Marina Vai

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Juan Carlos Moreno Garcia

Centre National de la Recherche Scientifique / French National Centre for Scientific Research

che pillay

University of KwaZulu-Natal

Jean-Luc Legras

Institut National de la Recherche Agronomique

Roma Siugzdaite

Ghent University

Francesca Stavrakopoulou

University of Exeter

Ilia Palaguta

Saint-Petersburg Stieglitz State Academy of Art and Design

Carlo Semenza

Università degli Studi di Padova

Gonzalo Bello

Fundação Oswaldo Cruz

Arif Celebi

Bezmialem Vakif University

Prof.Dr. Abdulkadir Koçer

Istanbul Medeniyet University

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Papers by Marina Vai

Three-dimensional structure of the catalytic domain of the yeast β-(1,3)-glucan transferase Gas1: a molecular modeling investigation

Journal of Molecular Modeling, Oct 21, 2005

Three-dimensional structure of the catalytic domain of chitinase Al Bacillus circulars WI-12 at a... more

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Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Journal of Visualized Experiments, Jun 13, 2018

Cover slips 24 x 50 mm thermo scientific 11911998

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A modular model integrating metabolism, growth, and cell cycle predicts that fermentation is required to modulate cell size in yeast populations

For unicellular organisms, the reproduction rate and growth are crucial determinants of fitness a... more For unicellular organisms, the reproduction rate and growth are crucial determinants of fitness and, therefore, essential functional manifestations of the organism genotype. Using the budding yeast Saccharomyces cerevisiae as a model organism, we integrated metabolism, which provides energy and building blocks for growth, with cell mass growth and cell cycle progression into a low-granularity, multiscale (from cell to population) computational model. This model predicted that cells with constitutive respiration do not modulate cell size according to the growth conditions. We experimentally validated the model predictions using mutants with defects in the upper part of glycolysis or glucose transport. Plugging in molecular details of cellular subsystems allowed us to refine predictions from the cellular to the molecular level. Our hybrid multiscale modeling approach provides a framework for structuring molecular knowledge and predicting cell phenotypes under various genetic and envir...

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Improved secretion of native human IGF-1 from gas1 Saccharomyces cerevisiae cells

InSaccharomyces cerevisiae overexpression of hybrid Virus-Like-Particles correlates with altered cell volume distributions

Biotechnology Letters, Nov 1, 1994

Overexpression of hybrid Virus-Like-Particles (VLP) was found to dramatically affect cell kinetic... more Overexpression of hybrid Virus-Like-Particles (VLP) was found to dramatically affect cell kinetics and morphology with a significant increase in doubling times, induction times and major alterations in cell volume distributions. Optimal expression (ca. 10 rag/l) was obtained with a two-step fermentation strategy (raffinose ~ galactose) coupled to GAL4 overexpression, resulting in partial cell lysis and VLP release.

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Probing Control Mechanisms of Cell Cycle and Ageing in Budding Yeast

Current Genomics, Dec 1, 2004

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Effects of temperature on the yeast cell cycle analyzed by flow cytometry

Cytometry, Sep 1, 1984

The effects of temperature (in the range 15–36°C) on growth and the nuclear and budding cycle hav... more The effects of temperature (in the range 15–36°C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30°C, and a transition point is apparent at about 20°C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21–36°C, while duration of G2+M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell‐cycle model for budding yeast.

Characterization and functional analysis of the Î²-1,3-glucanosyltransferase 3 of the human pathogenic fungusParacoccidioides brasiliensis

Fems Yeast Research, Feb 1, 2009

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous ... more The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, b-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The fulllength cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1D mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a b-1,3-glucan elongase capable of mediating fungal cell wall integrity.

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During yeast chronological aging resveratrol supplementation results in a short-lived phenotype Sir2-dependent

Redox biology, Aug 1, 2017

Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biologi... more Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD +-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension.

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Deletion or Overexpression of Mitochondrial NAD + Carriers in Saccharomyces cerevisiae Alters Cellular NAD and ATP Contents and Affects Mitochondrial Metabolism and the Rate of Glycolysis

Applied and Environmental Microbiology, Apr 1, 2011

The modification of enzyme cofactor concentrations can be used as a method for both studying and ... more The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

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Glycosylphosphatidylinositol-anchored Glucanosyltransferases Play an Active Role in the Biosynthesis of the Fungal Cell Wall

Journal of Biological Chemistry, May 1, 2000

A novel 1,3-␤-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was rece... more A novel 1,3-␤-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-␤-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1,3-␤-glucan molecule forming a 1,3-␤ linkage, resulting in the elongation of 1,3-␤-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-␤-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-␤-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.

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Identification of a glycoprotein involved in cell cycle progression in yeast

Journal of Biological Chemistry, Mar 1, 1986

The molecular events of start, the regulatory step that commits yeast cells to DNA replication, h... more The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated. One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity. Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae. On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1. Pulse and chase labeling of protein with [35S]methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular weight (Mr = 115,000). Peptide mapping analysis indicates that p100 and p100 yield identical maps and that both p100 and p100* are very much similar to p115. p115 is a glycosylated protein as shown by a labeling experiment with [3H]glucosamine and by the fact that the synthesis of both p100 and p115 is inhibited if cells are cultured in the presence of tunicamycin. A protein having the same heterogeneous aspect of migration on sodium dodecyl sulfate-polyacrylamide gel and the same apparent molecular weight and isoelectric point of p115 is abundantly present in a preparation of membranes from S. cerevisiae and the isolated radioactive p115 comigrates with it. Taken together these results favor the idea that terminal glycosylation of both p100 and p100* gives rise to the fully glycosylated p115 protein which appears to be a membrane-associated protein.

Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study

Microbiology, 2008

Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribo... more Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribosome biogenesis. During growth in shake flasks, a mutant deleted for SFP1 shows a small size phenotype and a reduced growth rate. We characterized the behaviour of an sfp1D mutant compared to an isogenic reference strain growing in chemostat cultures at the same specific growth rate. By studying glucose (anaerobic)-and ethanol (aerobic)-limited cultures we focused specifically on nutrient-dependent effects. Major differences in the genome-wide transcriptional profiles were observed during glucose-limited growth. In particular, Sfp1 appeared to be involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Flow cytometric analyses revealed size defects for the mutant under both growth conditions. Our results suggest that Sfp1 plays a role in transcriptional and cell size control, operating at two different levels of the regulatory network linking growth, metabolism and cell size.

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Defects in Assembly of the Extracellular Matrix Are Responsible for Altered Morphogenesis of a Candida albicans phr1 Mutant

Journal of Bacteriology, 1998

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Diel modulation of Hsp70 and Hsp60 in corals living in a shallow reef

Coral Reefs, Jun 1, 2018

In corals, different biological processes are associated with the daily variation of temperature ... more In corals, different biological processes are associated with the daily variation of temperature and light, which in shallow reefs can vary widely and regularly reach extreme levels. The expression of cellular defense mechanisms such as the heat shock proteins Hsp70 and Hsp60 was analyzed in coral colonies adapted to live in a shallow lagoon, during three consecutive days characterized by the same temperature/light trend. Each of the three species investigated showed a significant diel modulation of both Hsps, which also displayed a coordinated expression. However, the diel modulations of both Hsps were species specific and only that of the branching Acropora tenuis appeared correlated with the temperature. All the species displayed high Hsp levels at 13.00 and 16.00, probably as protection for the cellular stress produced by the elevated temperature and light. Taken together, the results may provide useful information about the cellular mechanisms involved in the corals adaptation to extreme conditions.

Lack of Ach1 CoA-Transferase Triggers Apoptosis and Decreases Chronological Lifespan in Yeast

Frontiers in Oncology, 2012

ACH1 encodes a mitochondrial enzyme of Saccharomyces cerevisiae endowed with CoAtransferase activ... more ACH1 encodes a mitochondrial enzyme of Saccharomyces cerevisiae endowed with CoAtransferase activity. It catalyzes the CoASH transfer from succinyl-CoA to acetate generating acetyl-CoA. It is known that ACH1 inactivation results in growth defects on media containing acetate as a sole carbon and energy source which are particularly severe at low pH. Here, we show that chronological aging ach1∆ cells which accumulate a high amount of extracellular acetic acid display a reduced chronological lifespan. The faster drop of cell survival is completely abrogated by alleviating the acid stress either by a calorie restricted regimen that prevents acetic acid production or by transferring chronologically aging mutant cells to water. Moreover, the short-lived phenotype of ach1∆ cells is accompanied by reactive oxygen species accumulation, severe mitochondrial damage, and an early insurgence of apoptosis. A similar pattern of endogenous severe oxidative stress is observed when ach1∆ cells are cultured using acetic acid as a carbon source under acidic conditions. On the whole, our data provide further evidence of the role of acetic acid as cell-extrinsic mediator of cell death during chronological aging and highlight a primary role of Ach1 enzymatic activity in acetic acid detoxification which is important for mitochondrial functionality.

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The susceptibility of corals to thermal stress by analyzing Hsp60 expression

Marine Environmental Research, Aug 1, 2014

Due to the increasing frequency and severity of the coral bleaching events in the context of glob... more Due to the increasing frequency and severity of the coral bleaching events in the context of global warming, there is an urgent need to improve our understanding of the susceptibility of corals to thermal stresses, particularly at the sub-cellular level. In this context, we examined the modulation of the polyp mitochondrial Hsp60 in three scleractinian coral species (Seriatopora hystrix, Montipora monasteriata and Acropora echinata) under simulated heat shock bleaching at 34 °C during a time course of 36 h. All three species displayed a similar initial increase of Hsp60 level which accompanies the increasing paleness of coral tissue. Afterwards, each of them showed a specific pattern of Hsp60 down-regulation which can be indicative of a different threshold of resistance, although it proceeded in synchrony with the complete bleaching of tissues. The finely branched S. hystrix was the species most susceptible to heat stress while the plating M. monasteriata was the most tolerant one, as its Hsp60 down-regulation was less rapid than the branching corals. On the whole, the Hsp60 modulation appears useful for providing information about the susceptibility of the different coral taxa to environmental disturbances.

Ethanol and Acetate Acting as Carbon/Energy Sources Negatively Affect Yeast Chronological Aging

Oxidative Medicine and Cellular Longevity, 2013

In Saccharomyces cerevisiae, the chronological lifespan (CLS) is defined as the length of time th... more In Saccharomyces cerevisiae, the chronological lifespan (CLS) is defined as the length of time that a population of nondividing cells can survive in stationary phase. In this phase, cells remain metabolically active, albeit at reduced levels, and responsive to environmental signals, thus simulating the postmitotic quiescent state of mammalian cells. Many studies on the main nutrient signaling pathways have uncovered the strong influence of growth conditions, including the composition of culture media, on CLS. In this context, two byproducts of yeast glucose fermentation, ethanol and acetic acid, have been proposed as extrinsic proaging factors. Here, we report that ethanol and acetic acid, at physiological levels released in the exhausted medium, both contribute to chronological aging. Moreover, this combined proaging effect is not due to a toxic environment created by their presence but is mainly mediated by the metabolic pathways required for their utilization as carbon/energy sources. In addition, measurements of key enzymatic activities of the glyoxylate cycle and gluconeogenesis, together with respiration assays performed in extreme calorie restriction, point to a long-term quiescent program favoured by glyoxylate/gluconeogenesis flux contrary to a proaging one based on the oxidative metabolism of ethanol/acetate via TCA and mitochondrial respiration.

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Towards understanding of the complex structure of growing yeast populations

Journal of Biotechnology, Feb 1, 2007

In a growing Saccharomyces cerevisiae population, cell size is finely modulated according to both... more In a growing Saccharomyces cerevisiae population, cell size is finely modulated according to both the chronological and genealogical ages. This generates the complex heterogeneous structure typical of budding yeast populations. In recent years, there has been a growing interest in developing mathematical models capable of faithfully describing population dynamics at the single cell level. A multistaged morphologically structured model has been lately proposed based on the population balance theory. The model was able to describe the dynamics of the generation of a heterogeneous growing yeast population starting from a sub-population of daughter unbudded cells. In this work, which aims at validating the model, the simulated experiment was performed by following the release of a homogeneous population of daughter unbudded cells. A biparametric flow cytometric approach allowed us to analyse the time course joint distribution of DNA and protein contents at the single cell level; this gave insights into the coupling between growth and cell cycle progression that generated the final population structure. The comparison between experimental and simulated size distributions revealed a strong agreement for some unexpected features as well. Therefore, the model can be considered as validated and extendable to more complex situations.

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Assays for Monitoring the Effects of Nicotinamide Supplementation on Mitochondrial Activity in Saccharomyces cerevisiae

Methods in molecular biology, 2020

The single-celled yeast Saccharomyces cerevisiae is one of the most valuable laboratory models th... more The single-celled yeast Saccharomyces cerevisiae is one of the most valuable laboratory models that has been used successfully to identify factors and pathways involved in several cellular processes, the counterparts of which are evolutionarily conserved. Furthermore, it is also a powerful tool for analyzing the effects of molecules of nutraceutical interest with the view of leading to human health benefits and improving the quality of aging. In this context, we present some of the methods that have allowed us to assess the beneficial influence of a form of vitamin B3, namely nicotinamide, on mitochondrial functionality during yeast chronological aging. Mitochondrial dysfunctions are considered to be hallmarks of aging, and of several metabolic and neurodegenerative diseases. More specifically, these methods concern the determination of the respiratory parameters in intact cells in order to evaluate the efficiency of mitochondrial respiration in concert with the risk of superoxide anion (O2-) production, which results from inefficient respiration. In addition, we describe fluorescent staining specific for O2- detection and mitochondrial membrane potential, as well as a simple clonogenic assay based on the ability of cells to grow on a carbon source that requires a functional mitochondrial metabolism.

Three-dimensional structure of the catalytic domain of the yeast β-(1,3)-glucan transferase Gas1: a molecular modeling investigation

Journal of Molecular Modeling, Oct 21, 2005

Three-dimensional structure of the catalytic domain of chitinase Al Bacillus circulars WI-12 at a... more

Download

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Journal of Visualized Experiments, Jun 13, 2018

Cover slips 24 x 50 mm thermo scientific 11911998

Download

A modular model integrating metabolism, growth, and cell cycle predicts that fermentation is required to modulate cell size in yeast populations

Download

Improved secretion of native human IGF-1 from gas1 Saccharomyces cerevisiae cells

InSaccharomyces cerevisiae overexpression of hybrid Virus-Like-Particles correlates with altered cell volume distributions