitative aspects of evolution, for example by determining how the rate of evolution depends on the mutation rate and the population size or asking whether the rate of mutation is uniform throughout the genome. The second is to try to evolve qualitatively novel, cell biologically interesting phenotypes and track the mutations that are responsible for the phenotype. Our efforts include trying to alter cell morphology, evolve multicellularity, and produce a biological oscillator.

  8. Synchronization and Arrest of the Budding Yeast Cell Cycle Using Chemical and Genetic Methods.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G 1 phase and at the M/G 1 transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G 2 /M by using hydroxyurea and nocodazole, respectively. © 2017 Cold Spring Harbor Laboratory Press.

  9. Methods for Synchronization and Analysis of the Budding Yeast Cell Cycle.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    Like other eukaryotes, budding yeast temporally separate cell growth and division. DNA synthesis is distinct from chromosome segregation. Storage carbohydrates are accumulated slowly and then rapidly liquidated once per cycle. Cyclin-dependent kinase associates with multiple different transcriptionally and posttranslationally regulated cyclins to drive the cell cycle. These and other crucial events of cellular growth and division are limited to narrow windows of the cell cycle. Many experiments in the yeast laboratory treat a culture of cells as a homogeneous mixture. Measurements of asynchronous cultures are, however, confounded by the presence of cells in various cell cycle stages; measuring a population average in unsynchronized cells provides at best a decreased signal and at worst an artifactual result. A number of experimentally tractable methods have been developed to generate populations of yeast cells that are synchronized with respect to cell cycle phase. Robust methods for determining cell cycle position have also been developed. These methods are introduced here. © 2017 Cold Spring Harbor Laboratory Press.

  10. Daughter-specific transcription factors regulate cell size control in budding yeast.

    Science.gov (United States)

    Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M; Rosebrock, Adam P; Futcher, Bruce; Cross, Frederick R

    2009-10-01

    In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.

  11. Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast

    Science.gov (United States)

    Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M.; Rosebrock, Adam P.; Futcher, Bruce; Cross, Frederick R.

    2009-01-01

    In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. PMID:19841732

  12. Daughter-specific transcription factors regulate cell size control in budding yeast.

    Directory of Open Access Journals (Sweden)

    Stefano Di Talia

    2009-10-01

    Full Text Available In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.

  13. A microfluidic system for studying ageing and dynamic single-cell responses in budding yeast.

    Directory of Open Access Journals (Sweden)

    Matthew M Crane

    Full Text Available Recognition of the importance of cell-to-cell variability in cellular decision-making and a growing interest in stochastic modeling of cellular processes has led to an increased demand for high density, reproducible, single-cell measurements in time-varying surroundings. We present ALCATRAS (A Long-term Culturing And TRApping System, a microfluidic device that can quantitatively monitor up to 1000 cells of budding yeast in a well-defined and controlled environment. Daughter cells are removed by fluid flow to avoid crowding allowing experiments to run for over 60 hours, and the extracellular media may be changed repeatedly and in seconds. We illustrate use of the device by measuring ageing through replicative life span curves, following the dynamics of the cell cycle, and examining history-dependent behaviour in the general stress response.

  14. Live cell imaging of mitochondrial movement along actin cables in budding yeast.

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    Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A

    2004-11-23

    Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

  15. 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.

  16. Genetic control of x-ray resistance in budding yeast cells

    International Nuclear Information System (INIS)

    Benathen, I.A.; Beam, C.A.

    1977-01-01

    Five x-ray-sensitive mutants were selected from 10,000 colonies arising from survivors of ultraviolet light. These were named XS5, XS6, XS7, XS8, and XS9. Mutant XS1 was donated by Nakai. These mutations affect the resistant budding cell survival component of the survival curve and, in diploids, the low-dose interdivisional cell shoulder. They are of two types: Class I, in which budding cells lack resistance; and Class II, in which budding cells show reduced resistance. When crossed with one another, they show a complex complementation pattern. Gene dosage effects are seen in XS1 heterozygotes, while budding but not between divisions. No direct correlation between radiation sensitivity, meiosis, and sporulation is observed; genes which influence radiation sensitivity do not affect meiotic recombination. A single mutation (XS1 or XS5) suppresses the shoulders of the survival curves of both budding haploid cells and diploid nonbudding cells

  17. Single-Cell Analysis of Growth in Budding Yeast and Bacteria Reveals a Common Size Regulation Strategy.

    Science.gov (United States)

    Soifer, Ilya; Robert, Lydia; Amir, Ariel

    2016-02-08

    To maintain a constant cell size, dividing cells have to coordinate cell-cycle events with cell growth. This coordination has long been supposed to rely on the existence of size thresholds determining cell-cycle progression [1]. In budding yeast, size is controlled at the G1/S transition [2]. In agreement with this hypothesis, the size at birth influences the time spent in G1: smaller cells have a longer G1 period [3]. Nevertheless, even though cells born smaller have a longer G1, the compensation is imperfect and they still bud at smaller cell sizes. In bacteria, several recent studies have shown that the incremental model of size control, in which size is controlled by addition of a constant volume (in contrast to a size threshold), is able to quantitatively explain the experimental data on four different bacterial species [4-7]. Here, we report on experimental results for the budding yeast Saccharomyces cerevisiae, finding, surprisingly, that cell size control in this organism is very well described by the incremental model, suggesting a common strategy for cell size control with bacteria. Additionally, we argue that for S. cerevisiae the "volume increment" is not added from birth to division, but rather between two budding events. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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    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

  19. Measuring mitotic spindle dynamics in budding yeast

    Science.gov (United States)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  20. The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

    Science.gov (United States)

    Chandler-Brown, Devon; Schmoller, Kurt M; Winetraub, Yonatan; Skotheim, Jan M

    2017-09-25

    Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Apoptosis at inflection point in liquid culture of budding yeasts.

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    Toshiyuki Hagiwara

    Full Text Available Budding yeasts are highly suitable for aging studies, because the number of bud scars (stage proportionally correlates with age. Its maximum stages are known to reach at 20-30 stages on an isolated agar medium. However, their stage dynamics in a liquid culture is virtually unknown. We investigate the population dynamics by counting scars in each cell. Here one cell division produces one new cell and one bud scar. This simple rule leads to a conservation law: "The total number of bud scars is equal to the total number of cells." We find a large discrepancy: extremely fewer cells with over 5 scars than expected. Almost all cells with 6 or more scars disappear within a short period of time in the late log phase (corresponds to the inflection point. This discrepancy is confirmed directly by the microscopic observations of broken cells. This finding implies apoptosis in older cells (6 scars or more.

  2. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    Science.gov (United States)

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B

    2014-05-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  3. 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.

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

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    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

  5. 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

    of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate...

  6. Effect of nagilactone E on cell morphology and glucan biosynthesis in budding yeast Saccharomyces cerevisiae.

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    Hayashi, Kengo; Yamaguchi, Yoshihiro; Ogita, Akira; Tanaka, Toshio; Kubo, Isao; Fujita, Ken-Ichi

    2018-05-14

    Nagilactones are norditerpene dilactones isolated from the root bark of Podocarpus nagi. Although nagilactone E has been reported to show antifungal activities, its activity is weaker than that of antifungals on the market. Nagilactone E enhances the antifungal activity of phenylpropanoids such as anethole and isosafrole against nonpathogenic Saccharomyces cerevisiae and pathogenic Candida albicans. However, the detailed mechanisms underlying the antifungal activity of nagilactone E itself have not yet been elucidated. Therefore, we investigated the antifungal mechanisms of nagilactone E using S. cerevisiae. Although nagilactone E induced lethality in vegetatively growing cells, it did not affect cell viability in non-growing cells. Nagilactone E-induced morphological changes in the cells, such as inhomogeneous thickness of the glucan layer and leakage of cytoplasm. Furthermore, a dose-dependent decrease in the amount of newly synthesized (1, 3)-β-glucan was detected in the membrane fractions of the yeast incubated with nagilactone E. These results suggest that nagilactone E exhibits an antifungal activity against S. cerevisiae by depending on cell wall fragility via the inhibition of (1, 3)-β-glucan biosynthesis. Additionally, we confirmed nagilactone E-induced morphological changes of a human pathogenic fungus Aspergillus fumigatus. Therefore, nagilactone E is a potential antifungal drug candidate with fewer adverse effects. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Energy landscape reveals that the budding yeast cell cycle is a robust and adaptive multi-stage process.

    Directory of Open Access Journals (Sweden)

    Cheng Lv

    2015-03-01

    Full Text Available Quantitatively understanding the robustness, adaptivity and efficiency of cell cycle dynamics under the influence of noise is a fundamental but difficult question to answer for most eukaryotic organisms. Using a simplified budding yeast cell cycle model perturbed by intrinsic noise, we systematically explore these issues from an energy landscape point of view by constructing an energy landscape for the considered system based on large deviation theory. Analysis shows that the cell cycle trajectory is sharply confined by the ambient energy barrier, and the landscape along this trajectory exhibits a generally flat shape. We explain the evolution of the system on this flat path by incorporating its non-gradient nature. Furthermore, we illustrate how this global landscape changes in response to external signals, observing a nice transformation of the landscapes as the excitable system approaches a limit cycle system when nutrients are sufficient, as well as the formation of additional energy wells when the DNA replication checkpoint is activated. By taking into account the finite volume effect, we find additional pits along the flat cycle path in the landscape associated with the checkpoint mechanism of the cell cycle. The difference between the landscapes induced by intrinsic and extrinsic noise is also discussed. In our opinion, this meticulous structure of the energy landscape for our simplified model is of general interest to other cell cycle dynamics, and the proposed methods can be applied to study similar biological systems.

  8. Sociobiology of the budding yeast

    Indian Academy of Sciences (India)

    2014-03-15

    Mar 15, 2014 ... The organisms taking part in social behaviours can be divided into actors ... behaviour that have been classified as social. .... tory strains on solid agar media. ..... scenario, PCD is beneficial for the young cells where effective.

  9. 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...

  10. 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; Lhoussaine, Cédric; Hersen, Pascal; Batt, Gregory

    2017-02-01

    With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar , a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. © 2017 The Authors.

  11. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    Science.gov (United States)

    Shibata, M.; Torigoe, M.; Matsumoto, Y.; Yamamoto, M.; Takizawa, N.; Hada, Y.; Mori, Y.; Takarabe, K.; Ono, F.

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  12. Synchronization of Budding Yeast by Centrifugal Elutriation.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    In yeast, cell size is normally tightly linked to cell cycle progression. Centrifugal elutriation is a method that fractionates cells based on the physical properties of cell size-fluid drag and buoyant density. Using a specially modified centrifuge and rotor system, cells can be physically separated into one or more cohorts of similar size and therefore cell cycle position. Small G 1 daughters are collected first, followed by successively larger cells. Elutriated populations can be analyzed immediately or can be returned to medium and permitted to synchronously progress through the cell cycle. This protocol describes two different elutriation methods. In the first, one or more fractions of synchronized cells are obtained from an asynchronous starting population, reincubated, and followed prospectively across a time series. In the second, an asynchronous starting population is separated into multiple fractions of similarly sized cells, and each cohort of similarly sized cells can be analyzed separately without further growth. © 2017 Cold Spring Harbor Laboratory Press.

  13. Actin and Endocytosis in Budding Yeast

    Science.gov (United States)

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  14. 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

  15. Sporulation in the Budding Yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Neiman, Aaron M.

    2011-01-01

    In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423

  16. Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

    OpenAIRE

    Huberts, Daphne H. E. W.; Janssens, Georges E.; Lee, Sung Sik; Vizcarra, Ima Avalos; Heinemann, Matthias

    2013-01-01

    We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 μm). After the loading procedure, culture medium is continuously flushed thro...

  17. Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

    NARCIS (Netherlands)

    Huberts, Daphne H. E. W.; Janssens, Georges E.; Lee, Sung Sik; Vizcarra, Ima Avalos; Heinemann, Matthias

    We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath

  18. Characterization of dependencies between growth and division in budding yeast.

    Science.gov (United States)

    Mayhew, Michael B; Iversen, Edwin S; Hartemink, Alexander J

    2017-02-01

    Cell growth and division are processes vital to the proliferation and development of life. Coordination between these two processes has been recognized for decades in a variety of organisms. In the budding yeast Saccharomyces cerevisiae , this coordination or 'size control' appears as an inverse correlation between cell size and the rate of cell-cycle progression, routinely observed in G 1 prior to cell division commitment. Beyond this point, cells are presumed to complete S/G 2 /M at similar rates and in a size-independent manner. As such, studies of dependence between growth and division have focused on G 1 Moreover, in unicellular organisms, coordination between growth and division has commonly been analysed within the cycle of a single cell without accounting for correlations in growth and division characteristics between cycles of related cells. In a comprehensive analysis of three published time-lapse microscopy datasets, we analyse both intra- and inter-cycle dependencies between growth and division, revisiting assumptions about the coordination between these two processes. Interestingly, we find evidence (i) that S/G 2 /M durations are systematically longer in daughters than in mothers, (ii) of dependencies between S/G 2 /M and size at budding that echo the classical G 1 dependencies, and (iii) in contrast with recent bacterial studies, of negative dependencies between size at birth and size accumulated during the cell cycle. In addition, we develop a novel hierarchical model to uncover inter-cycle dependencies, and we find evidence for such dependencies in cells growing in sugar-poor environments. Our analysis highlights the need for experimentalists and modellers to account for new sources of cell-to-cell variation in growth and division, and our model provides a formal statistical framework for the continued study of dependencies between biological processes. © 2017 The Author(s).

  19. Implication of Ca2+ in the Regulation of Replicative Life Span of Budding Yeast*

    OpenAIRE

    Tsubakiyama, Ryohei; Mizunuma, Masaki; Gengyo, Anri; Yamamoto, Josuke; Kume, Kazunori; Miyakawa, Tokichi; Hirata, Dai

    2011-01-01

    In eukaryotic cells, Ca2+-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca2+/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca2+ sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, ...

  20. Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

    NARCIS (Netherlands)

    Lee, Sung Sik; Avalos Vizcarra, Ima; Huberts, Daphne H E W; Lee, Luke P; Heinemann, Matthias

    2012-01-01

    Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field

  1. Spatial organization of the budding yeast genome in the cell nucleus and identification of specific chromatin interactions from multi-chromosome constrained chromatin model.

    Science.gov (United States)

    Gürsoy, Gamze; Xu, Yun; Liang, Jie

    2017-07-01

    Nuclear landmarks and biochemical factors play important roles in the organization of the yeast genome. The interaction pattern of budding yeast as measured from genome-wide 3C studies are largely recapitulated by model polymer genomes subject to landmark constraints. However, the origin of inter-chromosomal interactions, specific roles of individual landmarks, and the roles of biochemical factors in yeast genome organization remain unclear. Here we describe a multi-chromosome constrained self-avoiding chromatin model (mC-SAC) to gain understanding of the budding yeast genome organization. With significantly improved sampling of genome structures, both intra- and inter-chromosomal interaction patterns from genome-wide 3C studies are accurately captured in our model at higher resolution than previous studies. We show that nuclear confinement is a key determinant of the intra-chromosomal interactions, and centromere tethering is responsible for the inter-chromosomal interactions. In addition, important genomic elements such as fragile sites and tRNA genes are found to be clustered spatially, largely due to centromere tethering. We uncovered previously unknown interactions that were not captured by genome-wide 3C studies, which are found to be enriched with tRNA genes, RNAPIII and TFIIS binding. Moreover, we identified specific high-frequency genome-wide 3C interactions that are unaccounted for by polymer effects under landmark constraints. These interactions are enriched with important genes and likely play biological roles.

  2. The Genetic Requirements for Pentose Fermentation in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Karin Mittelman

    2017-06-01

    Full Text Available Cells grow on a wide range of carbon sources by regulating substrate flow through the metabolic network. Incoming sugar, for example, can be fermented or respired, depending on the carbon identity, cell type, or growth conditions. Despite this genetically-encoded flexibility of carbon metabolism, attempts to exogenously manipulate central carbon flux by rational design have proven difficult, suggesting a robust network structure. To examine this robustness, we characterized the ethanol yield of 411 regulatory and metabolic mutants in budding yeast. The mutants showed little variation in ethanol productivity when grown on glucose or galactose, yet diversity was revealed during growth on xylulose, a rare pentose not widely available in nature. While producing ethanol at high yield, cells grown on xylulose produced ethanol at high yields, yet induced expression of respiratory genes, and were dependent on them. Analysis of mutants that affected ethanol productivity suggested that xylulose fermentation results from metabolic overflow, whereby the flux through glycolysis is higher than the maximal flux that can enter respiration. We suggest that this overflow results from a suboptimal regulatory adjustment of the cells to this unfamiliar carbon source.

  3. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contr...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  4. The linear interplay of intrinsic and extrinsic noises ensures a high accuracy of cell fate selection in budding yeast

    Science.gov (United States)

    Li, Yongkai; Yi, Ming; Zou, Xiufen

    2014-01-01

    To gain insights into the mechanisms of cell fate decision in a noisy environment, the effects of intrinsic and extrinsic noises on cell fate are explored at the single cell level. Specifically, we theoretically define the impulse of Cln1/2 as an indication of cell fates. The strong dependence between the impulse of Cln1/2 and cell fates is exhibited. Based on the simulation results, we illustrate that increasing intrinsic fluctuations causes the parallel shift of the separation ratio of Whi5P but that increasing extrinsic fluctuations leads to the mixture of different cell fates. Our quantitative study also suggests that the strengths of intrinsic and extrinsic noises around an approximate linear model can ensure a high accuracy of cell fate selection. Furthermore, this study demonstrates that the selection of cell fates is an entropy-decreasing process. In addition, we reveal that cell fates are significantly correlated with the range of entropy decreases. PMID:25042292

  5. Taste bud cells and nerves

    OpenAIRE

    武田,正子/内田,暢彦/鈴木,裕子; タケダ,マサコ/ウチダ,ノブヒコ/スズキ,ユウコ; TAKEDA,Masako/UCHIDA,Nobuhiko/SUZUKI,Yuko

    2002-01-01

    Sectioning of glossopharyngeal nerves which innervate the taste buds in the circumvallate papillae caused apoptosis of taste buds, the numbers decreasing and the taste buds disappearing after 11 days. This indicates that gustatory nerves may release a trophic substance that induces and maintains taste buds. Taste bud cells contain neurotrophins, NCAM, NSE, PGP9.5, and NeuroD which are specific markers of neurons. The BDNF and GDNF of neurotrophins, and Trk B and GFRαl of their receptors were ...

  6. A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle

    NARCIS (Netherlands)

    Linke, C.; Chasapi, A.; González-Novo, A.; Al Sawad, I.; Tognetti, S.; Klipp, E.; Loog, M.; Krobitsch, S.; Posas, F.; Xenarios, I.; Barberis, M.

    2017-01-01

    Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates

  7. Naumovozyma castellii: an alternative model for budding yeast molecular biology.

    Science.gov (United States)

    Karademir Andersson, Ahu; Cohn, Marita

    2017-03-01

    Naumovozyma castellii (Saccharomyces castellii) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Owing to the suitable phylogenetic distance to S. cerevisiae, the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii, focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  8. A nutrient dependant switch explains mutually exclusive existence of meiosis and mitosis initiation in budding yeast.

    Science.gov (United States)

    Wannige, C T; Kulasiri, D; Samarasinghe, S

    2014-01-21

    Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways. © 2013 Elsevier Ltd. All rights reserved.

  9. The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

    Science.gov (United States)

    Meseroll, Rebecca A; Cohen-Fix, Orna

    2016-11-01

    In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  10. Implication of Ca2+ in the regulation of replicative life span of budding yeast.

    Science.gov (United States)

    Tsubakiyama, Ryohei; Mizunuma, Masaki; Gengyo, Anri; Yamamoto, Josuke; Kume, Kazunori; Miyakawa, Tokichi; Hirata, Dai

    2011-08-19

    In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.

  11. Identification of SUMO conjugation sites in the budding yeast proteome

    Directory of Open Access Journals (Sweden)

    Miguel Esteras

    2017-10-01

    Full Text Available Post-translational modification by the small ubiquitin-like modifier (SUMO is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

  12. Evolution of multinucleated Ashbya gossypii hyphae from a budding yeast-like ancestor.

    Science.gov (United States)

    Schmitz, Hans-Peter; Philippsen, Peter

    2011-06-01

    In the filamentous ascomycete Ashbya gossypii polarity establishment at sites of germ tube and lateral branch emergence depends on homologues of Saccharomyces cerevisiae factors controlling bud site selection and bud emergence. Maintenance of polar growth involves homologues of well-known polarity factors of budding yeast. To achieve the much higher rates of sustained polar surface expansion of hyphae compared to mainly non-polarly growing yeast buds five important alterations had to evolve. Permanent presence of the polarity machinery at a confined area in the rapidly expanding hyphal tip, increased cytoplasmic space with a much enlarged ER surface for generating secretory vesicles, efficient directed transport of secretory vesicles to and accumulation at the tip, increased capacity of the exocytosis system to process these vesicles, and an efficient endocytosis system for membrane and polarity factor recycling adjacent to the zone of exocytosis. Morphological, cell biological, and molecular aspects of this evolution are discussed based on experiments performed within the past 10 y. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  13. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

    Science.gov (United States)

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

    2012-01-01

    Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

  14. Focal accumulation of preribosomes outside the nucleolus during metaphase-anaphase in budding yeast.

    Science.gov (United States)

    Moriggi, Giulia; Gaspar, Sonia G; Nieto, Blanca; Bustelo, Xosé R; Dosil, Mercedes

    2017-09-01

    Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. © 2017 Moriggi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Focal accumulation of preribosomes outside the nucleolus during metaphase–anaphase in budding yeast

    Science.gov (United States)

    Moriggi, Giulia; Gaspar, Sonia G.; Nieto, Blanca; Bustelo, Xosé R.

    2017-01-01

    Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. PMID:28588079

  16. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  17. 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.

  18. The cellular robustness by genetic redundancy in budding yeast.

    Directory of Open Access Journals (Sweden)

    Jingjing Li

    2010-11-01

    Full Text Available The frequent dispensability of duplicated genes in budding yeast is heralded as a hallmark of genetic robustness contributed by genetic redundancy. However, theoretical predictions suggest such backup by redundancy is evolutionarily unstable, and the extent of genetic robustness contributed from redundancy remains controversial. It is anticipated that, to achieve mutual buffering, the duplicated paralogs must at least share some functional overlap. However, counter-intuitively, several recent studies reported little functional redundancy between these buffering duplicates. The large yeast genetic interactions released recently allowed us to address these issues on a genome-wide scale. We herein characterized the synthetic genetic interactions for ∼500 pairs of yeast duplicated genes originated from either whole-genome duplication (WGD or small-scale duplication (SSD events. We established that functional redundancy between duplicates is a pre-requisite and thus is highly predictive of their backup capacity. This observation was particularly pronounced with the use of a newly introduced metric in scoring functional overlap between paralogs on the basis of gene ontology annotations. Even though mutual buffering was observed to be prevalent among duplicated genes, we showed that the observed backup capacity is largely an evolutionarily transient state. The loss of backup capacity generally follows a neutral mode, with the buffering strength decreasing in proportion to divergence time, and the vast majority of the paralogs have already lost their backup capacity. These observations validated previous theoretic predictions about instability of genetic redundancy. However, departing from the general neutral mode, intriguingly, our analysis revealed the presence of natural selection in stabilizing functional overlap between SSD pairs. These selected pairs, both WGD and SSD, tend to have decelerated functional evolution, have higher propensities of co

  19. Quantitative analysis of taste bud cell numbers in fungiform and soft palate taste buds of mice.

    Science.gov (United States)

    Ohtubo, Yoshitaka; Yoshii, Kiyonori

    2011-01-07

    Mammalian taste bud cells (TBCs) consist of several cell types equipped with different taste receptor molecules, and hence the ratio of cell types in a taste bud constitutes the taste responses of the taste bud. Here we show that the population of immunohistochemically identified cell types per taste bud is proportional to the number of total TBCs in the taste bud or the area of the taste bud in fungiform papillae, and that the proportions differ among cell types. This result is applicable to soft palate taste buds. However, the density of almost all cell types, the population of cell types divided by the area of the respective taste buds, is significantly higher in soft palates. These results suggest that the turnover of TBCs is regulated to keep the ratio of each cell type constant, and that taste responsiveness is different between fungiform and soft palate taste buds. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Measurement of the volume growth rate of single budding yeast with the MOSFET-based microfluidic Coulter counter.

    Science.gov (United States)

    Sun, Jiashu; Stowers, Chris C; Boczko, Erik M; Li, Deyu

    2010-11-07

    We report on measurements of the volume growth rate of ten individual budding yeast cells using a recently developed MOSFET-based microfluidic Coulter counter. The MOSFET-based microfluidic Coulter counter is very sensitive, provides signals that are immune from the baseline drift, and can work with cell culture media of complex composition. These desirable features allow us to directly measure the volume growth rate of single cells of Saccharomyces cerevisiae LYH3865 strain budding yeast in YNB culture media over a whole cell cycle. Results indicate that all budding yeast follow a sigmoid volume growth profile with reduced growth rates at the initial stage before the bud emerges and the final stage after the daughter gets mature. Analysis of the data indicates that even though all piecewise linear, Gomperitz, and Hill's function models can fit the global growth profile equally well, the data strongly support local exponential growth phenomenon. Accurate volume growth measurements are important for applications in systems biology where quantitative parameters are required for modeling and simulation.

  1. 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

  2. The final cut: cell polarity meets cytokinesis at the bud neck in S. cerevisiae.

    Science.gov (United States)

    Juanes, Maria Angeles; Piatti, Simonetta

    2016-08-01

    Cell division is a fundamental but complex process that gives rise to two daughter cells. It includes an ordered set of events, altogether called "the cell cycle", that culminate with cytokinesis, the final stage of mitosis leading to the physical separation of the two daughter cells. Symmetric cell division equally partitions cellular components between the two daughter cells, which are therefore identical to one another and often share the same fate. In many cases, however, cell division is asymmetrical and generates two daughter cells that differ in specific protein inheritance, cell size, or developmental potential. The budding yeast Saccharomyces cerevisiae has proven to be an excellent system to investigate the molecular mechanisms governing asymmetric cell division and cytokinesis. Budding yeast is highly polarized during the cell cycle and divides asymmetrically, producing two cells with distinct sizes and fates. Many components of the machinery establishing cell polarization during budding are relocalized to the division site (i.e., the bud neck) for cytokinesis. In this review we recapitulate how budding yeast cells undergo polarized processes at the bud neck for cell division.

  3. The Budding Yeast “Saccharomyces cerevisiae” as a Drug Discovery Tool to Identify Plant-Derived Natural Products with Anti-Proliferative Properties

    Directory of Open Access Journals (Sweden)

    Bouchra Qaddouri

    2011-01-01

    Full Text Available The budding yeast Saccharomyces cerevisiae is a valuable system to study cell-cycle regulation, which is defective in cancer cells. Due to the highly conserved nature of the cell-cycle machinery between yeast and humans, yeast studies are directly relevant to anticancer-drug discovery. The budding yeast is also an excellent model system for identifying and studying antifungal compounds because of the functional conservation of fungal genes. Moreover, yeast studies have also contributed greatly to our understanding of the biological targets and modes of action of bioactive compounds. Understanding the mechanism of action of clinically relevant compounds is essential for the design of improved second-generation molecules. Here we describe our methodology for screening a library of plant-derived natural products in yeast in order to identify and characterize new compounds with anti-proliferative properties.

  4. An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Du, Zhiqiang; Valtierra, Stephanie; Li, Liming

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI(+)]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI(+)] and [PIN(+)] ([RNQ(+)]) (Genetics, Vol. 197, 685-700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI(+)] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ(+)] or [SWI(+)]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI(+)] prion stabilizes. Our finding provides strong evidence supporting the "cross-seeding" model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.

  5. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Richard C Silva

    Full Text Available The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

  6. Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

    OpenAIRE

    Lo, Hsiao-Chi; Wan, Lihong; Rosebrock, Adam; Futcher, Bruce; Hollingsworth, Nancy M.

    2008-01-01

    In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induct...

  7. Mek1/Mre4 is a master regulator of meiotic recombination in budding yeast

    Directory of Open Access Journals (Sweden)

    Nancy M. Hollingsworth

    2016-02-01

    Full Text Available Sexually reproducing organisms create gametes with half the somatic cell chromosome number so that fusion of gametes at fertilization does not change the ploidy of the cell. This reduction in chromosome number occurs by the specialized cell division of meiosis in which two rounds of chromosome segregation follow a single round of chromosome duplication. Meiotic crossovers formed between the non-sister chromatids of homologous chromosomes, combined with sister chromatid cohesion, physically connect homologs, thereby allowing proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs whose repair is highly regulated such that (1 there is a bias for recombination with homologs rather than sister chromatids, (2 crossovers are distributed throughout the genome by a process called interference, (3 crossover homeostasis regulates the balance between crossover and non-crossover repair to maintain a critical number of crossovers and (4 each pair of homologs receives at least one crossover. It was previously known that the imposition of interhomolog bias in budding yeast requires meiosis-specific modifications to the DNA damage response and the local activation of the meiosis-specific Mek1/Mre4 (hereafter Mek1 kinase at DSBs. However, because inactivation of Mek1 results in intersister, rather than interhomolog DSB repair, whether Mek1 had a role in interhomolog pathway choice was unknown. A recent study by Chen et al. (2015 reveals that Mek1 indirectly regulates the crossover/non-crossover decision between homologs as well as genetic interference. It does this by enabling phosphorylation of Zip1, the meiosis-specific transverse filament protein of the synaptonemal complex (SC, by the conserved cell cycle kinase, Cdc7-Dbf4 (DDK. These results suggest that Mek1 is a “master regulator” of meiotic recombination in budding yeast.

  8. The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

    2016-10-01

    Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Matuo, Youichirou; Nishijima, Shigehiro; Hase, Yoshihiro; Sakamoto, Ayako; Tanaka, Atsushi; Shimizu, Kikuo

    2006-01-01

    To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of γ-ray irradiation in the budding yeast Saccharomyces cerevisiae. The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by γ-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C → T:A, and all the transitions were G:C → A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5'-AC(A/T)-3' sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by γ-ray irradiation were located uniformly throughout the gene

  10. Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matuo, Youichirou [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Nishijima, Shigehiro [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Hase, Yoshihiro [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Sakamoto, Ayako [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Tanaka, Atsushi [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Shimizu, Kikuo [Radioisotope Research Center, Osaka University, Yamada-oka 2-4, Suita, Osaka 565-0871 (Japan)]. E-mail: shimizu@rirc.osaka-u.ac.jp

    2006-12-01

    To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of {gamma}-ray irradiation in the budding yeast Saccharomyces cerevisiae. The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by {gamma}-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C {sup {yields}} T:A, and all the transitions were G:C {sup {yields}} A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5'-AC(A/T)-3' sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by {gamma}-ray irradiation were located uniformly throughout the gene.

  11. Experimental testing of a new integrated model of the budding yeast Start transition.

    Science.gov (United States)

    Adames, Neil R; Schuck, P Logan; Chen, Katherine C; Murali, T M; Tyson, John J; Peccoud, Jean

    2015-11-05

    The cell cycle is composed of bistable molecular switches that govern the transitions between gap phases (G1 and G2) and the phases in which DNA is replicated (S) and partitioned between daughter cells (M). Many molecular details of the budding yeast G1-S transition (Start) have been elucidated in recent years, especially with regard to its switch-like behavior due to positive feedback mechanisms. These results led us to reevaluate and expand a previous mathematical model of the yeast cell cycle. The new model incorporates Whi3 inhibition of Cln3 activity, Whi5 inhibition of SBF and MBF transcription factors, and feedback inhibition of Whi5 by G1-S cyclins. We tested the accuracy of the model by simulating various mutants not described in the literature. We then constructed these novel mutant strains and compared their observed phenotypes to the model's simulations. The experimental results reported here led to further changes of the model, which will be fully described in a later article. Our study demonstrates the advantages of combining model design, simulation, and testing in a coordinated effort to better understand a complex biological network. © 2015 Adames et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

    Science.gov (United States)

    Minakawa, Tomoko; Ueda, Keiichi; Tanaka, Miyuu; Tanaka, Natsuki; Kuwamura, Mitsuru; Izawa, Takeshi; Konno, Toshihiro; Yamate, Jyoji; Itano, Eiko Nakagawa; Sano, Ayako; Wada, Shinpei

    2016-08-01

    Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

  13. Effects of intense magnetic fields on sedimentation pattern and gene expression profile in budding yeast

    Science.gov (United States)

    Ikehata, Masateru; Iwasaka, Masakazu; Miyakoshi, Junji; Ueno, Shoogo; Koana, Takao

    2003-05-01

    Effects of magnetic fields (MFs) on biological systems are usually investigated using biological indices such as gene expression profiles. However, to precisely evaluate the biological effects of MF, the effects of intense MFs on systematic material transport processes including experimental environment must be seriously taken into consideration. In this study, a culture of the budding yeast, Saccharomyces cerevisiae, was used as a model for an in vitro biological test system. After exposure to 5 T static vertical MF, we found a difference in the sedimentation pattern of cells depending on the location of the dish in the magnet bore. Sedimented cells were localized in the center of the dish when they were placed in the lower part of the magnet bore while the sedimentation of the cells was uniform in dishes placed in the upper part of the bore because of the diamagnetic force. Genome wide gene expression profile of the yeast cells after exposure to 5 T static MF for 2 h suggested that the MF did not affect the expression level of any gene in yeast cells although the sedimentation pattern was altered. In addition, exposure to 10 T for 1 h and 5 T for 24 h also did not affect the gene expression. On the other hand, a slight change in expressions of several genes which are related to respiration was observed by exposure to a 14 T static MF for 24 h. The necessity of estimating the indirect effects of MFs on a study of its biological effect of MF in vitro will be discussed.

  14. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    Science.gov (United States)

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  15. Simultaneous production of buds on mother and daughter cells of Saccharomyces cerevisiae in the presence of hydroxyurea

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, K; Michio, I

    1979-12-01

    Individual budding yeast cells, Saccharomyces cerevisiae, enclosed in small culture chambers were observed through two budding cycles to examine their behavior during growth and division. In the nutrient medium (YHG medium), the duration of the budding cycles was 77 minutes for mother cells and 90 minutes for daughter cells. Continuous exposure of cells to 16 or 32 mm hydroxyurea extended the duration of the cycles and increased the volume of cells, resulting in the formation of abnormally large and equal-sized mother-daughter pairs. Each cell of these pairs subsequently produced buds simultaneously. Stained cell nuclei showed simultaneous nuclear division. This synchronous budding on mother-daughter pairs was repeated in the next budding cycle. The coordination of growth with division is discussed in relation to these results.

  16. A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast.

    Science.gov (United States)

    Clarke, Jesse; Dephoure, Noah; Horecka, Ira; Gygi, Steven; Kellogg, Douglas

    2017-10-01

    In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth--dependent signals control cell growth and the cell cycle. © 2017 Clarke et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Cell to cell signalling during vertebrate limb bud development

    NARCIS (Netherlands)

    Panman, Lia

    2004-01-01

    Communication between cells is essential during embryonic development. The vertebrate limb bud provides us a model to study signalling interactions between cells during patterning of embryonic tissues and organogenesis. In chapter 1 I give an introduction about limb bud development that is focussed

  19. Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast.

    Science.gov (United States)

    Huberts, Daphne H E W; Sik Lee, Sung; Gonzáles, Javier; Janssens, Georges E; Vizcarra, Ima Avalos; Heinemann, Matthias

    2013-06-01

    This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50-100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ~7 h.

  20. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available east_seq_qual.zip File URL: ftp://ftp.biosciencedbc.jp/archive/yeast_cdna/LATEST/...yeast_seq_qual.zip File size: 59.9MB Simple search URL http://togodb.biosciencedbc.jp/togodb/view/budding_yeast_cdna

  1. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    International Nuclear Information System (INIS)

    Choi, Joon-Seok; Lee, Cheol-Koo

    2013-01-01

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA

  2. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

    2013-09-13

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

  3. Whole transcriptome profiling of taste bud cells.

    Science.gov (United States)

    Sukumaran, Sunil K; Lewandowski, Brian C; Qin, Yumei; Kotha, Ramana; Bachmanov, Alexander A; Margolskee, Robert F

    2017-08-08

    Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

  4. Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

    NARCIS (Netherlands)

    Novarina, Daniele; Mavrova, Sara N.; Janssens, Georges E.; Rempel, Irina L.; Veenhoff, Liesbeth M.; Chang, Michael

    The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast

  5. Visual screening for localized RNAs in yeast revealed novel RNAs at the bud-tip

    International Nuclear Information System (INIS)

    Andoh, Tomoko; Oshiro, Yukiko; Hayashi, Sachiko; Takeo, Hideki; Tani, Tokio

    2006-01-01

    Several RNAs, including rRNAs, snRNAs, snoRNAs, and some mRNAs, are known to be localized at specific sites in a cell. Although methods have been established to visualize RNAs in a living cell, no large-scale visual screening of localized RNAs has been performed. In this study, we constructed a genomic library in which random genomic fragments were inserted downstream of U1A-tag sequences under a GAL1 promoter. In a living yeast cell, transcribed U1A-tagged RNAs were visualized by U1A-GFP that binds the RNA sequence of the U1A-tag. In this screening, many RNAs showed nuclear signals. Since the nuclear signals of some RNAs were not seen when the U1A-tag was connected to the 3' ends of the RNAs, it is suggested that their nuclear signals correspond to nascent transcripts on GAL1 promoter plasmids. Using this screening method, we successfully identified two novel localized mRNAs, CSR2 and DAL81, which showed bud-tip localization

  6. SAGA complex and Gcn5 are necessary for respiration in budding yeast.

    Science.gov (United States)

    Canzonetta, Claudia; Leo, Manuela; Guarino, Salvatore Rocco; Montanari, Arianna; Francisci, Silvia; Filetici, Patrizia

    2016-12-01

    In budding yeast, growth through fermentation and/or respiration is dependent on the type of carbon source present in the medium. SAGA complex is the main acetylation complex and is required, together with Rtg factors, for nucleus-mitochondria communication and transcriptional activation of specific nuclear genes. Even though acetylation is necessary for mitochondria activity and respiratory pathways the direct role of histone acetyltransferases and SAGA complex has never been investigated directly. In this study we demonstrate, for the first time, that Gcn5 and SAGA are needed for respiratory metabolism and oxygen consumption. According to a central role for acetylation in respiration we find that the Gcn5 inhibitor CPTH2 had higher efficacy on cells grown in glycerol containing media. We also demonstrated that the opposing activities of Gcn5 and Hda1 modify selectively H3-AcK18 and are essential for respiration. Taken together our results suggest a novel paradigm coupling acetyltransferase activity to respiratory metabolism. Correspondingly we propose the selective utilization of KAT inhibitor CPTH2, combined to the modulation of the respiratory metabolism of the cell, as a promising novel tool of intervention in cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Anethole potentiates dodecanol's fungicidal activity by reducing PDR5 expression in budding yeast.

    Science.gov (United States)

    Fujita, Ken-Ichi; Ishikura, Takayuki; Jono, Yui; Yamaguchi, Yoshihiro; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2017-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the mechanism underlying this synergistic effect of anethole has not been characterized. We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux. Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5, in addition to a slight increase in PDR11, PDR12, and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δpdr5. In addition, Δpdr3 and Δlge1, lack transcription factors of PDR5 and PDR3, were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans. These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Positive Feedback of NDT80 Expression Ensures Irreversible Meiotic Commitment in Budding Yeast

    Science.gov (United States)

    Tsuchiya, Dai; Yang, Yang; Lacefield, Soni

    2014-01-01

    In budding yeast, meiotic commitment is the irreversible continuation of the developmental path of meiosis. After reaching meiotic commitment, cells finish meiosis and gametogenesis, even in the absence of the meiosis-inducing signal. In contrast, if the meiosis-inducing signal is removed and the mitosis-inducing signal is provided prior to reaching meiotic commitment, cells exit meiosis and return to mitosis. Previous work has shown that cells commit to meiosis after prophase I but before entering the meiotic divisions. Since the Ndt80 transcription factor induces expression of middle meiosis genes necessary for the meiotic divisions, we examined the role of the NDT80 transcriptional network in meiotic commitment. Using a microfluidic approach to analyze single cells, we found that cells commit to meiosis in prometaphase I, after the induction of the Ndt80-dependent genes. Our results showed that high-level expression of NDT80 is important for the timing and irreversibility of meiotic commitment. A modest reduction in NDT80 levels delayed meiotic commitment based on meiotic stages, although the timing of each meiotic stage was similar to that of wildtype cells. A further reduction of NDT80 resulted in the surprising finding of inappropriately uncommitted cells: withdrawal of the meiosis-inducing signal and addition of the mitosis-inducing signal to cells at stages beyond metaphase I caused return to mitosis, leading to multi-nucleate cells. Since Ndt80 enhances its own transcription through positive feedback, we tested whether positive feedback ensured the irreversibility of meiotic commitment. Ablating positive feedback in NDT80 expression resulted in a complete loss of meiotic commitment. These findings suggest that irreversibility of meiotic commitment is a consequence of the NDT80 transcriptional positive feedback loop, which provides the high-level of Ndt80 required for the developmental switch of meiotic commitment. These results also illustrate the

  9. Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

    Science.gov (United States)

    Chan, Kin

    2018-01-01

    Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

  10. Considerations for creating and annotating the budding yeast Genome Map at SGD: a progress report.

    Science.gov (United States)

    Chan, Esther T; Cherry, J Michael

    2012-01-01

    The Saccharomyces Genome Database (SGD) is compiling and annotating a comprehensive catalogue of functional sequence elements identified in the budding yeast genome. Recent advances in deep sequencing technologies have enabled for example, global analyses of transcription profiling and assembly of maps of transcription factor occupancy and higher order chromatin organization, at nucleotide level resolution. With this growing influx of published genome-scale data, come new challenges for their storage, display, analysis and integration. Here, we describe SGD's progress in the creation of a consolidated resource for genome sequence elements in the budding yeast, the considerations taken in its design and the lessons learned thus far. The data within this collection can be accessed at http://browse.yeastgenome.org and downloaded from http://downloads.yeastgenome.org. DATABASE URL: http://www.yeastgenome.org.

  11. The step-wise pathway of septin hetero-octamer assembly in budding yeast.

    Science.gov (United States)

    Weems, Andrew; McMurray, Michael

    2017-05-25

    Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3-Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11-Cdc12-Cdc3 or Shs1-Cdc12-Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.

  12. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    International Nuclear Information System (INIS)

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G.; Liskay, R.M.; Heffron, F.

    1991-01-01

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH 2 -terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily

  13. Genetic variation in adaptability and pleiotropy in budding yeast.

    Science.gov (United States)

    Jerison, Elizabeth R; Kryazhimskiy, Sergey; Mitchell, James Kameron; Bloom, Joshua S; Kruglyak, Leonid; Desai, Michael M

    2017-08-17

    Evolution can favor organisms that are more adaptable, provided that genetic variation in adaptability exists. Here, we quantify this variation among 230 offspring of a cross between diverged yeast strains. We measure the adaptability of each offspring genotype, defined as its average rate of adaptation in a specific environmental condition, and analyze the heritability, predictability, and genetic basis of this trait. We find that initial genotype strongly affects adaptability and can alter the genetic basis of future evolution. Initial genotype also affects the pleiotropic consequences of adaptation for fitness in a different environment. This genetic variation in adaptability and pleiotropy is largely determined by initial fitness, according to a rule of declining adaptability with increasing initial fitness, but several individual QTLs also have a significant idiosyncratic role. Our results demonstrate that both adaptability and pleiotropy are complex traits, with extensive heritable differences arising from naturally occurring variation.

  14. 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.

  15. A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

    Directory of Open Access Journals (Sweden)

    Krystina L Ho

    2015-03-01

    Full Text Available Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition - one to remove cohesin and the second to target Ty1-IN to chromatin.

  16. Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

    Science.gov (United States)

    Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

    2018-05-17

    Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division

    NARCIS (Netherlands)

    Kumar, Sanjeev; de Boer, Rinse; van der Klei, Ida J

    2018-01-01

    Here we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and younger peroxisomes. During budding the peroxisome

  18. Imaging of DNA Ultrafine Bridges in Budding Yeast

    DEFF Research Database (Denmark)

    Quevedo Rodriguez, Oliver; Lisby, Michael

    2018-01-01

    DNA ultrafine bridges (UFBs) are a type of chromatin-free DNA bridges that connect sister chromatids in anaphase and pose a threat to genome stability. However, little is known about the origin of these structures, and how they are sensed and resolved by the cell. In this chapter, we review tools...... and methods for studying UFBs by fluorescence microscopy including chemical and genetic approaches to induce UFBs in the model organism Saccharomyces cerevisiae....

  19. Imaging of DNA Ultrafine Bridges in Budding Yeast.

    Science.gov (United States)

    Quevedo, Oliver; Lisby, Michael

    2018-01-01

    DNA ultrafine bridges (UFBs) are a type of chromatin-free DNA bridges that connect sister chromatids in anaphase and pose a threat to genome stability. However, little is known about the origin of these structures, and how they are sensed and resolved by the cell. In this chapter, we review tools and methods for studying UFBs by fluorescence microscopy including chemical and genetic approaches to induce UFBs in the model organism Saccharomyces cerevisiae.

  20. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects.

    Directory of Open Access Journals (Sweden)

    Erica M Hildebrand

    2016-03-01

    Full Text Available The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4 is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4 for degradation. To identify additional mechanisms that prevent CENP-A(Cse4 misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4 in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4 is enriched at promoters that contain histone H2A.Z(Htz1 nucleosomes, but that H2A.Z(Htz1 is not required for CENP-A(Cse4 mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1 from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4. Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4. The down-regulated genes are enriched for CENP-A(Cse4 mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.

  1. License - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project License to Use This Database Last updated : 2010/02/15 You may use this databas...ional License described below. The Standard License specifies the license terms regarding the use of this database... and the requirements you must follow in using this database. The Additiona...n the Standard License. Standard License The Standard License for this database is the license specified in ...the Creative Commons Attribution-Share Alike 2.1 Japan . If you use data from this database

  2. Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

    2018-02-20

    The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

  3. Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Rakesh Kumar Singh

    Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

  4. Inflammation activates the interferon signaling pathways in taste bud cells.

    Science.gov (United States)

    Wang, Hong; Zhou, Minliang; Brand, Joseph; Huang, Liquan

    2007-10-03

    Patients with viral and bacterial infections or other inflammatory illnesses often experience taste dysfunctions. The agents responsible for these taste disorders are thought to be related to infection-induced inflammation, but the mechanisms are not known. As a first step in characterizing the possible role of inflammation in taste disorders, we report here evidence for the presence of interferon (IFN)-mediated signaling pathways in taste bud cells. IFN receptors, particularly the IFN-gamma receptor IFNGR1, are coexpressed with the taste cell-type markers neuronal cell adhesion molecule and alpha-gustducin, suggesting that both the taste receptor cells and synapse-forming cells in the taste bud can be stimulated by IFN. Incubation of taste bud-containing lingual epithelia with recombinant IFN-alpha and IFN-gamma triggered the IFN-mediated signaling cascades, resulting in the phosphorylation of the downstream STAT1 (signal transducer and activator of transcription protein 1) transcription factor. Intraperitoneal injection of lipopolysaccharide or polyinosinic:polycytidylic acid into mice, mimicking bacterial and viral infections, respectively, altered gene expression patterns in taste bud cells. Furthermore, the systemic administration of either IFN-alpha or IFN-gamma significantly increased the number of taste bud cells undergoing programmed cell death. These findings suggest that bacterial and viral infection-induced IFNs can act directly on taste bud cells, affecting their cellular function in taste transduction, and that IFN-induced apoptosis in taste buds may cause abnormal cell turnover and skew the representation of different taste bud cell types, leading to the development of taste disorders. To our knowledge, this is the first study providing direct evidence that inflammation can affect taste buds through cytokine signaling pathways.

  5. Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

    Directory of Open Access Journals (Sweden)

    Karen Voelkel-Meiman

    2015-06-01

    Full Text Available Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3 facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC. SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast

  6. Functional cell types in taste buds have distinct longevities.

    Directory of Open Access Journals (Sweden)

    Isabel Perea-Martinez

    Full Text Available Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

  7. Functional cell types in taste buds have distinct longevities.

    Science.gov (United States)

    Perea-Martinez, Isabel; Nagai, Takatoshi; Chaudhari, Nirupa

    2013-01-01

    Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

  8. Cdc7-Dbf4 regulates NDT80 transcription as well as reductional segregation during budding yeast meiosis.

    Science.gov (United States)

    Lo, Hsiao-Chi; Wan, Lihong; Rosebrock, Adam; Futcher, Bruce; Hollingsworth, Nancy M

    2008-11-01

    In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis.

  9. Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

    Science.gov (United States)

    Lo, Hsiao-Chi; Wan, Lihong; Rosebrock, Adam; Futcher, Bruce

    2008-01-01

    In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis. PMID:18768747

  10. Taste buds: cells, signals and synapses.

    Science.gov (United States)

    Roper, Stephen D; Chaudhari, Nirupa

    2017-08-01

    The past decade has witnessed a consolidation and refinement of the extraordinary progress made in taste research. This Review describes recent advances in our understanding of taste receptors, taste buds, and the connections between taste buds and sensory afferent fibres. The article discusses new findings regarding the cellular mechanisms for detecting tastes, new data on the transmitters involved in taste processing and new studies that address longstanding arguments about taste coding.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-11-27

    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.

  12. 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

  13. Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Ping Li

    2017-06-01

    Full Text Available Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.

  14. Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

    Science.gov (United States)

    Koch, Bailey A.; Han, Xuemei

    2017-01-01

    Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery. PMID:28609436

  15. FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast

    Science.gov (United States)

    Smukalla, Scott; Caldara, Marina; Pochet, Nathalie; Beauvais, Anne; Guadagnini, Stephanie; Yan, Chen; Vinces, Marcelo D.; Jansen, An; Prevost, Marie Christine; Latgé, Jean-Paul; Fink, Gerald R.; Foster, Kevin R.; Verstrepen, Kevin J.

    2008-01-01

    Summary The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait. PMID:19013280

  16. Lipid raft involvement in yeast cell growth and death

    Energy Technology Data Exchange (ETDEWEB)

    Mollinedo, Faustino, E-mail: fmollin@usal.es [Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas - Universidad de Salamanca, Salamanca (Spain)

    2012-10-10

    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{sup +}, K{sup +}, 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.

  17. 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.

  18. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

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

    Science.gov (United States)

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

    2016-04-01

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

  20. Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Yasuyuki Suda

    2018-01-01

    Full Text Available A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER. They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

  1. Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

    Science.gov (United States)

    Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

    2016-01-01

    The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

  2. Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

    Science.gov (United States)

    Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

    2018-05-04

    The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

  3. The protein expression landscape of mitosis and meiosis in diploid budding yeast.

    Science.gov (United States)

    Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

    2017-03-06

    Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

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

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  6. Role for cER and Mmr1p in anchorage of mitochondria at sites of polarized surface growth in budding yeast.

    Science.gov (United States)

    Swayne, Theresa C; Zhou, Chun; Boldogh, Istvan R; Charalel, Joseph K; McFaline-Figueroa, José Ricardo; Thoms, Sven; Yang, Christine; Leung, Galen; McInnes, Joseph; Erdmann, Ralf; Pon, Liza A

    2011-12-06

    Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Inducible nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers in the cell cycle of the budding yeast Saccharomyces cerevisiae: evidence that inducible NER is confined to the G1 phase of the mitotic cell cycle

    International Nuclear Information System (INIS)

    Scott, A.D.; Waters, R.

    1997-01-01

    We previously reported on an inducible component of nucleotide excision repair in Saccharomyces cerevisiae that is controlled by the RAD16 gene. Here we describe a study of this event at the MAT alpha and HML alpha mating-type loci and on the transcribed (TS) and nontranscribed (NTS) strands of the RAD16 gene. Events were examined at various stages of the mitotic cycle in cells synchronised by centrifugal elutriation. Repair of cyclobutane pyrimidine dimers (CPDs) following a single UV dose does not vary significantly in different stages of the mitotic cell cycle. CPDs are removed more rapidly from the transcriptionally active MAT alpha locus than from the silent HML alpha locus, and the TS of RAD16 is repaired faster than the NTS in all stages of the cycle following a single UV irradiation. Enhanced excision of CPDs at MAT alpha and HML alpha can be induced only in the G1 and early S stages of the cell cycle. Here prior irradiation of cells with 25 J/m 2 enhances the removal of CPDs following a second UV dose of 70 J/m 2 . The level of enhancement of repair does not differ significantly between MAT alpha and HML alpha in G1. Enhanced removal of CPDs is absent when cells receive the inducing dose in late S or G2/M. Repair of CPDs in both strands of RAD16 is similarly enhanced only if cells receive the initial irradiation in G1 and early S. The level of enhanced removal of CPDs is not significantly different in the TS and NTS of RAD16 either in asynchronous cells or in cells preirradiated in G1 and early S. It has been shown by others that UV-induced expression of RAD16 remains at high levels if cells are held in G1 by treatment with alpha factor. Therefore the increase in RAD16 transcript levels in G1 may be responsible for the ability to enhance NER solely in this stage of the cell cycle

  8. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

    Science.gov (United States)

    Li, Ping; Shao, Yize; Jin, Hui

    2015-01-01

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

  9. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

    Science.gov (United States)

    Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

    2018-02-27

    Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

  10. Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III-transcribed genes in budding yeast.

    Science.gov (United States)

    Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

    2016-10-15

    The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. © 2016 Belagal et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  11. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

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    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  12. Inflammation Activates the Interferon Signaling Pathways in Taste Bud Cells

    OpenAIRE

    Wang, Hong; Zhou, Minliang; Brand, Joseph; Huang, Liquan

    2007-01-01

    Patients with viral and bacterial infections or other inflammatory illnesses often experience taste dysfunctions. The agents responsible for these taste disorders are thought to be related to infection-induced inflammation, but the mechanisms are not known. As a first step in characterizing the possible role of inflammation in taste disorders, we report here evidence for the presence of interferon (IFN)-mediated signaling pathways in taste bud cells. IFN receptors, particularly the IFN-γ rece...

  13. 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.

  14. Timely activation of budding yeast APCCdh1 involves degradation of its inhibitor, Acm1, by an unconventional proteolytic mechanism.

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    Michael Melesse

    Full Text Available Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF complex or the anaphase-promoting complex (APC. Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20 in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell

  15. Timely Activation of Budding Yeast APCCdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

    Science.gov (United States)

    Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.

    2014-01-01

    Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles

  16. Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

    Science.gov (United States)

    Takagi, Hiroki; Seta, Yuji; Kataoka, Shinji; Nakatomi, Mitsushiro; Toyono, Takashi; Kawamoto, Tatsuo

    2018-03-10

    The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

  17. Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordial

    Science.gov (United States)

    Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to -50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D-reconstruction, supercooling and freezing patterns by infrared video thermog...

  18. Temporal Expression of a Master Regulator Drives Synchronous Sporulation in Budding Yeast

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    Minghao Chia

    2016-11-01

    Full Text Available Yeast cells enter and undergo gametogenesis relatively asynchronously, making it technically challenging to perform stage-specific genomic and biochemical analyses. Cell-to-cell variation in the expression of the master regulator of entry into sporulation, IME1, has been implicated to be the underlying cause of asynchronous sporulation. Here, we find that timing of IME1 expression is of critical importance for inducing cells to undergo sporulation synchronously. When we force expression of IME1 from an inducible promoter in cells incubated in sporulation medium for 2 hr, the vast majority of cells exhibit synchrony during premeiotic DNA replication and meiotic divisions. Inducing IME1 expression too early or too late affects the synchrony of sporulation. Surprisingly, our approach for synchronous sporulation does not require growth in acetate-containing medium, but can be achieved in cells grown in rich medium until saturation. Our system requires solely IME1, because the expression of the N6-methyladenosine methyltransferase IME4, another key regulator of early sporulation, is controlled by IME1 itself. The approach described here can be combined easily with other stage-specific synchronization methods, and thereby applied to study specific stages of sporulation, or the complete sporulation program.

  19. Temporal Expression of a Master Regulator Drives Synchronous Sporulation in Budding Yeast.

    Science.gov (United States)

    Chia, Minghao; van Werven, Folkert J

    2016-09-07

    Yeast cells enter and undergo gametogenesis relatively asynchronously, making it technically challenging to perform stage-specific genomic and biochemical analyses. Cell-to-cell variation in the expression of the master regulator of entry into sporulation IME1, has been implicated to be the underlying cause of asynchronous sporulation. Here we find that timing of IME1 expression is of critical importance for inducing cells to undergo sporulation synchronously. When we force expression of IME1 from an inducible promoter in cells incubated in sporulation medium for two hours, the vast majority of cells exhibit synchrony during pre-meiotic DNA replication and meiotic divisions. Inducing IME1 expression too early or too late affects the synchrony of sporulation. Surprisingly, our approach for synchronous sporulation does not require growth in acetate containing medium, but can be achieved in cells grown in rich medium until saturation. Our system solely requires IME1 because the expression of the N6-methyladenosine methyltransferase IME4, another key regulator of early sporulation, is controlled by IME1 itself. The approach described here can be easily combined with other stage specific synchronization methods, and thereby applied to study specific stages of sporulation or the complete sporulation program. Copyright © 2016 Author et al.

  20. Identification of She3 as an SCF(Grr1 substrate in budding yeast.

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    Ruiwen Wang

    Full Text Available The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF complex. Acting in concert with the substrate-binding F-box protein Grr1, SCF(Grr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.

  1. Voltage-gated sodium channels in taste bud cells.

    Science.gov (United States)

    Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

    2009-03-12

    Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  2. Sharing the cell's bounty - organelle inheritance in yeast.

    Science.gov (United States)

    Knoblach, Barbara; Rachubinski, Richard A

    2015-02-15

    Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.

  3. Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

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    Alexander Lichius

    Full Text Available A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk, whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.

  4. Spaceflight enhances cell aggregation and random budding in Candida albicans.

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    Aurélie Crabbé

    Full Text Available This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans-induced genes involved in cell aggregation (similar to flocculation, which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p. infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the

  5. Receptosecretory nature of type III cells in the taste bud.

    Science.gov (United States)

    Yoshie, Sumio

    2009-01-01

    Type III cells in taste buds form chemical synapses with intragemmal afferent nerve fibers and are characterized by the presence of membrane-bound vesicles in the cytoplasm. Although the vesicles differ in shape and size among species, they are primarily categorized into small clear (40 nm in diameter) and large dense-cored (90-200 nm) types. As such vesicles tend to be closely juxtaposed to the synaptic membrane of the cells, it is reasonable to consider that the vesicles include transmitter(s) towards the gustatory nerve. In the guinea-pig taste bud, stimulation with various taste substances (sucrose, sodium chloride, quinine hydrochloride, or monosodium L-glutamate) causes ultrastructural alterations of the type III cells. At the synapse, the presynaptic plasma membrane often displays invaginations of 90 nm in a mean diameter towards the cytoplasm, which indicates the dense-cored vesicles opening into the synaptic cleft by means of exocytosis. The vesicles are also exocytosed at the non-synaptic region into the intercellular space. These findings strongly suggest that the transmitters presumably contained in the vesicles are released to conduct the excitement of the type III cells to the nerves and also to exert their paracrine effects upon the surroundings, such as the Ebner's salivary gland, acting as local hormones.

  6. A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

    Science.gov (United States)

    Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

    2014-01-01

    In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009

  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...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... 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...

  8. A switch from a gradient to a threshold mode in the regulation of a transcriptional cascade promotes robust execution of meiosis in budding yeast.

    Directory of Open Access Journals (Sweden)

    Vyacheslav Gurevich

    Full Text Available Tight regulation of developmental pathways is of critical importance to all organisms, and is achieved by a transcriptional cascade ensuring the coordinated expression of sets of genes. We aimed to explore whether a strong signal is required to enter and complete a developmental pathway, by using meiosis in budding yeast as a model. We demonstrate that meiosis in budding yeast is insensitive to drastic changes in the levels of its consecutive positive regulators (Ime1, Ime2, and Ndt80. Entry into DNA replication is not correlated with the time of transcription of the early genes that regulate this event. Entry into nuclear division is directly regulated by the time of transcription of the middle genes, as premature transcription of their activator NDT80, leads to a premature entry into the first meiotic division, and loss of coordination between DNA replication and nuclear division. We demonstrate that Cdk1/Cln3 functions as a negative regulator of Ime2, and that ectopic expression of Cln3 delays entry into nuclear division as well as NDT80 transcription. Because Ime2 functions as a positive regulator for premeiotic DNA replication and NDT80 transcription, as well as a negative regulator of Cdk/Cln, we suggest that a double negative feedback loop between Ime2 and Cdk1/Cln3 promotes a bistable switch from the cell cycle to meiosis. Moreover, our results suggest a regulatory mode switch that ensures robust meiosis as the transcription of the early meiosis-specific genes responds in a graded mode to Ime1 levels, whereas that of the middle and late genes as well as initiation of DNA replication, are regulated in a threshold mode.

  9. Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast.

    Science.gov (United States)

    Johnson, Courtney R; Weems, Andrew D; Brewer, Jennifer M; Thorner, Jeremy; McMurray, Michael A

    2015-04-01

    Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its "G interface") cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un-expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its "unfoldase" activity, indicating a latent "holdase" activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases. © 2015 Johnson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. Shaping meiotic chromosomes with SUMO: a feedback loop controls the assembly of the synaptonemal complex in budding yeast

    Directory of Open Access Journals (Sweden)

    Hideo Tsubouchi

    2016-02-01

    Full Text Available The synaptonemal complex (SC is a meiosis-specific chromosomal structure in which homologous chromosomes are intimately linked through arrays of specialized proteins called transverse filaments (TF. Widely conserved in eukaryote meiosis, the SC forms during prophase I and is essential for accurate segregation of homologous chromosomes at meiosis I. However, the basic mechanism overlooking formation and regulation of the SC has been poorly understood. By using the budding yeast Saccharomyces cerevisiae, we recently showed that SC formation is controlled through the attachment of multiple molecules of small ubiquitin-like modifier (SUMO to a regulator of TF assembly. Intriguingly, this SUMOylation is activated by TF, implicating the involvement of a positive feedback loop in the control of SC assembly. We discuss the implication of this finding and possible involvement of a similar mechanism in regulating other processes.

  11. The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast.

    Science.gov (United States)

    Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi

    2012-07-30

    The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Voltage-gated sodium channels in taste bud cells

    Directory of Open Access Journals (Sweden)

    Williams Mark E

    2009-03-01

    Full Text Available Abstract Background Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. Results We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. Conclusion SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  13. Discrimination of taste qualities among mouse fungiform taste bud cells.

    Science.gov (United States)

    Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-09-15

    Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

  14. Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.

    Science.gov (United States)

    Okubo, Tadashi; Clark, Cheryl; Hogan, Brigid L M

    2009-02-01

    The epithelium of the mouse tongue and soft palate consists of at least three distinct epithelial cell populations: basal cells, keratinized cells organized into filiform and fungiform papillae, and taste receptor cells present in tight clusters known as taste buds in the fungiform and circumvallate papillae and soft palate. All three cell types develop from the simple epithelium of the embryonic tongue and palate, and are continually replaced in the adult by cell turnover. Previous studies using pulse-chase tritiated thymidine labeling in the adult mouse provided evidence for a high rate of cell turnover in the keratinocytes (5-7 days) and taste buds (10 days). However, little is known about the localization and phenotype of the long-term stem or progenitor cells that give rise to the mature taste bud cells and surrounding keratinocytes in these gustatory tissues. Here, we make use of a tamoxifen-inducible K14-CreER transgene and the ROSA26 LacZ reporter allele to lineage trace the mature keratinocytes and taste bud cells of the early postnatal and adult mouse tongue and soft palate. Our results support the hypothesis that both the pore keratinocytes and receptor cells of the taste bud are derived from a common K14(+)K5(+)Trp63(+)Sox2(+) population of bipotential progenitor cells located outside the taste bud. The results are also compatible with models in which the keratinocytes of the filiform and fungiform papillae are derived from basal progenitor cells localized at the base of these structures.

  15. Lgr5 Identifies Progenitor Cells Capable of Taste Bud Regeneration after Injury.

    Directory of Open Access Journals (Sweden)

    Norifumi Takeda

    Full Text Available Taste buds are composed of a variety of taste receptor cell types that develop from tongue epithelium and are regularly replenished under normal homeostatic conditions as well as after injury. The characteristics of cells that give rise to regenerating taste buds are poorly understood. Recent studies have suggested that Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5 identifies taste bud stem cells that contribute to homeostatic regeneration in adult circumvallate and foliate taste papillae, which are located in the posterior region of the tongue. Taste papillae in the adult anterior region of the tongue do not express Lgr5. Here, we confirm and extend these studies by demonstrating that Lgr5 cells give rise to both anterior and posterior taste buds during development, and are capable of regenerating posterior taste buds after injury induced by glossopharyngeal nerve transection.

  16. Lgr5 Identifies Progenitor Cells Capable of Taste Bud Regeneration after Injury.

    Science.gov (United States)

    Takeda, Norifumi; Jain, Rajan; Li, Deqiang; Li, Li; Lu, Min Min; Epstein, Jonathan A

    2013-01-01

    Taste buds are composed of a variety of taste receptor cell types that develop from tongue epithelium and are regularly replenished under normal homeostatic conditions as well as after injury. The characteristics of cells that give rise to regenerating taste buds are poorly understood. Recent studies have suggested that Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) identifies taste bud stem cells that contribute to homeostatic regeneration in adult circumvallate and foliate taste papillae, which are located in the posterior region of the tongue. Taste papillae in the adult anterior region of the tongue do not express Lgr5. Here, we confirm and extend these studies by demonstrating that Lgr5 cells give rise to both anterior and posterior taste buds during development, and are capable of regenerating posterior taste buds after injury induced by glossopharyngeal nerve transection.

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

    KAUST Repository

    Mangla, Karan; Dill, David L; Horowitz, Mark A

    2010-01-01

    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

  18. Extraction of the number of peroxisomes in yeast cells by automated image analysis.

    Science.gov (United States)

    Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

    2006-01-01

    An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

  19. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

    Science.gov (United States)

    Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F.; Mistretta, Charlotte M.; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC. PMID:26741369

  20. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Directory of Open Access Journals (Sweden)

    Kristin Boggs

    Full Text Available Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC. Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5 and young postnatal (P1-10 mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1 P0-Cre/R26-tdTomato (RFP to label NC, NC derived Schwann cells and derivatives; (2 Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3 Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

  1. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Science.gov (United States)

    Boggs, Kristin; Venkatesan, Nandakumar; Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F; Mistretta, Charlotte M; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

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

    International Nuclear Information System (INIS)

    Alsteens, David; Dupres, Vincent; Evoy, Kevin Mc; Dufrene, Yves F; Wildling, Linda; Gruber, Hermann J

    2008-01-01

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

  3. New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lin, Jyun-Liang; Yu, Hui-Chia; Chao, Ju-Lan; Wang, Chung; Cheng, Ming-Yuan

    2012-12-01

    BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

  4. 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.)

  5. Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Tamara Goldfarb

    2010-10-01

    Full Text Available Recombination between homologous chromosomes of different parental origin (homologs is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs] show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in

  6. During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages.

    Science.gov (United States)

    Nakayama, Ayumi; Miura, Hirohito; Ooki, Makoto; Harada, Shuitsu

    2015-03-01

    Sox2 is proposed to regulate the differentiation of bipotential progenitor cells into taste bud cells. However, detailed expression of Sox2 remains unclear. In this report, Sox2 expression during taste bud development in the fungiform (FF), circumvallate (CV) and soft palate (SP) areas is examined together with Prox1. First, we immunohistochemically checked Prox1 expression in adults and found that almost all taste bud cells are Prox1-positive. During FF development, intense Sox2 expression was restricted to taste bud primordia expressing Prox1 at E12.5. However, at E14.5, Sox2 was intensely expressed outside the developing taste buds resolving to perigemmal Sox2 expression in adults. In the SP, at E14.5, taste bud primordia emerged as Prox1-expressing cell clusters. However, intense Sox2 expression was not restricted to taste bud primordia but was detected widely in the epithelium. During development, Sox2 expression outside developing taste buds was generally down-regulated but was retained in the perigemmal region similarly to that in the FF. In the CV, the initial stage of taste bud development remained unclear because of the lack of taste bud primordia comparable to that in the FF and SP. Here, we show that Prox1-expressing cells appear in the apical epithelium at E12.5, in the inner trench wall at E17.5 and in the outer trench wall at E18.5. Sox2 was again not restricted to developing taste bud cells expressing Prox1 during CV development. The expression patterns support that Sox2 does not serve as a cell fate selector between taste bud cells and surrounding keratinocytes but rather may contribute to them both.

  7. [Functional properties of taste bud cells. Mechanisms of afferent neurotransmission in Type II taste receptor cells].

    Science.gov (United States)

    Romanov, R A

    2013-01-01

    Taste Bud cells are heterogeneous in their morphology and functionality. These cells are responsible for sensing a wide variety of substances and for associating detected compounds with a different taste: bitter, sweet, salty, sour and umami. Today we know that each of the five basic tastes corresponds to distinct cell populations organized into three basic morpho-functional cell types. In addition, some receptor cells of the taste bud demonstrate glia-related functions. In this article we expand on some properties of these three morphological receptor cell types. Main focus is devoted to the Type II cells and unusual mechanism for afferent neurotransmission in these cells. Taste cells of the Type II consist of three populations detecting bitter, sweet and umami tastes, and, thus, evoke a serious scientific interest.

  8. Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid.

    Science.gov (United States)

    Aihara, Eitaro; Mahe, Maxime M; Schumacher, Michael A; Matthis, Andrea L; Feng, Rui; Ren, Wenwen; Noah, Taeko K; Matsu-ura, Toru; Moore, Sean R; Hong, Christian I; Zavros, Yana; Herness, Scott; Shroyer, Noah F; Iwatsuki, Ken; Jiang, Peihua; Helmrath, Michael A; Montrose, Marshall H

    2015-11-24

    Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration.

  9. Cell kinetic study on the relation between irradiation hypogeusia and taste buds in rats

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Hideharu; Furumoto, Keiichi [Nippon Dental Univ., Tokyo (Japan)

    1998-12-01

    The present study was designed to elucidate the mechanism of hypogeusia caused by irradiation. X-ray treatment at 10 Gy or 20 Gy was given to the maxillofacial region including the tongue in rats, and the involvement of taste bud for hypogeusia was investigated. In addition, cytological kinetics were immunohistologically studied using bromodeoxyuridine in the taste bud and in the lingual mucosal epithelium. The following results were obtained: In the 10 Gy group, the number of taste bud become less after the exposure, but no hypogeusia was observed during the experimental period. In the 20 Gy group, any labeled taste bud was not observed on the 7th day, and all taste buds disappeared by the 10th day. In the lingual mucosal epithelium, the number of basal cells decreased to the minimum, and the body weight and total water intake decreased coincidently in the 20 Gy group, which were few in the 10 Gy group. (author)

  10. Cell kinetic study on the relation between irradiation hypogeusia and taste buds in rats

    International Nuclear Information System (INIS)

    Kubota, Hideharu; Furumoto, Keiichi

    1998-01-01

    The present study was designed to elucidate the mechanism of hypogeusia caused by irradiation. X-ray treatment at 10 Gy or 20 Gy was given to the maxillofacial region including the tongue in rats, and the involvement of taste bud for hypogeusia was investigated. In addition, cytological kinetics were immunohistologically studied using bromodeoxyuridine in the taste bud and in the lingual mucosal epithelium. The following results were obtained: In the 10 Gy group, the number of taste bud become less after the exposure, but no hypogeusia was observed during the experimental period. In the 20 Gy group, any labeled taste bud was not observed on the 7th day, and all taste buds disappeared by the 10th day. In the lingual mucosal epithelium, the number of basal cells decreased to the minimum, and the body weight and total water intake decreased coincidently in the 20 Gy group, which were few in the 10 Gy group. (author)

  11. Functional conservation of RNA polymerase II in fission and budding yeasts.

    Science.gov (United States)

    Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

    2000-02-04

    The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

  12. Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast [version 2; referees: 3 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Colette Fox

    2017-02-01

    Full Text Available Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, cyclin-dependent kinases (CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of

  13. Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: Import of foreign membrane microdomains

    Czech Academy of Sciences Publication Activity Database

    Vaškovičová, Katarína; Strádalová, Vendula; Efenberk, Aleš; Opekarová, Miroslava; Malínský, Jan

    2015-01-01

    Roč. 94, č. 1 (2015), s. 1-11 ISSN 0171-9335 R&D Projects: GA ČR(CZ) GAP302/11/0146 Institutional support: RVO:68378041 Keywords : plasma membrane * membrane microdomain * MCC Subject RIV: EA - Cell Biology Impact factor: 4.011, year: 2015

  14. Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast.

    Science.gov (United States)

    Lin, Kang-Wei; Yang, Chuan-Jun; Lian, Hui-Yong; Cai, Peng

    2016-01-01

    In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF.

  15. Gravity-induced buds formation from protonemata apical cells in the mosses

    Science.gov (United States)

    Kyyak, Natalia; Khorkavtsiv, Yaroslava

    The acceleration of moss protonemata development after the exit it to light from darkness is important gravidependent morphogenetic manifestation of the moss protonemata. The accelerated development of mosses shows in transformation of apical protonemata cells into the gametophores buds (Ripetskyj et al., 1999). In order to establish, that such reaction on gravitation is general property of gravisensity species, or its typical only for single moss species, experiments with the following moss species - Bryum intermedium (Ludw.) Brig., Bryum caespiticium Hedw., Bryum argenteum Hedw., Dicranodontium denudatum (Brid.) Britt. were carried out. All these species in response to influence of gravitation were capable to form rich bunches of gravitropical protonemata in darkness, that testified to their gravisensity. After the transference of Petri dishes with gravitropical protonemata from darkness on light was revealed, that in 3 of the investigated species the gametophores buds were absent. Only B. argenteum has reacted to action of gravitation by buds formation from apical cells of the gravitropical protonemata. With the purpose of strengthening of buds formation process, the experiments with action of exogenous kinetin (in concentration of 10 (-6) M) were carried out. Kinetin essentially stimulated apical buds formation of B. argenteum. The quantity of apical buds has increased almost in three times in comparison with the control. Besides, on separate stolons a few (3-4) buds from one apical cell were formed. Experimentally was established, that the gametophores buds formation in mosses is controlled by phytohormones (Bopp, 1985; Demkiv et al., 1991). In conditions of gravity influence its essentially accelerated. Probably, gravity essentially strengthened acropetal transport of phytohormones and formation of attractive center in the protonemata apical cell. Our investigations have allowed to make the conclusion, that gravi-dependent formation of the apical buds is

  16. Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

    Science.gov (United States)

    Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

    2004-11-01

    GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

  17. RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.

    Science.gov (United States)

    Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

    2012-04-18

    In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

  18. Caloric Restriction-Induced Extension of Chronological Lifespan Requires Intact Respiration in Budding Yeast.

    Science.gov (United States)

    Kwon, Young-Yon; Lee, Sung-Keun; Lee, Cheol-Koo

    2017-04-01

    Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.

  19. Four linked genes participate in controlling sporulation efficiency in budding yeast.

    Directory of Open Access Journals (Sweden)

    Giora Ben-Ari

    2006-11-01

    Full Text Available Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four "high" sporulation alleles are derived from the "low" sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one "QTL region" that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes.

  20. Expression of Podoplanin in the Mouse Tooth Germ and Apical Bud Cells

    Science.gov (United States)

    Sawa, Yoshihiko; Iwasawa, Kana; Ishikawa, Hiroyuki

    2008-01-01

    This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud. PMID:18989465

  1. Mechanisms of taste bud cell loss after head and neck irradiation.

    Science.gov (United States)

    Nguyen, Ha M; Reyland, Mary E; Barlow, Linda A

    2012-03-07

    Taste loss in human patients following radiotherapy for head and neck cancer is a common and significant problem, but the cellular mechanisms underlying this loss are not understood. Taste stimuli are transduced by receptor cells within taste buds, and like epidermal cells, taste cells are regularly replaced throughout adult life. This renewal relies on progenitor cells adjacent to taste buds, which continually supply new cells to each bud. Here we treated adult mice with a single 8 Gy dose of x-ray irradiation to the head and neck, and analyzed taste epithelium at 1-21 d postirradiation (dpi). We found irradiation targets the taste progenitor cells, which undergo cell cycle arrest (1-3 dpi) and apoptosis (within 1 dpi). Taste progenitors resume proliferation at 5-7 dpi, with the proportion of cells in S and M phase exceeding control levels at 5-6 and 6 dpi, respectively, suggesting that proliferation is accelerated and/or synchronized following radiation damage. Using 5-bromo-2-deoxyuridine birthdating to identify newborn cells, we found that the decreased proliferation following irradiation reduces the influx of cells at 1-2 dpi, while the robust proliferation detected at 6 dpi accelerates entry of new cells into taste buds. In contrast, the number of differentiated taste cells was not significantly reduced until 7 dpi. These data suggest a model where continued natural taste cell death, paired with temporary interruption of cell replacement, underlies taste loss after irradiation.

  2. Mechanisms of taste bud cell loss after head and neck irradiation

    Science.gov (United States)

    Nguyen, Ha M.; Reyland, Mary E.; Barlow, Linda A.

    2012-01-01

    Taste loss in human patients following radiotherapy for head and neck cancer is a common and significant problem, but the cellular mechanisms underlying this loss are not understood. Taste stimuli are transduced by receptor cells within taste buds, and like epidermal cells, taste cells are regularly replaced throughout adult life. This renewal relies on a progenitor cells adjacent to taste buds, which continually supply new cells to each bud. Here we treated adult mice with a single 8 Gy dose of X-ray irradiation to the head and neck, and analyzed taste epithelium at 1–21 days post-irradiation (dpi). We found irradiation targets the taste progenitor cells, which undergo cell cycle arrest (1–3 dpi) and apoptosis (within 1 dpi). Taste progenitors resume proliferation at 5–7 dpi, with the proportion of cells in S and M phase exceeding control levels at 5–6 and 6 dpi, respectively, suggesting that proliferation is accelerated and/or synchronized following radiation damage. Using BrdU birthdating to identify newborn cells, we found that the decreased proliferation following irradiation reduces the influx of cells at 1–2 dpi, while the robust proliferation detected at 6 dpi accelerates entry of new cells into taste buds. By contrast, the number of differentiated taste cells was not significantly reduced until 7 dpi. These data suggest a model where continued natural taste cell death, paired with temporary interruption of cell replacement underlies taste loss after irradiation. PMID:22399770

  3. Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds.

    Science.gov (United States)

    Ohtubo, Yoshitaka; Iwamoto, Masafumi; Yoshii, Kiyonori

    2012-06-01

    Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  4. Analytical model for macromolecular partitioning during yeast cell division

    International Nuclear Information System (INIS)

    Kinkhabwala, Ali; Khmelinskii, Anton; Knop, Michael

    2014-01-01

    Asymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell — which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles — can be achieved by both passive (e.g. diffusion, localized retention sites) and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate. Here we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports. In contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning

  5. 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...

  6. Yeast replicative aging: a paradigm for defining conserved longevity interventions

    OpenAIRE

    Wasko, Brian M.; Kaeberlein, Matt

    2013-01-01

    The finite replicative life span of budding yeast mother cells was demonstrated as early as 1959, but the idea that budding yeast could be used to model aging of multicellular eukaryotes did not enter the scientific mainstream until relatively recently. Despite continued skepticism by some, there are now abundant data that several interventions capable of extending yeast replicative life span have a similar effect in multicellular eukaryotes including nematode worms, fruit flies, and rodents....

  7. Cell apoptosis of taste buds in circumvallate papillae in diabetic rats.

    Science.gov (United States)

    Cheng, B; Pan, S; Liu, X; Zhang, S; Sun, X

    2011-09-01

    Diabetes mellitus may result in taste disturbance. The present study has revealed that cell apoptosis of taste buds in circumvallate papillae may contribute to the taste disturbance in a rat model of type2 diabetes. Type2 diabetes was induced in Wistar rats by feeding them with a high-fat diet (30% fat), and a single intraperitoneal injection of streptozotocin (30 mg/kg). The increased cell apoptosis of taste buds in circumvallate papilla sections was detected by TUNEL staining in diabetic rats, and the ultrastructure was further examined by transmission electronic microscopy. Immunohistochemical and Western blot analyses revealed the downregulation of Bcl-2, upregulation of Bax, and increased activation of caspase-9 and -3, in diabetic rats, indicating that the apoptosis of taste bud cells may be mediated via the intrinsic mitochondrial pathway in diabetics. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

  8. Delineating functional principles of the bow tie structure of a kinase-phosphatase network in the budding yeast.

    Science.gov (United States)

    Abd-Rabbo, Diala; Michnick, Stephen W

    2017-03-16

    Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted. We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae. Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package ( https://cran.r-project.org/web/packages/VertexSort/index.html ). The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes

  9. Yeast for virus research

    Science.gov (United States)

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  10. Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid

    OpenAIRE

    Aihara, Eitaro; Mahe, Maxime M.; Schumacher, Michael A.; Matthis, Andrea L.; Feng, Rui; Ren, Wenwen; Noah, Taeko K.; Matsu-ura, Toru; Moore, Sean R.; Hong, Christian I.; Zavros, Yana; Herness, Scott; Shroyer, Noah F.; Iwatsuki, Ken; Jiang, Peihua

    2015-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5+) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5+ cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells ...

  11. High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

    Science.gov (United States)

    Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

    2014-07-01

    Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

  12. Guidelines and recommendations on yeast cell death nomenclature

    OpenAIRE

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andres; Austriaco, Nicanor; Sigrist, Stephan J.

    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 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 mor...

  13. A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast

    Science.gov (United States)

    Kim, Junwon; Rose, Mark D.

    2012-01-01

    Yeast cells induce the genes required for mating prior to the completion of mitosis. To ensure proper cell cycle progression prior to mating differentiation, a key cytoplasmic regulator of cell fusion, Fus2p, is sequestered in the nucleus by cyclin-dependent kinase (Cdk). In response to pheromone signaling, the mitogen-activated protein kinase Fus3p phosphorylates Ser 84 in Fus2p to drive nuclear export. We found that Fus3p becomes active and phosphorylates S84 as early as S phase, raising the question of how Cdk prevents inappropriate activation of Fus2p. Countering Fus3p, Cdk and a p21-activated kinase, Cla4p, maintain Fus2p's nuclear localization by phosphorylating Ser 67, which drives nuclear import and inhibits nuclear export. When Cdk and Cla4p activities drop after cell division, Fus3p promotes Fus2p export both via S84 phosphorylation and by down-regulating S67 phosphorylation. Thus, potential premature activation of Fus2p in mitosis is prevented by cell cycle-dependent phosphorylation that overrides the mating pheromone-induced phosphorylation that drives nuclear export. PMID:22588722

  14. Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells

    Directory of Open Access Journals (Sweden)

    Brand Joseph

    2010-06-01

    Full Text Available Abstract Background The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues. Results Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF-α, interferon (IFN-γ, and interleukin (IL-6, in mouse circumvallate and foliate papillae. TNF-α and IFN-γ immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds

  15. Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells.

    Science.gov (United States)

    Cohn, Zachary J; Kim, Agnes; Huang, Liquan; Brand, Joseph; Wang, Hong

    2010-06-10

    The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS)-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues. Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6, in mouse circumvallate and foliate papillae. TNF-alpha and IFN-gamma immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds, the niche for taste progenitor

  16. Prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Che K

    2017-02-01

    Full Text Available Keying Che,1,* Yang Zhao,2,3,* Xiao Qu,1 Zhaofei Pang,1 Yang Ni,4 Tiehong Zhang,4 Jiajun Du,1,5 Hongchang Shen4 1Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 2Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Collaborative Innovation Center of Cancer Medicine, Fudan University Shanghai Cancer Center, 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 4Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, 5Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China *These authors contributed equally to this work Purpose: Gastric carcinoma (GC is a highly aggressive cancer and one of the leading causes of cancer-related deaths worldwide. Histopathological evaluation pertaining to invasiveness is likely to provide additional information in relation to patient outcome. In this study, we aimed to evaluate the prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma.Materials and methods: Hematoxylin and eosin-stained slides generated from 296 gastric adenocarcinoma patients with full clinical and pathological and follow-up information were systematically reviewed. The patients were grouped on the basis of tumor budding, single cell invasion, large cell invasion, mitotic count, and fibrosis. The association between histopathological parameters, different classification systems, and overall survival (OS was statistically analyzed.Results: Among the 296 cases that were analyzed, high-grade tumor budding was observed in 49.0% (145 of them. Single cell invasion and large cell invasion were observed in 62.8% (186 and 16.9% (50 of the cases, respectively. Following univariate analysis, patients with high-grade tumor budding had shorter OS than those with low-grade tumor budding (hazard ratio [HR]: 2.260, P<0

  17. Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Takanori Takebe

    2017-12-01

    Full Text Available Summary: Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC. By conducting massive “reverse” screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108. Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. : With the goal of clinical translation of liver bud transplant therapy, Takebe et al. established a massive organoid production platform from endoderm, endothelial, and mesenchymal progenitor populations specified entirely from human iPSCs, reproducibly demonstrating functionality both in vitro and in vivo. Keywords: iPSC, liver bud, organoid, transplantation, self-organization, endothelial, mesenchymal, liver failure, clinical grade

  18. UV-sensitivity of bromodeoxyuridine (BUdR)-substituted chromosomes in Chinese hamster cells

    International Nuclear Information System (INIS)

    Antoshchina, M.M.; Luchnik, N.V.

    1990-01-01

    Chinese hamster cells with chromosomes differently substituted for BUdR (TT-TT, TT-TB, TB-TB, TB-BB, where T is thymidine containing chromatid and B is BUdR substituted chromatid) were exposed to UV-light in phase G 2 and chromosome aberrations (mainly chromatid breaks) were analysed. Breaks frequency per chromosome was proportional to BUdR content. No breaks were found in TT-TT chromosomes. The frequency of breaks per TB chromatid was similar with TT-TB and TB-BB chromosomes. In TB-BB chromosomes, however, virtually no breaks occured in TB chromatids whereas in BB chromatids, their frequency was much higher than was expected

  19. 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

  20. Cellular radiation effects and hyperthermia: Cytokinetic investigations with stationary phase yeast cells

    International Nuclear Information System (INIS)

    Fingerhut, R.; Otto, F.; Oldiges, H.; Kiefer, J.

    1980-01-01

    Wild type diploid yeast, Saccharomyces cerevisiae strain 211, was subjected to 250 kV X-rays or 50 0 C heat treatment for 30 min or to a combination of both. X-ray exposure took place either in air or in nitrogen. Cell number, percentage of budding cells and cell cycle progression was followed for up to 12 h post irradiation. The distribution of cell cycle stages was determined by flow cytofluorometry. All treatments cause a retardation of cell division rate. Hyperthermia leads mainly to a lengthening of G 1 , whereas X-rays arrest the cells reversibly in G 2 . The effect of the combined treatment appears to be merely additive. No selective action of hyperthermia on hypoxic cells was found. (orig.) [de

  1. A Novel Family of Cell Wall-Related Proteins Regulated Differently during the Yeast Life Cycle

    Science.gov (United States)

    Rodríguez-Peña, José Manuel; Cid, Víctor J.; Arroyo, Javier; Nombela, César

    2000-01-01

    The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial β-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Δ crh2Δ strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1–green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle. PMID:10757808

  2. Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

    Science.gov (United States)

    Nasu, Masanori; Nakahara, Taka; Tominaga, Noriko; Tamaki, Yuichi; Ide, Yoshiaki; Tachibana, Toshiaki; Ishikawa, Hiroshi

    2013-03-01

    The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

  3. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    Directory of Open Access Journals (Sweden)

    Constance Mehlgarten

    Full Text Available Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative, which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Debashis Barik

    2016-12-01

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

  6. Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

    Science.gov (United States)

    Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

    2009-05-01

    Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

  7. Tumor budding cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer

    International Nuclear Information System (INIS)

    Karamitopoulou, Eva

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4) and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with Wingless-INT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial–mesenchymal transition (EMT). Emerging evidence has demonstrated that cancer stem cells (CSCs), small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion, and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5) of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric, and ampullary) carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs, and EMT-type cells in PDAC.

  8. Tumor budding cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer.

    Science.gov (United States)

    Karamitopoulou, Eva

    2012-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4) and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with Wingless-INT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial-mesenchymal transition (EMT). Emerging evidence has demonstrated that cancer stem cells (CSCs), small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion, and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5) of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric, and ampullary) carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs, and EMT-type cells in PDAC.

  9. Tumor Budding Cells, Cancer Stem Cells and Epithelial-Mesenchymal Transition-type Cells in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Eva eKaramitopoulou

    2013-01-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4 and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with WNT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial-mesenchymal transition (EMT. Emerging evidence has demonstrated that cancer stem cells (CSCs, small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5 of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric and ampullary carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs and EMT-type cells in PDAC.

  10. Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats.

    Science.gov (United States)

    Lee, Sang-Bok; Lee, Cil-Han; Kim, Se-Nyun; Chung, Ki-Myung; Cho, Young-Kyung; Kim, Kyung-Nyun

    2009-12-01

    Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

  11. Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

    Science.gov (United States)

    Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

    2016-11-01

    Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

  12. Neurochemical characterization of sea lamprey taste buds and afferent gustatory fibers: presence of serotonin, calretinin, and CGRP immunoreactivity in taste bud bi-ciliated cells of the earliest vertebrates.

    Science.gov (United States)

    Barreiro-Iglesias, Antón; Villar-Cerviño, Verona; Villar-Cheda, Begoña; Anadón, Ramón; Rodicio, María Celina

    2008-12-01

    Neuroactive substances such as serotonin and other monoamines have been suggested to be involved in the transmission of gustatory signals from taste bud cells to afferent fibers. Lampreys are the earliest vertebrates that possess taste buds, although these differ in structure from taste buds in jawed vertebrates, and their neurochemistry remains unknown. We used immunofluorescence methods with antibodies raised against serotonin, tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), glutamate, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), calretinin, and acetylated alpha-tubulin to characterize the neurochemistry and innervation of taste buds in the sea lamprey, Petromyzon marinus L. For localization of proliferative cells in taste buds we used bromodeoxyuridine labeling and proliferating cell nuclear antigen immunohistochemistry. Results with both markers indicate that proliferating cells are restricted to a few basal cells and that almost all cells in taste buds are nonproliferating. A large number of serotonin-, calretinin-, and CGRP-immunoreactive bi-ciliated cells were revealed in lamprey taste buds. This suggests that serotonin participates in the transmission of gustatory signals and indicates that this substance appeared early on in vertebrate evolution. The basal surface of the bi-ciliated taste bud cells was contacted by tubulin-immunoreactive fibers. Some of the fibers surrounding the taste bud were calretinin immunoreactive. Lamprey taste bud cells or afferent fibers did not exhibit TH, GABA, glutamate, or NPY immunoreactivity, which suggests that expression of these substances evolved in taste buds of some gnathostomes lines after the separation of gnathostomes and lampreys. (c) 2008 Wiley-Liss, Inc.

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

    Science.gov (United States)

    Palková, Zdena; Váchová, Libuše

    2016-09-01

    Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Armstrong, Christine A; Tomita, Kazunori

    2017-03-01

    Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments. © 2017 The Authors.

  15. Calorie Restriction-Mediated Replicative Lifespan Extension in Yeast Is Non-Cell Autonomous

    Science.gov (United States)

    Mei, Szu-Chieh; Brenner, Charles

    2015-01-01

    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. PMID:25633578

  16. 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.

  17. MicroRNA-200b is downregulated in colon cancer budding cells

    DEFF Research Database (Denmark)

    Knudsen, Kirsten Nguyen; Lindebjerg, Jan; Nielsen, Boye Schnack

    2017-01-01

    BACKGROUND: The microRNA-200 (miR-200) family acts as a major suppressor of epithelial-mesenchymal transition (EMT). Impaired miR-200 expression may lead to EMT initiation and eventually cancer dissemination. The presence of tumor budding cells (TBC) is associated with metastasis and poor prognosis......, and molecular similarities to EMT indicate that these cells may reflect ongoing EMT. The aim of this study was to investigate the expression of miR-200b in budding cells of colon cancer and the relationship with the EMT-markers E-cadherin, β-catenin and laminin-5γ2. MATERIAL & METHODS: MiR-200b was investigated...... by in situ hybridization in 58 cases of stage II (n = 36) and III colon (n = 22) cancers with tumor budding. Expression of E-cadherin, β-catenin and laminin-5γ2 was examined by immunohistochemistry. A multiplex fluorescence assay combining miR-200b with cytokeratin and laminin-5γ2 was employed on a subset...

  18. 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.

  19. Presynaptic (Type III) cells in mouse taste buds sense sour (acid) taste.

    Science.gov (United States)

    Huang, Yijen A; Maruyama, Yutaka; Stimac, Robert; Roper, Stephen D

    2008-06-15

    Taste buds contain two types of cells that directly participate in taste transduction - receptor (Type II) cells and presynaptic (Type III) cells. Receptor cells respond to sweet, bitter and umami taste stimulation but until recently the identity of cells that respond directly to sour (acid) tastants has only been inferred from recordings in situ, from behavioural studies, and from immunostaining for putative sour transduction molecules. Using calcium imaging on single isolated taste cells and with biosensor cells to identify neurotransmitter release, we show that presynaptic (Type III) cells specifically respond to acid taste stimulation and release serotonin. By recording responses in cells isolated from taste buds and in taste cells in lingual slices to acetic acid titrated to different acid levels (pH), we also show that the active stimulus for acid taste is the membrane-permeant, uncharged acetic acid moiety (CH(3)COOH), not free protons (H(+)). That observation is consistent with the proximate stimulus for acid taste being intracellular acidification, not extracellular protons per se. These findings may also have implications for other sensory receptors that respond to acids, such as nociceptors.

  20. Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Anthony Arlia-Ciommo

    2014-05-01

    Full Text Available A body of evidence supports the view that the signaling pathways governing cellular aging – as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae. Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific ′′master regulator′′ proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest, the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast.

  1. Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I

    2014-05-27

    A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae . Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific "master regulator" proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast.

  2. β-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

    Directory of Open Access Journals (Sweden)

    Dany Gaillard

    2017-08-01

    Full Text Available Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional β-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest β-catenin plays a greater role in renewal of anterior versus posterior taste buds.

  3. β-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

    Science.gov (United States)

    Gaillard, Dany; Bowles, Spencer G; Salcedo, Ernesto; Xu, Mingang; Millar, Sarah E; Barlow, Linda A

    2017-08-01

    Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional β-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest β-catenin plays a greater role in renewal of anterior versus posterior taste buds.

  4. β-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice

    Science.gov (United States)

    Gaillard, Dany; Xu, Mingang; Millar, Sarah E.

    2017-01-01

    Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional β-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest β-catenin plays a greater role in renewal of anterior versus posterior taste buds. PMID:28846687

  5. Radiosensitivity of yeast cells as a function of radiation LET

    International Nuclear Information System (INIS)

    Lobachevskij, P.N.; Krasavin, E.A.

    1988-01-01

    A model is proposed for interpreting the radiosensitivity of yeast cells as a function of linear energy transfer (LET) of ionizing radiation. The model takes into account the role of repair processes in sensitivity of yeast cells to ionizing radiation of different LET. Two types of repair are discussed: (1) a nonspecific repair (characteristic of both haploid and diploid cells), and (2) a diploid - soecific repair (characteristic of diploid cells only)

  6. 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.

  7. Phosphorylation of the Budding Yeast 9-1-1 Complex Is Required for Dpb11 Function in the Full Activation of the UV-Induced DNA Damage Checkpoint▿ †

    Science.gov (United States)

    Puddu, Fabio; Granata, Magda; Di Nola, Lisa; Balestrini, Alessia; Piergiovanni, Gabriele; Lazzaro, Federico; Giannattasio, Michele; Plevani, Paolo; Muzi-Falconi, Marco

    2008-01-01

    Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G1 phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Δ dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase. PMID:18541674

  8. X-ray irradiation of yeast cells

    Science.gov (United States)

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

    1997-10-01

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

  9. A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks.

    Directory of Open Access Journals (Sweden)

    Teeraphan Laomettachit

    Full Text Available To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a "standard component" modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with "standard components" can capture in quantitative detail many essential properties of cell cycle control in budding yeast.

  10. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

    Science.gov (United States)

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

    2015-09-01

    Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

  11. Evidence that a recombinationless strain, rad 51, of Saccharomyces cerevisiae lacks the budding cell resistance to γ-rays

    International Nuclear Information System (INIS)

    Hama-Inaba, Hiroko; Saeki, Tetsuya

    1975-01-01

    The radiosensitivities of a wild-type and x-ray sensitive mutant, rad 51 (defective in genetic recombination) of Saccharomyces cerevisiae to γ-rays were compared, using non-synchronized and partially synchronized cultures. The rad 51 cells, either haploid or diploid, showed only very small changes in radiosensitivity during cell growth, whereas the wild-type cells, especially haploid, showed the well-known budding resistance. The heterozygous (wild/rad 51) diploid cells showed in a survival curve a remarkable budding resistance and sigmoidal inactivation kinetics similar to those of wild-type homozygous diploid cells. (author)

  12. NaCl responsive taste cells in the mouse fungiform taste buds.

    Science.gov (United States)

    Yoshida, R; Horio, N; Murata, Y; Yasumatsu, K; Shigemura, N; Ninomiya, Y

    2009-03-17

    Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells ( approximately 44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells ( approximately 56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.

  13. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

    Science.gov (United States)

    Kim, Dong Kyun; Kim, Song Ja; Kang, Shin Sung; Jin, Eun Jung

    2009-09-30

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

  14. Mitochondrial fission proteins regulate programmed cell death in yeast.

    Science.gov (United States)

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  15. The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

    Science.gov (United States)

    2011-01-01

    Background Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency. Results In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection. Conclusions The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw

  16. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    Science.gov (United States)

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  17. Yeast cell factories on the horizon

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2015-01-01

    For thousands of years, yeast has been used for making beer, bread, and wine. In modern times, it has become a commercial workhorse for producing fuels, chemicals, and pharmaceuticals such as insulin, human serum albumin, and vaccines against hepatitis virus and human papillomavirus. Yeast has also...... 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...

  18. Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

    Science.gov (United States)

    Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

    2013-05-01

    Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.

  19. Differing sensitivity to fluorescent light in Chinese hamster cells containing equally incorporated quantities of BUdR versus IUdR

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Morstyn, G.; Russo, A.; Kinsella, T.J.; Fornace, A. Jr.; McPherson, S.; Glatstein, E.

    1984-01-01

    Chinese hamster V79 cells that had incorporated approximately equal levels of either BUdR or IUdR into their DNA were found to be equal sensitizers to x rays. However, BUdR-substituted cells were much more sensitive to fluorescent light than IUdR-substituted cells, both on a cell survival basis and by the initial number of single strand DNA breaks induced. Since a major toxicity to the use of BUdR clinically has been light-induced skin rash, these data indicate that the use of IUdR clinically might cause less untoward toxicity but yet provide the same radiosensitization as BUdR

  20. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    Directory of Open Access Journals (Sweden)

    Shieh Grace S

    2011-12-01

    Full Text Available Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1 marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3, which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  1. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    LENUS (Irish Health Repository)

    Shieh, Grace S.

    2011-12-22

    Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3\\' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  2. Functional expression of ionotropic purinergic receptors on mouse taste bud cells.

    Science.gov (United States)

    Hayato, Ryotaro; Ohtubo, Yoshitaka; Yoshii, Kiyonori

    2007-10-15

    Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

  3. Correlating yeast cell stress physiology to changes in the cell surface morphology: atomic force microscopic studies.

    Science.gov (United States)

    Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

    2006-07-06

    Atomic Force Microscopy (AFM) has emerged as a powerful biophysical tool in biotechnology and medicine to investigate the morphological, physical, and mechanical properties of yeasts and other biological systems. However, properties such as, yeasts' response to environmental stresses, metabolic activities of pathogenic yeasts, cell-cell/cell-substrate adhesion, and cell-flocculation have rarely been investigated so far by using biophysical tools. Our recent results obtained by AFM on one strain each of Saccharomyces cerevisiae and Schizosaccharomyces pombe show a clear correlation between the physiology of environmentally stressed yeasts and the changes in their surface morphology. The future directions of the AFM related techniques in relation to yeasts are also discussed.

  4. 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

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    2014-08-20

    This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Guidelines and recommendations on yeast cell death nomenclature

    Science.gov (United States)

    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 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. PMID:29354647

  9. Trp53 activity is repressed in radio-adapted cultured murine limb bud cells

    International Nuclear Information System (INIS)

    Vares, Guillaume; Wang, Bing; Tanaka, Kaoru; Shang, Yi; Fujita, Kazuko; Hayata, Isamu; Nenoi, Mitsuru

    2011-01-01

    Understanding the effects of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity. (author)

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

    Science.gov (United States)

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

    2016-08-01

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

  11. DNA is structured as a linear "jigsaw puzzle" in the genomes of Arabidopsis, rice, and budding yeast.

    Science.gov (United States)

    Liu, Yun-Hua; Zhang, Meiping; Wu, Chengcang; Huang, James J; Zhang, Hong-Bin

    2014-01-01

    Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice.

  12. PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

    Science.gov (United States)

    Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

    2016-12-15

    During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

  13. The role of mitochondria in yeast programmed cell death

    International Nuclear Information System (INIS)

    Guaragnella, Nicoletta; Ždralević, Maša; Antonacci, Lucia; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2012-01-01

    Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this mini-review we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

  14. 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

  15. 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

  16. Mitochondrial fission proteins regulate programmed cell death in yeast

    OpenAIRE

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J.; Qi, Bing; Pevsner, Jonathan; McCaffery, J. Michael; Hill, R. Blake; Basañez, Gorka; Hardwick, J. Marie

    2004-01-01

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we fo...

  17. 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

  18. In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

    Science.gov (United States)

    Yuri, Shunsuke; Nishikawa, Masaki; Yanagawa, Naomi; Jo, Oak D; Yanagawa, Norimoto

    2017-02-14

    A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells

    Directory of Open Access Journals (Sweden)

    Shunsuke Yuri

    2017-02-01

    Full Text Available A method to maintain and rebuild ureteric bud (UB-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.

  20. Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors

    Directory of Open Access Journals (Sweden)

    Yuliang Liu

    2011-01-01

    Full Text Available Virus-host interactions play key roles in promoting efficient egress of many RNA viruses, including Ebola virus (EBOV or “e” and Marburg virus (MARV or “m”. Late- (L- domains conserved in viral matrix proteins recruit specific host proteins, such as Tsg101 and Nedd4, to facilitate the budding process. These interactions serve as attractive targets for the development of broad-spectrum budding inhibitors. A major gap still exists in our understanding of the mechanism of filovirus budding due to the difficulty in detecting virus-host complexes and mapping their trafficking patterns in the natural environment of the cell. To address this gap, we used a bimolecular complementation (BiMC approach to detect, localize, and follow the trafficking patterns of eVP40-Tsg101 complexes in live mammalian cells. In addition, we used the BiMC approach along with a VLP budding assay to test small molecule inhibitors identified by in silico screening for their ability to block eVP40 PTAP-mediated interactions with Tsg101 and subsequent budding of eVP40 VLPs. We demonstrated the potential broad spectrum activity of a lead candidate inhibitor by demonstrating its ability to block PTAP-dependent binding of HIV-1 Gag to Tsg101 and subsequent egress of HIV-1 Gag VLPs.

  1. Generation of branching ureteric bud tissues from human pluripotent stem cells.

    Science.gov (United States)

    Mae, Shin-Ichi; Ryosaka, Makoto; Toyoda, Taro; Matsuse, Kyoko; Oshima, Yoichi; Tsujimoto, Hiraku; Okumura, Shiori; Shibasaki, Aya; Osafune, Kenji

    2018-01-01

    Recent progress in kidney regeneration research is noteworthy. However, the selective and robust differentiation of the ureteric bud (UB), an embryonic renal progenitor, from human pluripotent stem cells (hPSCs) remains to be established. The present study aimed to establish a robust induction method for branching UB tissue from hPSCs towards the creation of renal disease models. Here, we found that anterior intermediate mesoderm (IM) differentiates from anterior primitive streak, which allowed us to successfully develop an efficient two-dimensional differentiation method of hPSCs into Wolffian duct (WD) cells. We also established a simplified procedure to generate three-dimensional WD epithelial structures that can form branching UB tissues. This system may contribute to hPSC-based regenerative therapies and disease models for intractable disorders arising in the kidney and lower urinary tract. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Budding yeast ATM/ATR control meiotic double-strand break (DSB levels by down-regulating Rec114, an essential component of the DSB-machinery.

    Directory of Open Access Journals (Sweden)

    Jesús A Carballo

    2013-06-01

    Full Text Available An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs. Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  3. Budding Yeast ATM/ATR Control Meiotic Double-Strand Break (DSB) Levels by Down-Regulating Rec114, an Essential Component of the DSB-machinery

    Science.gov (United States)

    Carballo, Jesús A.; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L.; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S.

    2013-01-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or “DSB homeostasis”, might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks. PMID:23825959

  4. Yeast mother cell-specific aging

    Czech Academy of Sciences Publication Activity Database

    Breitenbach, M.; Laun, P.; Pichová, Alena; Madeo, F.; Heeren, G.; Kohlwein, S. D.; Froehlich, K. U.; Dawes, I.

    2001-01-01

    Roč. 18, - (2001), s. 21 ISSN 0749-503X. [International Conference on Yeast Genetics and Molecular Biology /20./. 26.08.2001-31.08.2001, Prague] Institutional research plan: CEZ:AV0Z5020903 Subject RIV: EB - Genetics ; Molecular Biology

  5. A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls.

    Science.gov (United States)

    Kitagaki, Hiroshi; Ito, Kiyoshi; Shimoi, Hitoshi

    2004-10-01

    Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

  6. Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

    Science.gov (United States)

    Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

    2018-05-01

    In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

  7. Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1

    Directory of Open Access Journals (Sweden)

    Perna Marla K

    2011-04-01

    Full Text Available Abstract Background Mammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene. Results Histological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1-null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types. Conclusions p27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia

  8. 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.

  9. The neuropeptides CCK and NPY and the changing view of cell-to-cell communication in the taste bud.

    Science.gov (United States)

    Herness, Scott; Zhao, Fang-Li

    2009-07-14

    The evolving view of the taste bud increasingly suggests that it operates as a complex signal processing unit. A number of neurotransmitters and neuropeptides and their corresponding receptors are now known to be expressed in subsets of taste receptor cells in the mammalian bud. These expression patterns set up hard-wired cell-to-cell communication pathways whose exact physiological roles still remain obscure. As occurs in other cellular systems, it is likely that neuropeptides are co-expressed with neurotransmitters and function as neuromodulators. Several neuropeptides have been identified in taste receptor cells including cholecystokinin (CCK), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and glucagon-like peptide 1 (GLP-1). Of these, CCK and NPY are the best studied. These two peptides are co-expressed in the same presynaptic cells; however, their postsynaptic actions are both divergent and antagonistic. CCK and its receptor, the CCK-1 subtype, are expressed in the same subset of taste receptor cells and the autocrine activation of these cells produces a number of excitatory physiological actions. Further, most of these cells are responsive to bitter stimuli. On the other hand, NPY and its receptor, the NPY-1 subtype, are expressed in different cells. NPY, acting in a paracrine fashion on NPY-1 receptors, results in inhibitory actions on the cell. Preliminary evidence suggests the NPY-1 receptor expressing cell co-expresses T1R3, a member of the T1R family of G-protein coupled receptors thought to be important in detection of sweet and umami stimuli. Thus the neuropeptide expressing cells co-express CCK, NPY, and CCK-1 receptor. Neuropeptides released from these cells during bitter stimulation may work in concert to both modulate the excitation of bitter-sensitive taste receptor cells while concurrently inhibiting sweet-sensitive cells. This modulatory process is similar to the phenomenon of lateral inhibition that occurs in other sensory systems.

  10. ESCRT-independent budding of HIV-1 gag virus-like particles from Saccharomyces cerevisiae spheroplasts.

    Directory of Open Access Journals (Sweden)

    Andrew P Norgan

    Full Text Available Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated in the generation of VLPs. Our data reveal: 1 characterized Gag-ESCRT interaction motifs (late domains are not required for VLP budding, 2 loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3 ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.

  11. 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.

  12. Synaptic communication and signal processing among sensory cells in taste buds.

    Science.gov (United States)

    Chaudhari, Nirupa

    2014-08-15

    Taste buds (sensory structures embedded in oral epithelium) show a remarkable diversity of transmitters synthesized and secreted locally. The known transmitters accumulate in a cell type selective manner, with 5-HT and noradrenaline being limited to presynaptic cells, GABA being synthesized in both presynaptic and glial-like cells, and acetylcholine and ATP used for signalling by receptor cells. Each of these transmitters participates in local negative or positive feedback circuits that target particular cell types. Overall, the role of ATP is the best elucidated. ATP serves as a principal afferent transmitter, and also is the key trigger for autocrine positive feedback and paracrine circuits that result in potentiation (via adenosine) or inhibition (via GABA or 5-HT). While many of the cellular receptors and mechanisms for these circuits are known, their impact on sensory detection and perception remains to be elaborated in most instances. This brief review examines what is known, and some of the open questions and controversies surrounding the transmitters and circuits of the taste periphery. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  13. A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship.

    Science.gov (United States)

    Caudron, Fabrice; Barral, Yves

    2013-12-05

    Cellular behavior is frequently influenced by the cell's history, indicating that single cells may memorize past events. We report that budding yeast permanently escape pheromone-induced cell-cycle arrest when experiencing a deceptive mating attempt, i.e., not reaching their putative partner within reasonable time. This acquired behavior depends on super-assembly and inactivation of the G1/S inhibitor Whi3, which liberates the G1 cyclin Cln3 from translational inhibition. Super-assembly of Whi3 is a slow response to pheromone, driven by polyQ and polyN domains, counteracted by Hsp70, and stable over generations. Unlike prion aggregates, Whi3 super-assemblies are not inherited mitotically but segregate to the mother cell. We propose that such polyQ- and polyN-based elements, termed here mnemons, act as cellular memory devices to encode previous environmental conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

    Science.gov (United States)

    Liu, Qicai; Li, Ermao; Huang, Long; Cheng, Minsheng; Li, Li

    2018-01-01

    Background: The limb-bud and heart gene ( LBH ) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M. Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay. Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells. Conclusions : Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might

  15. A study on immobilized ethanol yeast cells by radiation technique

    International Nuclear Information System (INIS)

    Li Zhengkui; Zhang Bosen

    1994-01-01

    Hydrophilic monomer 2-hydroxyethyl acrylate (HEA) and a series of polyethylene glycol dimethacrylate monomers were copolymerized by radiation technique at low temperature (-78 degree C) and hydrophilic hydrogels were obtained. The immobilization of yeast cells with these copolymer carriers led to a higher ethanol productivity than free cells. Of all copolymer carriers, the ethanol yield with poly (HEA-14 G) was the highest, about 2.45 times as high as that of free yeast cells. In addition, the ethanol productivity of 12 batch repeated reactions with poly (HEA-14G) carrier was all higher than that of free yeast cells. The ethanol productivity of immobilized yeast cells was dependent on the proportion of hydrophilic monomer to other monomers in copolymer systems, the chain length of the bifunctional monomer, the degree of hydration of copolymer carriers, the structure of copolymer carriers and porosity in the internal structure of carriers. The ethanol yield of immobilized cells depended on swelling ability and porosity of copolymer carriers

  16. 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.

  17. Targeted taste cell-specific overexpression of brain-derived neurotrophic factor in adult taste buds elevates phosphorylated TrkB protein levels in taste cells, increases taste bud size, and promotes gustatory innervation.

    Science.gov (United States)

    Nosrat, Irina V; Margolskee, Robert F; Nosrat, Christopher A

    2012-05-11

    Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system.

  18. Targeted Taste Cell-specific Overexpression of Brain-derived Neurotrophic Factor in Adult Taste Buds Elevates Phosphorylated TrkB Protein Levels in Taste Cells, Increases Taste Bud Size, and Promotes Gustatory Innervation*

    Science.gov (United States)

    Nosrat, Irina V.; Margolskee, Robert F.; Nosrat, Christopher A.

    2012-01-01

    Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system. PMID:22442142

  19. The digestion of yeast cell wall polysaccharides in veal calves

    NARCIS (Netherlands)

    Gaillard, B.D.E.; Weerden, van E.J.

    1976-01-01

    1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their ‘normal’, milk-substitute

  20. 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

  1. 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 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 ...

  2. 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...

  3. The impact of metabolism on aging and cell size in single yeast cells

    NARCIS (Netherlands)

    Huberts, Daphne

    2015-01-01

    The aim of this thesis was to determine how metabolism affects yeast aging in single yeast cells using a novel microfluidic device. We first review how cells are able to sense nutrients in their environment and then describe the use of the microfluidic dissection platform that greatly improves our

  4. The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

    Directory of Open Access Journals (Sweden)

    Ralf Kist

    2014-10-01

    Full Text Available In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

  5. The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

    Science.gov (United States)

    Kist, Ralf; Watson, Michelle; Crosier, Moira; Robinson, Max; Fuchs, Jennifer; Reichelt, Julia; Peters, Heiko

    2014-10-01

    In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

  6. Zizyphin modulates calcium signalling in human taste bud cells and fat taste perception in the mouse.

    Science.gov (United States)

    Murtaza, Babar; Berrichi, Meryem; Bennamar, Chahid; Tordjmann, Thierry; Djeziri, Fatima Z; Hichami, Aziz; Leemput, Julia; Belarbi, Meriem; Ozdener, Hakan; Khan, Naim A

    2017-10-01

    Zizyphin, isolated from Zizyphus sps. leaf extracts, has been shown to modulate sugar taste perception, and the palatability of a sweet solution is increased by the addition of fatty acids. We, therefore, studied whether zizyphin also modulates fat taste perception. Zizyphin was purified from edible fruit of Zizyphus lotus L. Zizyphin-induced increases in [Ca 2+ ]i in human taste bud cells (hTBC). Zizyphin shared the endoplasmic reticulum Ca 2+ pool and also recruited, in part, Ca 2+ from extracellular environment via the opening of store-operated Ca 2+ channels. Zizyphin exerted additive actions on linoleic acid (LA)-induced increases in [Ca 2+ ]i in these cells, indicating that zizyphin does not exert its action via fatty acid receptors. However, zizyphin seemed to exert, at least in part, its action via bile acid receptor Takeda-G-protein-receptor-5 in hTBC. In behavioural tests, mice exhibited preference for both LA and zizyphin. Interestingly, zizyphin increased the preference for a solution containing-LA. This study is the first evidence of the modulation of fat taste perception by zizyphin at the cellular level in hTBC. Our study might be helpful for considering the synthesis of zizyphin analogues as 'taste modifiers' with a potential in the management of obesity and lipid-mediated disorders. © 2017 Société Française de Pharmacologie et de Thérapeutique.

  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. 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)

  9. A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

    Science.gov (United States)

    Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

    2018-05-01

    Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

  10. Arenavirus Budding

    Directory of Open Access Journals (Sweden)

    Shuzo Urata

    2011-01-01

    Full Text Available Several arenaviruses cause hemorrhagic fever disease in humans and pose a significant public health concern in their endemic regions. On the other hand, the prototypic arenavirus LCMV is a superb workhorse for the investigation of virus-host interactions and associated disease. The arenavirus small RING finger protein called Z has been shown to be the main driving force of virus budding. The budding activity of Z is mediated by late (L domain motifs, PT/SAP, and PPXY, located at the C-terminus of Z. This paper will present the current knowledge on arenavirus budding including the diversity of L domain motifs used by different arenaviruses. We will also discuss how improved knowledge of arenavirus budding may facilitate the development of novel antiviral strategies to combat human pathogenic arenaviruses.

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

    International Nuclear Information System (INIS)

    Demicheli, Marina C.; Andrade, Antero S.R.; Goes, Alfredo Miranda

    2007-01-01

    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)

  12. 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......The yeast Saccharomyces cerevisiae is well a characterized microorganism and widely used as eukaryotic model organism as well as a key cell factory for bioproduction of various products. The latter comprise a large variety of scientifically and industrially relevant products such as low-value bulk...... chemicals and biofuels, food additives, high-value chemicals and recombinant proteins. Despite the recent achievements in the fields of systems biology and metabolic engineering together with availability of broad genetic engineering toolbox, the full potential of S. cerevisiae as a cell factory is not yet...

  13. Sorption of strontium by magnetically modified yeast cells

    International Nuclear Information System (INIS)

    Hu Yantao; Ji Yanqin; Tian Qing; Shao Xianzhang; Shi Jianhe; Ivo Safarik; Zhang Shengdong; Li Jinying

    2008-01-01

    Magnetically modified fodder's yeast (Kluyveromyces fragilis) cells using water based magnetic fluid, were characterized by scanning electron microscopy (SEM) and Vibrating Sample Magnetometer (VSM). The sorption-desorption properties of Sr 2+ by these yeast cells from nitrate salt of Sr 2+ were studied. The results demonstrated that the Sr 2+ sorption volume by these cells enhanced with increasing pH and reached a plateau between pH 4.0 and 7.0. A minor effect by temperature was observed. The sorption volumes are 19.5 mg/g and 53.5 mg/g from 10 ppm and 40 ppm Sr 2+ solution respectively within 20 min. The sorption of Sr 2+ in these cells can be desorbed under 0.1 mol/L HNO 3 solution. The maximum Sr 2+ sorption volume is 96.7 mg/g at 20℃. The sorption characteristic fits Langmuir model well with 140.8 mg/g calculated maximum sorption volume by these yeast cells. (authors)

  14. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

    Science.gov (United States)

    Ren, Wenwen; Lewandowski, Brian C; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A; Margolskee, Robert F; Jiang, Peihua

    2014-11-18

    Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

  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. Digital Image Analysis of Yeast Single Cells Growing in Two Different Oxygen Concentrations to Analyze the Population Growth and to Assist Individual-Based Modeling.

    Science.gov (United States)

    Ginovart, Marta; Carbó, Rosa; Blanco, Mónica; Portell, Xavier

    2017-01-01

    Nowadays control of the growth of Saccharomyces to obtain biomass or cellular wall components is crucial for specific industrial applications. The general aim of this contribution is to deal with experimental data obtained from yeast cells and from yeast cultures to attempt the integration of the two levels of information, individual and population, to progress in the control of yeast biotechnological processes by means of the overall analysis of this set of experimental data, and to assist in the improvement of an individual-based model, namely, INDISIM- Saccha . Populations of S. cerevisiae growing in liquid batch culture, in aerobic and microaerophilic conditions, were studied. A set of digital images was taken during the population growth, and a protocol for the treatment and analyses of the images obtained was established. The piecewise linear model of Buchanan was adjusted to the temporal evolutions of the yeast populations to determine the kinetic parameters and changes of growth phases. In parallel, for all the yeast cells analyzed, values of direct morphological parameters, such as area, perimeter, major diameter, minor diameter, and derived ones, such as circularity and elongation, were obtained. Graphical and numerical methods from descriptive statistics were applied to these data to characterize the growth phases and the budding state of the yeast cells in both experimental conditions, and inferential statistical methods were used to compare the diverse groups of data achieved. Oxidative metabolism of yeast in a medium with oxygen available and low initial sugar concentration can be taken into account in order to obtain a greater number of cells or larger cells. Morphological parameters were analyzed statistically to identify which were the most useful for the discrimination of the different states, according to budding and/or growth phase, in aerobic and microaerophilic conditions. The use of the experimental data for subsequent modeling work was then

  17. Different characteristics between menadione and menadione sodium bisulfite as redox mediator in yeast cell suspension

    OpenAIRE

    Yamashoji, Shiro

    2016-01-01

    Menadione promoted the production of active oxygen species (AOS) in both yeast cell suspension and the crude enzymes from the cells, but menadione sodium bisulfite (MSB) had little effect on the production of AOS in the cell suspension. MSB kept the stable increase in the electron transfer from intact yeast cells to anode compared to menadione, but the electron transfer promoted by MSB was inhibited in permeabilized yeast cell suspension. Menadione promoted oxidation of NAD(P)H much faster th...

  18. 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

  19. Yeast cells proliferation on various strong static magnetic fields and temperatures

    International Nuclear Information System (INIS)

    Otabe, E S; Kuroki, S; Nikawa, J; Matsumoto, Y; Ooba, T; Kiso, K; Hayashi, H

    2009-01-01

    The effect of strong magnetic fields on activities of yeast cells were investigated. Experimental yeast cells were cultured in 5 ml of YPD(Yeast extract Peptone Dextrose) for the number density of yeast cells of 5.0 ±0.2 x 10 6 /ml with various temperatures and magnetic fields up to 10 T. Since the yeast cells were placed in the center of the superconducting magnet, the effect of magnetic force due to the diamagnetism and magnetic gradient was negligibly small. The yeast suspension was opened to air and cultured in shaking condition. The number of yeast cells in the yeast suspension was counted by a counting plate with an optical microscope, and the time dependence of the number density of yeast cells was measured. The time dependence of the number density of yeast cells, ρ, of initial part is analyzed in terms of Malthus equation as given by ρ = ρo exp(kt), where k is the growth coefficient. It is found that, the growth coefficient under the magnetic field is suppressed compared with the control. The growth coefficient decreasing as increasing magnetic field and is saturated at about 5 T. On the other hand, it is found that the suppression of growth of yeast cells by the magnetic field is diminished at high temperatures.

  20. Arenavirus Budding

    OpenAIRE

    Urata, Shuzo; de la Torre, Juan Carlos

    2011-01-01

    Several arenaviruses cause hemorrhagic fever disease in humans and pose a significant public health concern in their endemic regions. On the other hand, the prototypic arenavirus LCMV is a superb workhorse for the investigation of virus-host interactions and associated disease. The arenavirus small RING finger protein called Z has been shown to be the main driving force of virus budding. The budding activity of Z is mediated by late (L) domain motifs, PT/SAP, and PPXY, located at the C-termin...

  1. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials (“aging factors”) through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms. PMID:26351681

  2. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals "aging factors" and mechanism of lifespan asymmetry.

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K; Li, Hao

    2015-09-22

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.

  3. The yeast replicative aging model.

    Science.gov (United States)

    He, Chong; Zhou, Chuankai; Kennedy, Brian K

    2018-03-08

    It has been nearly three decades since the budding yeast Saccharomyces cerevisiae became a significant model organism for aging research and it has emerged as both simple and powerful. The replicative aging assay, which interrogates the number of times a "mother" cell can divide and produce "daughters", has been a stalwart in these studies, and genetic approaches have led to the identification of hundreds of genes impacting lifespan. More recently, cell biological and biochemical approaches have been developed to determine how cellular processes become altered with age. Together, the tools are in place to develop a holistic view of aging in this single-celled organism. Here, we summarize the current state of understanding of yeast replicative aging with a focus on the recent studies that shed new light on how aging pathways interact to modulate lifespan in yeast. Copyright © 2018. Published by Elsevier B.V.

  4. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Science.gov (United States)

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  5. A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

    Science.gov (United States)

    Huberman, Lori B.; Murray, Andrew W.

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  6. Expression and secretion of TNF-α in mouse taste buds: a novel function of a specific subset of type II taste cells.

    Science.gov (United States)

    Feng, Pu; Zhao, Hang; Chai, Jinghua; Huang, Liquan; Wang, Hong

    2012-01-01

    Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno-colocalization experiments using taste-cell-type markers, the TNF-α-producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2(-/-)/TLR4(-/-) double-gene-knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF-α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions.

  7. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    Science.gov (United States)

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

    Science.gov (United States)

    Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

    2012-08-01

    Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Science.gov (United States)

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

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

  11. 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......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...... 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...

  12. 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)

  13. Molecular profiling of tumour budding implicates TGFβ-mediated epithelial-mesenchymal transition as a therapeutic target in oral squamous cell carcinoma.

    Science.gov (United States)

    Jensen, D H; Dabelsteen, E; Specht, L; Fiehn, A M K; Therkildsen, M H; Jønson, L; Vikesaa, J; Nielsen, F C; von Buchwald, C

    2015-08-01

    Although tumour budding is an adverse prognostic factor for many cancer types, the molecular mechanisms governing this phenomenon are incompletely understood. Therefore, understanding the molecular basis of tumour budding may provide new therapeutic and diagnostic options. We employ digital image analysis to demonstrate that the number of tumour buds in cytokeratin-stained sections correlates with patients having lymph node metastases at diagnosis. The tumour bud count was also a predictor of overall survival, independent of TNM stage. Tumour buds and paired central tumour areas were subsequently collected from oral squamous cell carcinoma (OSCC) specimens, using laser capture microdissection, and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature, comprising factors involved in epithelial-mesenchymal transition (EMT) and activated TGFβ signalling. Transcription factors ZEB1 and PRRX1 were up-regulated concomitantly with the decreased expression of mesenchymal-epithelial (MET) transcription factors (eg OVOL1) in addition to Krüppel-like factors and Grainyhead-like factors. Moreover, miR-200 family members were down-regulated in budding tumour cells. We used immunohistochemistry to validate five markers of the EMT/MET process in 199 OSCC tumours, as well as in situ hybridization in 20 OSCC samples. Given the strong relationship between tumour budding and the development of lymph node metastases and an adverse prognosis, therapeutics based on inhibiting the activation of TGFβ signalling may prove useful in the treatment of OSCC. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    Ying Luo

    Full Text Available 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.

  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. Diploid yeast cells yield homozygous spontaneous mutations

    Science.gov (United States)

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

    1993-01-01

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

  17. Expression of synaptogyrin-1 in T1R2-expressing type II taste cells and type III taste cells of rat circumvallate taste buds.

    Science.gov (United States)

    Kotani, Takeshi; Toyono, Takashi; Seta, Yuji; Kitou, Ayae; Kataoka, Shinji; Toyoshima, Kuniaki

    2013-09-01

    Synaptogyrins are conserved components of the exocytic apparatus and function as regulators of Ca(2+)-dependent exocytosis. The synaptogyrin family comprises three isoforms: two neuronal (synaptogyrin-1 and -3) and one ubiquitous (synaptogyrin-2) form. Although the expression patterns of the exocytic proteins synaptotagmin-1, SNAP-25, synaptobrevin-2 and synaptophysin have been elucidated in taste buds, the function and expression pattern of synaptogyrin-1 in rat gustatory tissues have not been determined. Therefore, we examined the expression patterns of synaptogyrin-1 and several cell-specific markers of type II and III cells in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays and immunoblot analysis revealed the expression of synaptogyrin-1 mRNA and its protein in circumvallate papillae. In fungiform, foliate and circumvallate papillae, the antibody against synaptogyrin-1 immunolabeled a subset of taste bud cells and intra- and subgemmal nerve processes. Double-labeling experiments revealed the expression of synaptogyrin-1 in most taste cells immunoreactive for aromatic L-amino acid decarboxylase and the neural cell adhesion molecule. A subset of synaptogyrin-1-immunoreactive taste cells also expressed phospholipase Cβ2, gustducin, or sweet taste receptor (T1R2). In addition, most synaptogyrin-1-immunoreactive taste cells expressed synaptobrevin-2. These results suggest that synaptogyrin-1 plays a regulatory role in transmission at the synapses of type III cells and is involved in exocytic function with synaptobrevin-2 in a subset of type II cells in rat taste buds.

  18. Use of the micronucleus assay for the selective detection of radiosensitivity in BUdR-unincorporated cells after pulse-labelling of exponentially growing tumour cells

    Energy Technology Data Exchange (ETDEWEB)

    Masunaga, S.; Ono, K.; Fushiki, M.; Abe, M. (Kyoto Univ. (Japan). Faculty of Medicine); Wandl, E.O. (Vienna Univ. (Austria). Klinik fuer Strahlentherapie und Strahlenbiologie)

    1990-08-01

    To determine the radiosensitivity of non S-phase tumour cells in vitro, survival curves of SCC VII tumour cells were obtained after a short block with hydroxyurea. Dose-response curves of micronucleus (MN) frequency appearing in non-S-phase cells were also determined by excluding S-phase cells with immunofluorescence staining to 5-bromo-2'-deoxyuridine (BUdR). Both the dose response curves of MN frequency and survival curves were analysed by a linear-quadratic model (surviving fraction =exp (-{alpha}D-{beta}D{sup 2}), MN frequency =aD+bD{sup 2}+c). A good correlation between the {alpha}/{beta} and a/b ratios was observed. In both BUdR-unincorporated and asynchronous cell cultures, the regression lines between the surviving fraction and micronucleus frequency were statistically identical. (author).

  19. Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

    Science.gov (United States)

    Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

    2013-07-01

    Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

  20. Research of aquatic organism addition influence on the reproduction of yeast cells in the dough

    Directory of Open Access Journals (Sweden)

    Дмитро Павлович Крамаренко

    2016-12-01

    Full Text Available The analysis of the research results of influence of various amounts of aquatic organism additions on the reproduction of yeast cells is given. A positive impact of aquatic organism addition of animal and plant origin in investigated quantities on the reproduction of yeast cells is revealed. The influence of the chemical composition of the aquatic organism additives on the reproduction of yeast cells is proved

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

    Science.gov (United States)

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

    2014-03-03

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

  2. Binding kinetics of magnetic nanoparticles on latex beads and yeast cells studied by magnetorelaxometry

    International Nuclear Information System (INIS)

    Eberbeck, Dietmar; Bergemann, Christian; Hartwig, Stefan; Steinhoff, Uwe; Trahms, Lutz

    2005-01-01

    The ion exchange mediated binding of magnetic nanoparticles (MNP) to modified latex spheres and yeast cells was quantified using magnetorelaxometry. By fitting subsequently recorded relaxation curves, the kinetics of the binding reactions was extracted. The signal of MNP with weak ion exchanger groups bound to latex and yeast cells scales linearly with the concentration of latex beads or yeast cells whereas that of MNP with strong ion exchanger groups is proportional to the square root of concentration. The binding of the latter leads to a much stronger aggregation of yeast cells than the former MNP

  3. Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

    Science.gov (United States)

    Melloy, Patricia G; Rose, Mark D

    2017-09-15

    Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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.

  5. Molecular profiling of tumour budding implicates TGFβ-mediated epithelial–mesenchymal transition as a therapeutic target in oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Jensen, David Hebbelstrup; Dabelsteen, Erik; Specht, Lena

    2015-01-01

    collected from oral squamous cell carcinoma (OSCC) specimens using laser capture microdissection and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature comprising factors involved...

  6. Interleukin-10 is produced by a specific subset of taste receptor cells and critical for maintaining structural integrity of mouse taste buds.

    Science.gov (United States)

    Feng, Pu; Chai, Jinghua; Zhou, Minliang; Simon, Nirvine; Huang, Liquan; Wang, Hong

    2014-02-12

    Although inflammatory responses are a critical component in defense against pathogens, too much inflammation is harmful. Mechanisms have evolved to regulate inflammation, including modulation by the anti-inflammatory cytokine interleukin-10 (IL-10). Previously we have shown that taste buds express various molecules involved in innate immune responses, including the proinflammatory cytokine tumor necrosis factor (TNF). Here, using a reporter mouse strain, we show that taste cells also express the anti-inflammatory cytokine IL-10. Remarkably, IL-10 is produced by only a specific subset of taste cells, which are different from the TNF-producing cells in mouse circumvallate and foliate taste buds: IL-10 expression was found exclusively in the G-protein gustducin-expressing bitter receptor cells, while TNF was found in sweet and umami receptor cells as reported previously. In contrast, IL-10R1, the ligand-binding subunit of the IL-10 receptor, is predominantly expressed by TNF-producing cells, suggesting a novel cellular hierarchy for regulating TNF production and effects in taste buds. In response to inflammatory challenges, taste cells can increase IL-10 expression both in vivo and in vitro. These findings suggest that taste buds use separate populations of taste receptor cells that coincide with sweet/umami and bitter taste reception to modulate local inflammatory responses, a phenomenon that has not been previously reported. Furthermore, IL-10 deficiency in mice leads to significant reductions in the number and size of taste buds, as well as in the number of taste receptor cells per taste bud, suggesting that IL-10 plays critical roles in maintaining structural integrity of the peripheral gustatory system.

  7. Fibrin glue mixed with platelet-rich fibrin as a scaffold seeded with dental bud cells for tooth regeneration.

    Science.gov (United States)

    Yang, Kai-Chiang; Wang, Chun-Hao; Chang, Hao-Hueng; Chan, Wing P; Chi, Chau-Hwa; Kuo, Tzong-Fu

    2012-11-01

    Odontogenesis is a complex process with a series of epithelial-mesenchymal interactions and odontogenic molecular cascades. In tissue engineering of teeth from stem cells, platelet-rich fibrin (PRF), which is rich in growth factors and cytokines, may improve regeneration. Accordingly, PRF was added into fibrin glue to enrich the microenvironment with growth factors. Unerupted second molar tooth buds were harvested from miniature swine and cultured in vitro for 3 weeks to obtain dental bud cells (DBCs). Whole blood was collected for the preparation of PRF and fibrin glue before surgery. DBCs were suspended in fibrin glue and then enclosed with PRF, and the DBC-fibrin glue-PRF composite was autografted back into the original alveolar sockets. Radiographic and histological examinations were used to identify the regenerated tooth structure 36 weeks after implantation. Immunohistochemical staining was used to detect proteins specific to tooth regeneration. One pig developed a complete tooth with crown, root, pulp, enamel, dentin, odontoblast, cementum, blood vessels, and periodontal ligaments in indiscriminate shape. Another animal had an unerupted tooth that expressed cytokeratin 14, dentin matrix protein-1, vascular endothelial growth factor, and osteopontin. This study demonstrated, using autogenic cell transplantation in a porcine model, that DBCs seeded into fibrin glue-PRF could regenerate a complete tooth. Copyright © 2011 John Wiley & Sons, Ltd.

  8. ABA Represses the Expression of Cell Cycle Genes and May Modulate the Development of Endodormancy in Grapevine Buds

    Directory of Open Access Journals (Sweden)

    Ricardo Vergara

    2017-05-01

    Full Text Available Recently, the plant hormone abscisic acid (ABA has been implicated as a key player in the regulation of endodormancy (ED in grapevine buds (Vitis vinifera L. In this study, we show that in the vine, the expression of genes related to the biosynthesis of ABA (VvNCED1; VvNCED2 and the content of ABA are significantly higher in the latent bud than at the shoot apex, while the expression of an ABA catabolic gene (VvA8H3 showed no significant difference between either organ. A negative correlation between the content of ABA and transcript levels of cell cycle genes (CCG was found in both tissues. This result suggested that ABA may negatively regulate the expression of CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of CCG was analyzed in two meristematic tissues of the vine: somatic embryos and shoot apexes. The results indicated that cell cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding cyclin-dependent kinases (VvCDKB1, VvCDKB2 and genes encoding cyclins of type A (VvCYCA1, VvCYCA2, VvCYCA3, B (VvCYCB, and D (VvCYCD3.2a and up-regulated the expression of VvICK5, a gene encoding an inhibitor of CDKs. During ED, the content of ABA increased, and the expression of CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC reduced the content of ABA and up-regulated the expression of CCG, this last effect was abolished when HC and ABA were co-applied. Taken together, these results suggest that ABA-mediated repression of CCG transcription may be part of the mechanism through which ABA modulates the development of ED in grapevine buds.

  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. Synergistic reduction of toluylene blue induced by acetaldehyde and menadione in yeast cell suspension: Application to determination of yeast cell activity

    Directory of Open Access Journals (Sweden)

    Shiro Yamashoji

    2017-03-01

    Full Text Available Membrane permeant acetaldehyde and menadione induced the synergistic reduction of toluylene blue (TB acting as non-membrane permeant redox indicator in yeast cell suspension. NADH and acetaldehyde also induced the synergistic TB reduction in permeabilized yeast cells and phosphate buffer, but menadione had no ability to promote TB reduction. The pre-incubation of acetaldehyde inhibited the above synergistic reduction of TB in intact and permeabilized yeast cell suspension. The pre-incubation of acetaldehyde might promote NADH oxidation by alcohol dehydrogenase, because acetaldehyde decreased the intracellular NAD(PH concentration. The above facts indicate that the synergistic reduction of TB is controlled by the order of addition of menadione and acetaldehyde. The synergistic reduction of TB by menadione and acetaldehyde was proportional to viable yeast cell number from 104 to 2×106 cells/ml, and this assay was applicable to cytotoxicity test. The time required for the above assay was only 2 min.

  11. Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope

    International Nuclear Information System (INIS)

    De, Tathagata; Chettoor, Antony M.; Agarwal, Pranav; Salapaka, Murti V.; Nettikadan, Saju

    2010-01-01

    The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells (Kasas and Ikai, 1995), (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.

  12. Arecoline Alters Taste Bud Cell Morphology, Reduces Body Weight, and Induces Behavioral Preference Changes in Gustatory Discrimination in C57BL/6 Mice.

    Science.gov (United States)

    Peng, Wei-Hau; Chau, Yat-Pang; Lu, Kuo-Shyan; Kung, Hsiu-Ni

    2016-01-01

    Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCβ2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

    DEFF Research Database (Denmark)

    Benghezal, Mohammed; Roubaty, Carole; Veepuri, Vijayanath

    2007-01-01

    Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal...

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

    Directory of Open Access Journals (Sweden)

    Ida M Smith

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

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

    Science.gov (United States)

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

    2014-01-01

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

  16. Ca2+ signaling in taste bud cells and spontaneous preference for fat: unresolved roles of CD36 and GPR120.

    Science.gov (United States)

    Abdoul-Azize, Souleymane; Selvakumar, Subramaniam; Sadou, Hassimi; Besnard, Philippe; Khan, Naim Akhtar

    2014-01-01

    Recent compelling evidences from rodent and human studies raise the possibility for an additional sixth taste modality devoted to oro-gustatory perception of dietary lipids. Understanding the mechanisms underlying oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. A number of studies have suggested that lingual CD36, a glycoprotein, highly expressed by circumvallate papillae of the tongue, is implicated in the perception of dietary fat taste. G protein-coupled receptors (GPCRs) are important signaling molecules for many aspects of cellular functions. It has been shown that these receptors, particularly GPR120, are also involved in lipid taste perception. We have shown that dietary long-chain fatty acids (LCFAs), in CD36-positive taste bud cells (TBC), induce increases in free intracellular Ca(2+) concentrations, [Ca(2+)]i, by recruiting Ca(2+) from endoplasmic reticulum (ER) pool via inositol 1,4,5-triphosphate production, followed by Ca(2+) influx via opening of store-operated Ca(2+) (SOC) channels. GPR120 is also coupled to increases in [Ca(2+)]i by dietary fatty acids. We observed that stromal interaction molecule 1 (STIM1), a sensor of Ca(2+) depletion in the ER, mediated fatty acid-induced Ca(2+) signaling and spontaneous preference for fat in the mouse. In this review article, we discuss the recent advances and unresolved roles of CD36 and GPR120 in lipid taste signaling in taste bud cells. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

    International Nuclear Information System (INIS)

    Asanuma, N.

    1990-01-01

    X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues

  18. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    Science.gov (United States)

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.

  19. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    Science.gov (United States)

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lamoolphak, Wiwat [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Goto, Motonobu [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Sasaki, Mitsuru [Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 850-8555 (Japan); Suphantharika, Manop [Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400 (Thailand); Muangnapoh, Chirakarn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Prommuag, Chattip [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand); Shotipruk, Artiwan [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Patumwan, Payathai Road, Bangkok 10330 (Thailand)]. E-mail: artiwan.s@chula.ac.th

    2006-10-11

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  2. 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.

  3. 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 .

  4. 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}.

  5. DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

    Science.gov (United States)

    Li, Chun Mei; Zheng, Lin Ling; Yang, Xiao Xi; Wan, Xiao Yan; Wu, Wen Bi; Zhen, Shu Jun; Li, Yuan Fang; Luo, Ling Fei; Huang, Cheng Zhi

    2016-01-01

    Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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).

  7. Taste bud cells of adult mice are responsive to Wnt/β-catenin signaling: implications for the renewal of mature taste cells.

    Science.gov (United States)

    Gaillard, Dany; Barlow, Linda A

    2011-04-01

    Wnt/β-catenin signaling initiates taste papilla development in mouse embryos, however, its involvement in taste cell turnover in adult mice has not been explored. Here we used the BATGAL reporter mouse model, which carries an engineered allele in which the LacZ gene is expressed in the presence of activated β-catenin, to determine the responsiveness of adult taste bud cells to canonical Wnt signaling. Double immunostaining with markers of differentiated taste cells revealed that a subset of Type I, II, and III taste cells express β-galactosidase. Using in situ hybridization, we showed that β-catenin activates the transcription of the LacZ gene mainly in intragemmal basal cells that are immature taste cells, identified by their expression of Sonic Hedgehog (Shh). Finally, we showed that β-catenin activity is significantly reduced in taste buds of 25-week-old mice compared with 10-week-old animals. Our data suggest that Wnt/β-catenin signaling may influence taste cell turnover by regulating cell differentiation. Reduced canonical Wnt signaling in older mice could explain in part the loss of taste sensitivity with aging, implicating a possible deficiency in the rate of taste cell renewal. More investigations are now necessary to understand if and how Wnt signaling regulates adult taste cell turnover. Copyright © 2011 Wiley-Liss, Inc.

  8. Taste bud cells of adult mice are responsive to Wnt/β-catenin signaling: implications for the renewal of mature taste cells

    Science.gov (United States)

    Gaillard, Dany; Barlow, Linda A.

    2012-01-01

    Wnt/β-catenin signaling initiates taste papilla development in mouse embryos, however, its involvement in taste cell turnover in adult mice has not been explored. Here we used the BATGAL reporter mouse model, which carries an engineered allele in which the LacZ gene is expressed in the presence of activated β-catenin, to determine the responsiveness of adult taste bud cells to canonical Wnt signaling. Double immunostaining with markers of differentiated taste cells revealed that a subset of type I, II and III taste cells express β-galactosidase. Using in situ hybridization, we showed that β-catenin activates the transcription of the LacZ gene mainly in intragemmal basal cells that are immature taste cells, identified by their expression of Sonic Hedgehog (Shh). Finally, we showed that β-catenin activity is significantly reduced in taste buds of 25 week-old mice compared to 10 week-old animals. Our data suggest that Wnt/β-catenin signaling may influence taste cell turnover by regulating cell differentiation. Reduced canonical Wnt signaling in older mice could explain in part the loss of taste sensitivity with aging, implicating a possible deficiency in the rate of taste cell renewal. More investigations are now necessary to understand if and how Wnt signaling regulates adult taste cell turnover. PMID:21328519

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

  10. Expression of the voltage-gated potassium channel KCNQ1 in mammalian taste bud cells and the effect of its null-mutation on taste preferences.

    Science.gov (United States)

    Wang, Hong; Iguchi, Naoko; Rong, Qi; Zhou, Minliang; Ogunkorode, Martina; Inoue, Masashi; Pribitkin, Edmund A; Bachmanov, Alexander A; Margolskee, Robert F; Pfeifer, Karl; Huang, Liquan

    2009-01-20

    Vertebrate taste buds undergo continual cell turnover. To understand how the gustatory progenitor cells in the stratified lingual epithelium migrate and differentiate into different types of mature taste cells, we sought to identify genes that were selectively expressed in taste cells at different maturation stages. Here we report the expression of the voltage-gated potassium channel KCNQ1 in mammalian taste buds of mouse, rat, and human. Immunohistochemistry and nuclear staining showed that nearly all rodent and human taste cells express this channel. Double immunostaining with antibodies against type II and III taste cell markers validated the presence of KCNQ1 in these two types of cells. Co-localization studies with cytokeratin 14 indicated that KCNQ1 is also expressed in type IV basal precursor cells. Null mutation of the kcnq1 gene in mouse, however, did not alter the gross structure of taste buds or the expression of taste signaling molecules. Behavioral assays showed that the mutant mice display reduced preference to some umami substances, but not to any other taste compounds tested. Gustatory nerve recordings, however, were unable to detect any significant change in the integrated nerve responses of the mutant mice to umami stimuli. These results suggest that although it is expressed in nearly all taste bud cells, the function of KCNQ1 is not required for gross taste bud development or peripheral taste transduction pathways, and the reduced preference of kcnq1-null mice in the behavioral assays may be attributable to the deficiency in the central nervous system or other organs.

  11. 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....... In addition to plant biomass hydrolysates, glycerol is of interest here, being available in amounts relevant for industrial scale bioprocesses due to increased production of biodiesel. The well characterised cell factory Saccharomyces cerevisiae exhibits a clear preference for glucose as a carbon source......, and is highly adapted to its utilisation. Although there have been several studies on glycerol metabolism in S. cerevisiae, many industrially used strains grow poorly on glycerol (μmax = 0.01h-1). On the other hand, several non-conventional yeast species are efficient in utilization of glycerol, some...

  12. 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.

  13. 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

  14. Biotechnological Applications of Dimorphic Yeasts

    Science.gov (United States)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  15. 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.

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

    Science.gov (United States)

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

    2015-01-01

    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.

  17. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

    Science.gov (United States)

    Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

    2011-07-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  18. Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa.

    Science.gov (United States)

    Liu, J; Kipreos, E T

    2000-07-01

    Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs. Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date. To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans. We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes. The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans. In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85. One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence. Interestingly, CAK1 and csk1 branch together with high bootstrap support values. We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction. This result suggests that CAK1 and csk1 are orthologs and that a

  19. 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 (...

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

    NARCIS (Netherlands)

    Wloch-Salamon, D.M.; Bem, A.E.

    2013-01-01

    The occurrence of programmed cell death in unicellular organisms is a subject that arouses great interest of theoreticians and experimental scientists. Already found evolutionarily conserved genes and metabolic pathways confirmed its existence in yeast, protozoa and even bacteria. In the yeast

  1. Study on immobilized yeast cells with hydrophilic polymer carrier by radiation-induced copolymerization

    International Nuclear Information System (INIS)

    Li Zhengkui; Zhang Bosen

    1993-01-01

    Various kinds of monomers 2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl acrylate (HEA), hydroxypropyl methacrylate (HPMA) and methoxy polyethylene glycol methylacrylate (M-23G) are copolymerized by radiation technique at low temperature (-78 degree C) and several kinds of copolymer carriers were obtained. Yeast cells are immobilized through adhesion and multiplication of yeast cells themselves on these carriers. The ethanol productivity of immobilized yeast cells with these carriers was related to the monomer composition and water content of copolymer carriers and the optimum monomer composition was 20%:10% in poly (HEA-M23G). In this case, the ethanol productivity of immobilized yeast cells was 26 mg/(ml · h), which was 4 times as high as that of free cells. Effect of adding crosslinking reagent (4G) in lower monomer composition of poly(HEA-M23G) on the ethanol productivity of immobilized cells was better than that in higher one in this work

  2. 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.

  3. The putative Notch ligand HyJagged is a transmembrane protein present in all cell types of adult Hydra and upregulated at the boundary between bud and parent

    Directory of Open Access Journals (Sweden)

    Tischer Susanne

    2011-09-01

    Full Text Available Abstract Background The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian Hydra it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in Hydra Notch is activated by presenilin through proteolytic cleavage at the S3 site as in all animals. However, the endogenous ligand for HvNotch has not been described yet. Results We have cloned a cDNA from Hydra, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. Hyjagged mRNA is undetectable in adult Hydra by in situ hybridisation but is strongly upregulated and easily visible at the border between bud and parent shortly before bud detachment. In contrast, HyJagged protein is found in all cell types of an adult hydra, where it localises to membranes and endosomes. Co-localisation experiments showed that it is present in the same cells as HvNotch, however not always in the same membrane structures. Conclusions The putative Notch ligand HyJagged is conserved in Cnidarians. Together with HvNotch it may be involved in the formation of the parent-bud boundary in Hydra. Moreover, protein distribution of both, HvNotch receptor and HyJagged indicate a more widespread function for these two transmembrane proteins in the adult hydra, which may be regulated by additional factors, possibly involving endocytic pathways.

  4. 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.

  5. 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.

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

    International Nuclear Information System (INIS)

    Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi

    2013-01-01

    Highlights: ► NO is produced from L-arginine in response to elevated temperature in yeast. ► Tah18 was first identified as the yeast protein involved in NO synthesis. ► 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.

  7. 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....

  8. Use of non-conventional cell disruption method for extraction of proteins from black yeasts

    Directory of Open Access Journals (Sweden)

    Maja eLeitgeb

    2016-04-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. 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.

  11. In vitro ochratoxin A adsorption by commercial yeast cell walls

    Directory of Open Access Journals (Sweden)

    Carina Maricel Pereyra

    2015-03-01

    Full Text Available ABSTRACT. Pereyra C.M., Cavaglieri L.R., Keller K.M., Chiacchiera, S.M., Rosa C.A.R. & Dalcero A.M. In vitro ochratoxin A adsorption by commercial yeast cell walls. [Adsorção in vitro de ocratoxina A por paredes celulares de levedura comercial.] Revista Brasileira de Medicina Veterinária, 37(1:25-28, 2015. Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, (5800 Río Cuarto, Córdoba, Argentina. E-mail: lcavaglieri@exa.unrc.edu.ar The aim of the present study was to evaluate the ochratoxin A (OTA adsorption capacity by two kinds of commercial yeast cell walls (YCW1 and YCW2 used as dietary supplements. An in vitro test was designed to mimic the temperature (37ºC, pH (2 and passage time (30 min through the stomach of a monogastric animal. The test was performed using different concentrations of YWC (100 and 200 µg/mL and OTA (10; 80; 160 and 1000 ng/mL and extracts were quantified by HPLC. For each OTA concentration, two independent trials varying the concentration of each YCW were performed. The two YCW assayed containing different percentages of polysaccharides, were able to adsorb similar amounts of OTA. Furthermore, the relationship mannans/β-glucans does not influence the rate of adsorption of OTA. In general, it was observed that the adsorption capacity increased with decreasing OTA concentration. Results from this work related to adsorption capacity of OTA with YCW allow predicting that other factor than 3D-structure and β-glucans and mannans could be involved. Future studies could be conducted to test the in vivo binding ability to alleviate the toxic effects of OTA under field conditions. Both YCW are a promising mycotoxin adsorbent to be used in animal feed to prevent mycotoxicoses.

  12. Cell Size Influences the Reproductive Potential and Total Lifespan of the Saccharomyces cerevisiae Yeast as Revealed by the Analysis of Polyploid Strains

    Directory of Open Access Journals (Sweden)

    Renata Zadrag-Tecza

    2018-01-01

    Full Text Available The total lifespan of the yeast Saccharomyces cerevisiae may be divided into two phases: the reproductive phase, during which the cell undergoes mitosis cycles to produce successive buds, and the postreproductive phase, which extends from the last division to cell death. These phases may be regulated by a common mechanism or by distinct ones. In this paper, we proposed a more comprehensive approach to reveal the mechanisms that regulate both reproductive potential and total lifespan in cell size context. Our study was based on yeast cells, whose size was determined by increased genome copy number, ranging from haploid to tetraploid. Such experiments enabled us to test the hypertrophy hypothesis, which postulates that excessive size achieved by the cell—the hypertrophy state—is the reason preventing the cell from further proliferation. This hypothesis defines the reproductive potential value as the difference between the maximal size that a cell can reach and the threshold value, which allows a cell to undergo its first cell cycle and the rate of the cell size to increase per generation. Here, we showed that cell size has an important impact on not only the reproductive potential but also the total lifespan of this cell. Moreover, the maximal cell size value, which limits its reproduction capacity, can be regulated by different factors and differs depending on the strain ploidy. The achievement of excessive size by the cell (hypertrophic state may lead to two distinct phenomena: the cessation of reproduction without “mother” cell death and the cessation of reproduction with cell death by bursting, which has not been shown before.

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

    KAUST Repository

    Vella, D.; Ajdari, A.; Vaziri, A.; Boudaoud, A.

    2011-01-01

    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

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

    Science.gov (United States)

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

    2011-01-01

    The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  15. 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

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved....... 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.......g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts....

  16. Stable current outputs and phytate degradation by yeast-based biofuel cell.

    Science.gov (United States)

    Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

    2014-09-01

    In this paper, we report for the first time that Candida melibiosica 2491 yeast strain expresses enhanced phytase activity when used as a biocatalyst in biofuel cells. The polarization also results in an increase of the yeast biomass. Higher steady-state electrical outputs, assigned to earlier production of an endogenous mediator, were achieved at continuous polarization under constant load. The obtained results prove that the C. melibiosica yeast-based biofuel cell could be used for simultaneous electricity generation and phytate bioremediation. In addition, the higher phytase activity obtained by interruptive polarization suggests a new method for increasing the phytase yield from microorganisms. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

    International Nuclear Information System (INIS)

    Ji, Jiayao; Li, Qinggang; Xie, Yuansheng; Zhang, Xueguang; Cui, Shaoyuan; Shi, Suozhu; Chen, Xiangmei

    2012-01-01

    Highlights: ► Overexpression of Robo2 caused reduced UB branching and glomerular number. ► Fewer MM cells surrounding the UB after overexpression of Robo2 in vitro. ► No abnormal Epithelial Morphology of UB or apoptosis of mm cells in the kidney. ► Overexpression of Robo2 affected MM cells migration and caused UB deficit. ► The reduced glomerular number can also be caused by fewer MM cells. -- Abstract: Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2–Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2–Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.

  18. 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.)

  19. 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....

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

    Science.gov (United States)

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

    2006-01-01

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

  1. Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

    Science.gov (United States)

    Choi, Jinsu; Lee, Hojae; Choi, Insung S.; Yang, Sung Ho

    2017-09-01

    Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.

  2. Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

    Science.gov (United States)

    Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

    2014-02-01

    The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

  3. Cell-type-dependent action potentials and voltage-gated currents in mouse fungiform taste buds.

    Science.gov (United States)

    Kimura, Kenji; Ohtubo, Yoshitaka; Tateno, Katsumi; Takeuchi, Keita; Kumazawa, Takashi; Yoshii, Kiyonori

    2014-01-01

    Taste receptor cells fire action potentials in response to taste substances to trigger non-exocytotic neurotransmitter release in type II cells and exocytotic release in type III cells. We investigated possible differences between these action potentials fired by mouse taste receptor cells using in situ whole-cell recordings, and subsequently we identified their cell types immunologically with cell-type markers, an IP3 receptor (IP3 R3) for type II cells and a SNARE protein (SNAP-25) for type III cells. Cells not immunoreactive to these antibodies were examined as non-IRCs. Here, we show that type II cells and type III cells fire action potentials using different ionic mechanisms, and that non-IRCs also fire action potentials with either of the ionic mechanisms. The width of action potentials was significantly narrower and their afterhyperpolarization was deeper in type III cells than in type II cells. Na(+) current density was similar in type II cells and type III cells, but it was significantly smaller in non-IRCs than in the others. Although outwardly rectifying current density was similar between type II cells and type III cells, tetraethylammonium (TEA) preferentially suppressed the density in type III cells and the majority of non-IRCs. Our mathematical model revealed that the shape of action potentials depended on the ratio of TEA-sensitive current density and TEA-insensitive current one. The action potentials of type II cells and type III cells under physiological conditions are discussed. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  4. 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

  5. Protective and detrimental bystander effects induced by x-irradiation in the limb bud cell cultures of fetal mice

    International Nuclear Information System (INIS)

    Wang, B.; Ohyama, H.; Shang, Y.; Yukawa, O.; Aizawa, S.; Hayata, I.

    2003-01-01

    Full text: Radioadaptive response and bystander effect represent important phenomena in radiobiology with an impact on the novel bioresponse mechanisms and risk estimates. The micromass cultures of limb bud cells (LBCs) provide an in vitro cellular maturation system, in which progress of cellular proliferation and differentiation is comparably paralleled to that of in vivo. This paper reports for the first time the evidential correlation and interaction, which simultaneously exist in the micromass culture system, between radioadaptive response and bystander effect. A radioadaptive response was induced in LBCs of embryonic day 11 (E11) ICR mice. Conditioning irradiation of the E11 cells with 30 cGy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cellular proliferation and differentiation induced by a challenging dose of 5 Gy given next day. Both protective and detrimental bystander effects were observed, namely, irradiating 50% of the E11 cells with 30 cGy led to a successful induction of radioadaptive response, and irradiating 70% of the E12 cells with 5 Gy produced comparably the detrimental effect to that of when all the cells were irradiated. Further, the bystander effects were markedly vanished by pretreatment of the cells with inhibitors to block the gap junction-mediated intracellular communication. These results indicated that the bystander effect played an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect by the challenging irradiation. Concerning the underlying mechanism, the gap junction-mediated intracellular communication was suggested being involved in the induction of the bystander effects

  6. Characterisation of the nascent polypeptide-associated complex in fission yeast

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Semple, Colin A; Hartmann-Petersen, Rasmus

    2007-01-01

    with other cell proteins, but has also been found to associate with DNA junctions, and to be involved in other processes including transcription regulation and mitochondrial protein import.Here, we characterize NAC in fission yeast. We find that NAC is associated with ribosomes, while a significant fraction...... defects in protein degradation. Accordingly, we find that the NAC UBA domain belongs to an ancient and distinct subgroup of the UBA family. In contrast to the situation with budding yeast, fission yeast cells devoid of NAC were not temperature sensitive. However, they displayed resistance to the amino...

  7. 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

  8. 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

  9. Adhesion of yeast cells on surface of polymers produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu, Zhaoxin; Takehisa, Masaaki; Xie Zongchuan.

    1995-01-01

    The adhesion of yeast (Saccharomyces formesences) cells on polymers was studied thermodynamically. The polymers were laminally prepared by means of radiation polymerization. By measuring contact angles, we calculated dispersion component and polar component of surface free energy of the polymers and the cells, and interfacial free energy between the polymer and the cells. Then interfacial free energy change of the cell adhesion to surface of the polymer was evaluated. The adhesion behavior of yeast cells on the polymers was observed by optical microscope. From above results, we conclude that the initial adhesion of the cells is related to the surface free energy of the polymer, but the irreversible adhesion may be close to the polar component in surface free energy. The high polar component is favourable the irreversible adhesion of yeast cells. (author)

  10. Checkpoint independence of most DNA replication origins in fission yeast.

    Science.gov (United States)

    Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

    2007-12-19

    In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint

  11. Checkpoint independence of most DNA replication origins in fission yeast

    Science.gov (United States)

    Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

    2007-01-01

    Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in

  12. Checkpoint independence of most DNA replication origins in fission yeast

    Directory of Open Access Journals (Sweden)

    Scott Donna

    2007-12-01

    Full Text Available Abstract Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU, which limits the pool of deoxyribonucleoside triphosphates (dNTPs and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR or cds1 (which encodes the fission yeast homologue of Chk2. Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3% behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild

  13. Taste Bud-Derived BDNF Is Required to Maintain Normal Amounts of Innervation to Adult Taste Buds123

    Science.gov (United States)

    Meng, Lingbin; Ohman-Gault, Lisa; Ma, Liqun

    2015-01-01

    Abstract Gustatory neurons transmit chemical information from taste receptor cells, which reside in taste buds in the oral cavity, to the brain. As adult taste receptor cells are renewed at a constant rate, nerve fibers must reconnect with new taste receptor cells as they arise. Therefore, the maintenance of gustatory innervation to the taste bud is an active process. Understanding how this process is regulated is a fundamental concern of gustatory system biology. We speculated that because brain-derived neurotrophic factor (BDNF) is required for taste bud innervation during development, it might function to maintain innervation during adulthood. If so, taste buds should lose innervation when Bdnf is deleted in adult mice. To test this idea, we first removed Bdnf from all cells in adulthood using transgenic mice with inducible CreERT2 under the control of the Ubiquitin promoter. When Bdnf was removed, approximately one-half of the innervation to taste buds was lost, and taste buds became smaller because of the loss of taste bud cells. Individual taste buds varied in the amount of innervation each lost, and those that lost the most innervation also lost the most taste bud cells. We then tested the idea that that the taste bud was the source of this BDNF by reducing Bdnf levels specifically in the lingual epithelium and taste buds. Taste buds were confirmed as the source of BDNF regulating innervation. We conclude that BDNF expressed in taste receptor cells is required to maintain normal levels of innervation in adulthood. PMID:26730405

  14. Taste Bud-Derived BDNF Is Required to Maintain Normal Amounts of Innervation to Adult Taste Buds.

    Science.gov (United States)

    Meng, Lingbin; Ohman-Gault, Lisa; Ma, Liqun; Krimm, Robin F

    2015-01-01

    Gustatory neurons transmit chemical information from taste receptor cells, which reside in taste buds in the oral cavity, to the brain. As adult taste receptor cells are renewed at a constant rate, nerve fibers must reconnect with new taste receptor cells as they arise. Therefore, the maintenance of gustatory innervation to the taste bud is an active process. Understanding how this process is regulated is a fundamental concern of gustatory system biology. We speculated that because brain-derived neurotrophic factor (BDNF) is required for taste bud innervation during development, it might function to maintain innervation during adulthood. If so, taste buds should lose innervation when Bdnf is deleted in adult mice. To test this idea, we first removed Bdnf from all cells in adulthood using transgenic mice with inducible CreERT2 under the control of the Ubiquitin promoter. When Bdnf was removed, approximately one-half of the innervation to taste buds was lost, and taste buds became smaller because of the loss of taste bud cells. Individual taste buds varied in the amount of innervation each lost, and those that lost the most innervation also lost the most taste bud cells. We then tested the idea that that the taste bud was the source of this BDNF by reducing Bdnf levels specifically in the lingual epithelium and taste buds. Taste buds were confirmed as the source of BDNF regulating innervation. We conclude that BDNF expressed in taste receptor cells is required to maintain normal levels of innervation in adulthood.

  15. A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

    Directory of Open Access Journals (Sweden)

    Elgjo Kjell

    2009-07-01

    Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

  16. Processing umami and other tastes in mammalian taste buds.

    Science.gov (United States)

    Roper, Stephen D; Chaudhari, Nirupa

    2009-07-01

    Neuroscientists are now coming to appreciate that a significant degree of information processing occurs in the peripheral sensory organs of taste prior to signals propagating to the brain. Gustatory stimulation causes taste bud cells to secrete neurotransmitters that act on adjacent taste bud cells (paracrine transmitters) as well as on primary sensory afferent fibers (neurocrine transmitters). Paracrine transmission, representing cell-cell communication within the taste bud, has the potential to shape the final signal output that taste buds transmit to the brain. The following paragraphs summarize current thinking about how taste signals generally, and umami taste in particular, are processed in taste buds.

  17. Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase.

    Science.gov (United States)

    Odagiri, Nao; Seki, Masayuki; Onoda, Fumitoshi; Yoshimura, Akari; Watanabe, Sei; Enomoto, Takemi

    2003-03-01

    MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.

  18. Nanoscopic morphological changes in yeast cell surfaces caused by oxidative stress: an atomic force microscopic study.

    Science.gov (United States)

    Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

    2009-06-01

    Nanoscopic changes in the cell surface morphology of the yeasts Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354), due to their exposure to varying concentrations of hydrogen peroxide (oxidative stress), were investigated using an atomic force microscope (AFM). Increasing hydrogen peroxide concentration led to a decrease in cell viabilities and mean cell volumes, and an increase in the surface roughness of the yeasts. In addition, AFM studies revealed that oxidative stress caused cell compression in both S. cerevisiae and Schiz. pombe cells and an increase in the number of aged yeasts. These results confirmed the importance and usefulness of AFM in investigating the morphology of stressed microbial cells at the nanoscale. The results also provided novel information on the relative oxidative stress tolerance of S. cerevisiae and Schiz. pombe.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  20. WARBURG EFFECT AND TRANSLOCATION-INDUCED GENOMIC INSTABILITY: TWO YEAST MODELS FOR CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Valentina eTosato

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Tosato, Valentina [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Grüning, Nana-Maria [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Breitenbach, Michael [Division of Genetics, Department of Cell Biology, University of Salzburg, Salzburg (Austria); Arnak, Remigiusz [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Ralser, Markus [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Bruschi, Carlo V., E-mail: bruschi@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy)

    2013-01-18

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

  2. Transgenic labeling of higher order neuronal circuits linked to phospholipase C-β2-expressing taste bud cells in medaka fish.

    Science.gov (United States)

    Ieki, Takashi; Okada, Shinji; Aihara, Yoshiko; Ohmoto, Makoto; Abe, Keiko; Yasuoka, Akihito; Misaka, Takumi

    2013-06-01

    The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-β2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-β2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

  3. Discrete innervation of murine taste buds by peripheral taste neurons.

    Science.gov (United States)

    Zaidi, Faisal N; Whitehead, Mark C

    2006-08-09

    The peripheral taste system likely maintains a specific relationship between ganglion cells that signal a particular taste quality and taste bud cells responsive to that quality. We have explored a measure of the receptoneural relationship in the mouse. By injecting single fungiform taste buds with lipophilic retrograde neuroanatomical markers, the number of labeled geniculate ganglion cells innervating single buds on the tongue were identified. We found that three to five ganglion cells innervate a single bud. Injecting neighboring buds with different color markers showed that the buds are primarily innervated by separate populations of geniculate cells (i.e., multiply labeled ganglion cells are rare). In other words, each taste bud is innervated by a population of neurons that only connects with that bud. Palate bud injections revealed a similar, relatively exclusive receptoneural relationship. Injecting buds in different regions of the tongue did not reveal a topographic representation of buds in the geniculate ganglion, despite a stereotyped patterned arrangement of fungiform buds as rows and columns on the tongue. However, ganglion cells innervating the tongue and palate were differentially concentrated in lateral and rostral regions of the ganglion, respectively. The principal finding that small groups of ganglion cells send sensory fibers that converge selectively on a single bud is a new-found measure of specific matching between the two principal cellular elements of the mouse peripheral taste system. Repetition of the experiments in the hamster showed a more divergent innervation of buds in this species. The results indicate that whatever taste quality is signaled by a murine geniculate ganglion neuron, that signal reflects the activity of cells in a single taste bud.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  5. Genetic control of yeast cell radiosensitivity modification by oxygen and hypoxic sensitizers

    International Nuclear Information System (INIS)

    Zhuranovskaya, G.P.; Petin, V.G.

    1984-01-01

    Diploid yeast cells Saccharomyces cerevisiae ''of the wild type'', individual mutants, homozygous in rad 2 and rad 54 and double mutants, containing both these loci in homozygous state are considered to prove genetic determination of radiosensitivity modification of hypoxic cells by oxygen and electron acceptor compounds previously demonstrated on yeast cells of other genotypes. It is shown that both ''oxygen effect'' and the effect of hypoxic sensitizers depend on the activity of repair systems. The possible mechanism of participation of post-radiation restoration processes in the modification of cell radiosensitivity, is discussed

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    Science.gov (United States)

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

  8. 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.

  9. 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.

  10. Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Jiayao [Institute of Nephrology, State Key Laboratory of Kidney Disease (2011DAV00088), The Chinese PLA General Hospital, Beijing 100853 (China); Medical College of NanKai University, Tianjin (China); Li, Qinggang; Xie, Yuansheng; Zhang, Xueguang; Cui, Shaoyuan; Shi, Suozhu [Institute of Nephrology, State Key Laboratory of Kidney Disease (2011DAV00088), The Chinese PLA General Hospital, Beijing 100853 (China); Chen, Xiangmei, E-mail: xmchen301@126.com [Institute of Nephrology, State Key Laboratory of Kidney Disease (2011DAV00088), The Chinese PLA General Hospital, Beijing 100853 (China); Medical College of NanKai University, Tianjin (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Overexpression of Robo2 caused reduced UB branching and glomerular number. Black-Right-Pointing-Pointer Fewer MM cells surrounding the UB after overexpression of Robo2 in vitro. Black-Right-Pointing-Pointer No abnormal Epithelial Morphology of UB or apoptosis of mm cells in the kidney. Black-Right-Pointing-Pointer Overexpression of Robo2 affected MM cells migration and caused UB deficit. Black-Right-Pointing-Pointer The reduced glomerular number can also be caused by fewer MM cells. -- Abstract: Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that

  11. Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation.

    OpenAIRE

    Witt, M; Reutter, K; Ganchrow, D; Ganchrow, J R

    2000-01-01

    Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primor...

  12. 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

  13. 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

  14. Aged yeast mother cells show markers of apoptosis

    Czech Academy of Sciences Publication Activity Database

    Laun, P.; Pichová, Alena; Madeo, F.; Heeren, G.; Kohlwein, S. D.; Fröhlich, K. W.; Dawes, I.; Breitenbach, M.

    2001-01-01

    Roč. 18, S1 (2001), s. S160 ISSN 0749-503X. [International Conference on Yeast Genetics and Molecular Biology /20./. 26.08.2001-31.08.2001, Prague] Institutional research plan: CEZ:AV0Z5020903 Subject RIV: EB - Genetics ; Molecular Biology

  15. 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.

  16. Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction

    DEFF Research Database (Denmark)

    Styrkársdóttir, U; Egel, R; Nielsen, O

    1992-01-01

    in signal transduction in budding yeast. Expression of the S. cerevisiae STE11 gene in S. pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone. Also, such cells become capable of conjugation and sporulation. When mat1-Pm is artifically expressed from a heterologous promoter...

  17. Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

    Science.gov (United States)

    Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

    2017-05-01

    In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

  18. 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

  19. 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.

  20. Drying enhances immunoactivity of spent brewer's yeast cell wall β-D-glucans.

    Science.gov (United States)

    Liepins, Janis; Kovačova, Elena; Shvirksts, Karlis; Grube, Mara; Rapoport, Alexander; Kogan, Grigorij

    2015-07-20

    Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast β-D-glucans as BRM, and drying as an efficient pretreatment to increase β-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified β-D-glucan. However, drying increased purified β-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted β-D-glucan. This is corroborated with FT-IR analyses of the β-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality β-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast β-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. High similarity of phylogenetic profiles of rate-limiting enzymes with inhibitory relation in Human, Mouse, Rat, budding Yeast and E. coli.

    Science.gov (United States)

    Zhao, Min; Qu, Hong

    2011-11-30

    The phylogenetic profile is widely used to characterize functional linkage and conservation between proteins without amino acid sequence similarity. To survey the conservative regulatory properties of rate-limiting enzymes (RLEs) in metabolic inhibitory network across different species, we define the enzyme inhibiting pair as: where the first enzyme in a pair is the inhibitor provider and the second is the target of the inhibitor. Phylogenetic profiles of enzymes in the inhibiting pairs are further generated to measure the functional linkage of these enzymes during evolutionary history. We find that the RLEs generate, on average, over half of all in vivo inhibitors in each surveyed model organism. And these inhibitors inhibit on average over 85% targets in metabolic inhibitory network and cover the majority of targets of cross-pathway inhibiting relations. Furthermore, we demonstrate that the phylogenetic profiles of the enzymes in inhibiting pairs in which at least one enzyme is rate-limiting often show higher similarities than those in common inhibiting enzyme pairs. In addition, RLEs, compared to common metabolic enzymes, often tend to produce ADP instead of AMP in conservative inhibitory networks. Combined with the conservative roles of RLEs in their efficiency in sensing metabolic signals and transmitting regulatory signals to the rest of the metabolic system, the RLEs may be important molecules in balancing energy homeostasis via maintaining the ratio of ATP to ADP in living cells. Furthermore, our results indicate that similarities of phylogenetic profiles of enzymes in the inhibiting enzyme pairs are not only correlated with enzyme topological importance, but also related with roles of the enzymes in metabolic inhibitory network.

  2. Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme.

    Science.gov (United States)

    Arques, Carlos G; Doohan, Roisin; Sharpe, James; Torres, Miguel

    2007-10-01

    Regionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.

  3. Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

    International Nuclear Information System (INIS)

    Singh, Bhag; Gupta, Radhey S.

    2006-01-01

    Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase

  4. 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...

  5. 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.

  6. The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast

    Directory of Open Access Journals (Sweden)

    Ana Belén Sanz

    2017-12-01

    Full Text Available Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies.

  7. 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

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  8. Taste buds as peripheral chemosensory processors.

    Science.gov (United States)

    Roper, Stephen D

    2013-01-01

    Taste buds are peripheral chemosensory organs situated in the oral cavity. Each taste bud consists of a community of 50-100 cells that interact synaptically during gustatory stimulation. At least three distinct cell types are found in mammalian taste buds - Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Type I cells appear to be glial-like cells. Receptor cells express G protein-coupled taste receptors for sweet, bitter, or umami compounds. Presynaptic cells transduce acid stimuli (sour taste). Cells that sense salt (NaCl) taste have not yet been confidently identified in terms of these cell types. During gustatory stimulation, taste bud cells secrete synaptic, autocrine, and paracrine transmitters. These transmitters include ATP, acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and GABA. Glutamate is an efferent transmitter that stimulates Presynaptic cells to release 5-HT. This chapter discusses these transmitters, which cells release them, the postsynaptic targets for the transmitters, and how cell-cell communication shapes taste bud signaling via these transmitters. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Evaluation of yeast cell wall on the performance of broiles fed diets with or without mycotoxins

    Directory of Open Access Journals (Sweden)

    E Santin

    2006-12-01

    Full Text Available This experiment aimed at evaluating the effects of the interactions between aflatoxin (500 or 250 ppb and ochratoxin (500 or 250 ppb, and the possible benefits of adding yeast cell wall to prevent the effects of these mycotoxins in broiler chickens. Relative organ weight gain and live performance were evaluated at 21 and 42 days of age. Results indicated that at the levels of mycotoxins included in the experimental diets, ochratoxin reduced feed intake and body weight gain, and aflatoxin only affect feed intake of 21-day-old birds. No interaction was observed between aflatoxin and ochratoxin at the levels used in experimental study. Yeast cell wall did not significantly reduced the deleterious effects of ochratoxins. No significant differences were observed in relative organ weight gain. Yeast cell wall improved feed conversion ratio when birds were fed either contaminated or non-contaminated feeds.

  10. Sexual differentiation in fission yeast

    DEFF Research Database (Denmark)

    Egel, R; Nielsen, O; Weilguny, D

    1990-01-01

    The regulation of sexual reproduction in yeast constitutes the highest level of differentiation observed in these unicellular organisms. The various ramifications of this system involve DNA rearrangement, transcriptional control, post-translational modification (such as protein phosphorylation) a......) and receptor/signal processing. A few basic similarities are common to both fission and budding yeasts. The wiring of the regulatory circuitry, however, varies considerably between these divergent yeast groups....

  11. 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.

  12. 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.

  13. 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.

  14. Breadth of Tuning and Taste Coding in Mammalian Taste Buds

    OpenAIRE

    Tomchik, Seth M.; Berg, Stephanie; Kim, Joung Woul; Chaudhari, Nirupa; Roper, Stephen D.

    2007-01-01

    A longstanding question in taste research concerns taste coding and, in particular, how broadly are individual taste bud cells tuned to taste qualities (sweet, bitter, umami, salty, and sour). Taste bud cells express G-protein-coupled receptors for sweet, bitter, or umami tastes but not in combination. However, responses to multiple taste qualities have been recorded in individual taste cells. We and others have shown previously there are two classes of taste bud cells directly involved in gu...

  15. Processing Umami and Other Tastes in Mammalian Taste Buds

    OpenAIRE

    Roper, Stephen D.; Chaudhari, Nirupa

    2009-01-01

    Neuroscientists are now coming to appreciate that a significant degree of information processing occurs in the peripheral sensory organs of taste prior to signals propagating to the brain. Gustatory stimulation causes taste bud cells to secrete neurotransmitters that act on adjacent taste bud cells (paracrine transmitters) as well as on primary sensory afferent fibers (neurocrine transmitters). Paracrine transmission, representing cell-cell communication within the taste bud, has the potentia...

  16. Immobilization of yeast cells on hydrogel carriers obtained by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Luzhao Xin; Carenza, M.; Kaetsu, Isao; Kumakura, Minoru; Yoshida, Masaru; Fujimura, Takashi

    1992-01-01

    Polymer hydrogels were obtained by radiation-induced copolymerization at -78 o C of aqueous solutions of acrylic and methacrylic esters. The matrices were characterized by equilibrium water content measurements, by optical microscopy observations and by scanning electron microscopy analysis. Yeast cells were immobilized on these hydrogels and the ethanol productivity by batch fermentation was determined. Matrix hydrophilicity and porosity were found to deeply influence the adhesion of yeast cells and, hence, the ethanol productivity. The latter as well as other physico-chemical properties were also affected by the presence of a crosslinking agent added in small amounts to the polymerizating mixture. (author)

  17. Soft x-ray-controlled dose deposition in yeast cells: techniques, model, and biological assessment

    Science.gov (United States)

    Milani, Marziale; Batani, Dimitri; Conti, Aldo; Masini, Alessandra; Costato, Michele; Pozzi, Achille; Turcu, I. C. Edmond

    1996-12-01

    A procedure is presented to release soft x-rays onto yeast cell membrane allegedly damaging the resident enzymatic processes connected with fermentation. The damage is expected to be restricted to regulating fermentation processes without interference with respiration. By this technique fermentation is followed leading to CO2 production, and respiration resulting in global pressure measurements. A solid state pressure sensor system has been developed linked to a data acquisition system. Yeast cells cultures have been investigated at different concentrations and with different nutrients. A non-monotone response in CO2 production as a function of the delivered x-ray dose is observed.

  18. Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway.

    Science.gov (United States)

    Serrano, Raquel; Martín, Humberto; Casamayor, Antonio; Ariño, Joaquín

    2006-12-29

    Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.

  19. Radiation induced formation of giant cells (Saccharomyces uvarum). Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Baumstark-Khan, C; Schnitzler, L; Rink, H

    1984-02-01

    X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using Calcofluor White, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis.

  20. Radiation induced formation of giant cells (Saccharomyces uvarum). Pt. 1

    International Nuclear Information System (INIS)

    Baumstark-Khan, C.; Schnitzler, L.; Rink, H.

    1984-01-01

    X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using Calcofluor White, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis. (orig.)

  1. Immobilization of yeast cells with ionic hydrogel produced by radiation polymerization

    International Nuclear Information System (INIS)

    Lu Zhaoxin; Fujimura, T.

    1990-01-01

    The mixture of an ionic monomer of 2-acrylamido 2-methylpropane-sulfonic acid and a series of polyethylene glycol dimethacrylate monomer were polymerized at-78 deg C with 60 Co γ-rays and were used for immobilization of yeast cells. The immobilized yeast cells with these carriers had higher ethanol productivity than that without any carriers. The yield of ethanol with poly TBAS-14G carrier was the highest, and increased by 3.5 times compared with the free yeast cells. It was found that the ethanol yield increased with the increase of the glycol number in polyethylene glycol dimethacrylate. The state of the immobilized cells was observed with microscope and it was found that the difference in the ethanol productivity was mainly due to the difference in the internal structure and the properties of polymer carrier. It was considered that the polymer carrier had a proper hydrophilicity, swelling ability, cation in the surface and porousity in the internal structure for immobilizing yeast cells

  2. Introduction of the yeast DNA repair gene PHR1 into normal and xeroderma pigmentosum human cells

    International Nuclear Information System (INIS)

    Whyte, D.B.

    1988-01-01

    The goal of the work described herein is to determine how UV light kills and mutates human cells. Specifically, the hypothesis to be tested states that the major cause of cell death is the cyclobutane dimer. The yeast (S. cerevisiae) enzyme photolyase provides an elegant means of dissecting the biological effects of the two lesions. Photolyase, the product of the PHR1 gene, catalyzes the visible light-dependent reversal of cyclobutane pyrimidine dimers. Introducing the gene for photolyase into human cells, which do not have a functional photoreactivation mechanism, should allow specific repair of cyclobutane pyrimidine dimers. To express the yeast DNA repair gene in human cells, the yeast PHR1 coding sequence was cloned into the mammalian expression vector pRSV4NEO-I. The resulting plasmid, pRSVPHR1, contains the coding sequence of the yeast gene, under control of transcription signals recognized by mammalian cells, and the dominant selectable gene neo. pRSVPHR1 was introduced into normal and XP SV40-transformed fibroblasts by the calcium phosphate coprecipitation technique, and G418-resistant clones were isolated. The level of PHR1 expression was determined by cytoplasmic RNA dot blots. Two clones, XP-3B and GM-20A, had high levels of expression

  3. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    Directory of Open Access Journals (Sweden)

    Elham Aslankoohi

    Full Text Available Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.

  4. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    Science.gov (United States)

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.

  5. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection.

    Science.gov (United States)

    Li, Yi-Ke; Yang, Juan-Mei; Huang, Yi-Bo; Ren, Dong-Dong; Chi, Fang-Lu

    2015-06-01

    The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

  6. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection

    Directory of Open Access Journals (Sweden)

    Yi-ke Li

    2015-01-01

    Full Text Available The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

  7. 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.

  8. Propagation of Mammalian Prions in Yeast

    National Research Council Canada - National Science Library

    Harris, David A

    2006-01-01

    ...: the budding yeast Saccharomyces cerevisiae. This unicellular organism offers a number of potential advantages for the study of prion biology, including rapid generation time, ease of culturing, and facile genetics...

  9. Modifying infrared scattering effects of single yeast cells with plasmonic metal mesh

    Science.gov (United States)

    Malone, Marvin A.; Prakash, Suraj; Heer, Joseph M.; Corwin, Lloyd D.; Cilwa, Katherine E.; Coe, James V.

    2010-11-01

    The scattering effects in the infrared (IR) spectra of single, isolated bread yeast cells (Saccharomyces cerevisiae) on a ZnSe substrate and in metal microchannels have been probed by Fourier transform infrared imaging microspectroscopy. Absolute extinction [(3.4±0.6)×10-7 cm2 at 3178 cm-1], scattering, and absorption cross sections for a single yeast cell and a vibrational absorption spectrum have been determined by comparing it to the scattering properties of single, isolated, latex microspheres (polystyrene, 5.0 μm in diameter) on ZnSe, which are well modeled by the Mie scattering theory. Single yeast cells were then placed into the holes of the IR plasmonic mesh, i.e., metal films with arrays of subwavelength holes, yielding "scatter-free" IR absorption spectra, which have undistorted vibrational lineshapes and a rising generic IR absorption baseline. Absolute extinction, scattering, and absorption spectral profiles were determined for a single, ellipsoidal yeast cell to characterize the interplay of these effects.

  10. 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…

  11. 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

  12. 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.

  13. Obtaining sorbents of metal ions based on yeast cells Rhodotorula glutinis

    Directory of Open Access Journals (Sweden)

    Zh. Tattibayeva

    2013-05-01

    Full Text Available Ability to separate Cu2+ and Pb2+ ions from solution using yeast cells Rhodotorulа glutinis were considered. The degree of water purification in this case is of 60-70%. To increase the degree of binding of metal ions with cells and facilitate separation processes of water sorbents their immobilization on the surface of the water in the presence of polyethyleneimine was carried out. It is shown that under optimal conditions on the surface of 1 g diatomite 18 ∙ 106 cells is adsorbed. The high sorption capacity of diatomite justified its porosity. IR spectroscopic study of the interaction of the ions Cu2+ and Pb2+ with cell surface showed that high affinity Pb2 + ions to the surface of yeast cells is connected with form of slightly soluble compounds with the phosphate ions.

  14. Nutrient sensing and TOR signaling in yeast and mammals.

    Science.gov (United States)

    González, Asier; Hall, Michael N

    2017-02-15

    Coordinating cell growth with nutrient availability is critical for cell survival. The evolutionarily conserved TOR (target of rapamycin) controls cell growth in response to nutrients, in particular amino acids. As a central controller of cell growth, mTOR (mammalian TOR) is implicated in several disorders, including cancer, obesity, and diabetes. Here, we review how nutrient availability is sensed and transduced to TOR in budding yeast and mammals. A better understanding of how nutrient availability is transduced to TOR may allow novel strategies in the treatment for mTOR-related diseases. © 2017 The Authors.

  15. Histologic assessment of tumor budding in preoperative biopsies to predict nodal metastasis in squamous cell carcinoma of the tongue and floor of the mouth.

    Science.gov (United States)

    Seki, Mai; Sano, Takaaki; Yokoo, Satoshi; Oyama, Tetsunari

    2016-04-01

    In squamous cell carcinoma (SCC) of the tongue and the floor of the mouth (FOM), it is important to predict lymph node metastasis, including occult metastasis, before operating. The purpose of this study was for us to determine practical histopathologic parameters as predictive factors for lymph node metastasis in preoperative SCC biopsy specimens. We examined 91 cases of SCC for conventional histopathologic assessment and a new factor, tumor budding, and their relationship with lymph node metastasis. Significant factors via univariate analysis (p factoring into the decision as to whether neck dissection is indicated. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1582-E1590, 2016. © 2015 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Smith, Ida Mosbech; Baker, A; Arneborg, Nils

    2015-01-01

    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, 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......). 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. SIGNIFICANCE AND IMPACT...

  17. What Are Taste Buds?