Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells.

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation.

Reactive oxygen species (ROS), play important roles in cellular signaling, nonetheless are toxic at higher concentrations. Cells have many interconnected, overlapped or backup systems to neutralize ROS, but their regulatory mechanisms remain poorly understood. Here, we reveal an essential role for mitochondrial AMPylase Fmp40 from budding yeast in regulating the redox states of the mitochondrial 1-Cys peroxiredoxin Prx1, which is the only protein shown to neutralize H2O2 with the oxidation of the mitochondrial glutathione and the thioredoxin Trx3, directly involved in the reduction of Prx1. Deletion of FMP40 impacts a cellular response to H2O2 treatment that leads to programmed cell death (PCD) induction and an adaptive response involving up or down regulation of genes encoding, among others the catalase Cta1, PCD inducing factor Aif1, and mitochondrial redoxins Trx3 and Grx2. This ultimately perturbs the reduced glutathione and NADPH cellular pools. We further demonstrated that Fmp40 AMPylates Prx1, Trx3, and Grx2 in vitro and interacts with Trx3 in vivo. AMPylation of the threonine residue 66 in Trx3 is essential for this protein's proper endogenous level and its precursor forms' maturation under oxidative stress conditions. Additionally, we showed the Grx2 involvement in the reduction of Trx3 in vivo. Taken together, Fmp40, through control of the reduction of mitochondrial redoxins, regulates the hydrogen peroxide, GSH and NADPH signaling influencing the yeast cell survival.

Microbial cell autofluorescence as a method for measuring the intracellular content of B2 and B6 vitamins.

Vitamins are important organic compound required for the proper functioning of cells and organisms. Vitamins of special industrial and pharmaceutical interests include riboflavin (vitamin B2) and pyridoxine (vitamin B6). Commercial production of those biological compounds has increasingly relied on microorganisms and requires simple methods for detecting and estimating their level of synthesis during the biotechnological process. In the case of yeast, methods based on autofluorescence, i.e. natural fluorescence emitted by several cellular compounds, including vitamins, may be useful. Considering that the intensity of emitted light is proportional to the intracellular concentration of riboflavin and pyridoxine, autofluorescence may be a convenient method for their quantification. In this report, we demonstrate a simple, rapid, and sufficiently trustworthy spectrofluorimetric method for determining the content of vitamins B2 and B6 in yeast cells which consists of cells growing, harvesting, washing, and resuspending in a buffer, and then measuring the emitted visible light using specific wavelength of excitation (λex=340 nm and λem=385 nm for pyridoxine; λex=460 nm and λem=535 nm for riboflavin). The limits of detection (LOD) and quantification (LOQ) estimated through measurements of vitamin fluorescence were below 0.005 µg/ml for riboflavin and below 0.05 µg/ml for pyridoxine, respectively. In turn, the smallest credible cell density for measuring autofluorescence was set at 1×108 yeast cells/ml. The relative level of the cell's autofluorescence can be expressed in mass units by applying proper calculation formulas. A comparison of the autofluorescence-based method with the reference HPLC-UV method shows that autofluorescence measurement can be used in the screening analysis of vitamin content (especially riboflavin) in microbial cells.

Redox perturbations in yeast cells lacking glutathione reductase.

Cellular redox homeostasis has a major effect on cell functions and its maintenance is supported by glutathione and protein thiols which serve as redox buffers in cells. The regulation of the glutathione biosynthetic pathway is a focus of a lot of scientific research. However, still little is known about how complex cellular networks influence glutathione homeostasis. In this work was used an experimental system based on an S. cerevisiae yeast mutant with a lack of the glutathione reductase enzyme and allyl alcohol as a precursor of acrolein inside the cell to determine the cellular processes influencing glutathione homeostasis. The absence of Glr1p slows down the growth rate of the cell population, especially in the presence of allyl alcohol, but does not lead to complete inhibition of the cell's reproductive capacity. It also amends the GSH/GSSG ratio and the share of NADPH and NADP+ in the total NADP(H) pool. The obtained results show that potential pathways involved in the maintenance of redox homeostasis are based from one side on de novo synthesis of GSH as indicated by increased activity of γ-GCS and increased expression of GSH1 gene in the Δglr1 mutant, from the other hand, on increased the level of NADPH. This is because the lower ratio of GSH/GSSG can be counterbalanced with the NADPH/NADP+ alternative system. The higher level of NADPH can be used by the thioredoxin system and other enzymes requiring NADPH to reduce cytosolic GSSG and maintain glutathione redox potential.

Different life strategies in genetic backgrounds of the Saccharomyces cerevisiae yeast cells.

Changes in the natural environment require an organism to make constant adaptations enabling efficient use of environmental resources and ensuring its success in competition with other organisms. Such adaptations are expressed through various life strategies, largely determined by the rate of consumption and use of available resources, affecting the life-history traits and the related trade-offs. Allocation of available resources must take into consideration the costs of cell maintenance as well as reproduction. Given that carbon metabolism plays a crucial role in resource allocation, yeast living in different ecological niches show various life-history traits. There are a lot of data about life-history strategies in yeast living in various ecological niches; however, the question is whether different life strategies will be noted for yeast strains growing under strictly controlled conditions. Our studies based on three laboratory yeast strains representing different genetic backgrounds show that each of these strains has specified life strategies which are mainly determined by the glucose uptake rate and its intracellular usage. These results suggest that specific life strategies and related differences in the physiological and metabolic parameters of the cell are the key aspects that may explain various features of cells from different yeast strains, either industrial or laboratory.

Unbalance between Pyridine Nucleotide Cofactors in The SOD1 Deficient Yeast Saccharomyces cerevisiae Causes Hypersensitivity to Alcohols and Aldehydes.

Alcohol and aldehyde dehydrogenases are especially relevant enzymes involved in metabolic and detoxification reactions that occur in living cells. The comparison between the gene expression, protein content, and enzymatic activities of cytosolic alcohol and aldehyde dehydrogenases of the wild-type strain and the Δsod1 mutant lacking superoxide dismutase 1, which is hypersensitive to alcohols and aldehydes, shows that the activity of these enzymes is significantly higher in the Δsod1 mutant, but this is not a mere consequence of differences in the enzymatic protein content nor in the expression levels of genes. The analysis of the NAD(H) and NADP(H) content showed that the higher activity of alcohol and aldehyde dehydrogenases in the Δsod1 mutant could be a result of the increased availability of pyridine nucleotide cofactors. The higher level of NAD+ in the Δsod1 mutant is not related to the higher level of tryptophan; in turn, a higher generation of NADPH is associated with the upregulation of the pentose phosphate pathway. It is concluded that the increased sensitivity of the Δsod1 mutant to alcohols and aldehydes is not only a result of the disorder of redox homeostasis caused by the induction of oxidative stress but also a consequence of the unbalance between pyridine nucleotide cofactors.

Multifaceted role of glucose and its metabolism in the regulation of physiological parameters and reproductive potential of the cells on the example of research using the yeast Saccharomyces cerevisiae / Wieloaspektowa rola glukozy i jej metabolizmu w regulacji parametrów fizjologicznych i potencjalu reprodukcyjnego komórek na podstawie badan z uzyciem drozdzy Saccharomyces cerevisiae.

Glucose is not only the primary source of energy, but also a compound which plays an important role in the metabolism and maintenance of the proper physiological state of the cell. This is particularly pronounced in the case of yeasts, in which the influence of glucose on the physiological state of the cell is directly manifested. Among other by obtaining energy through fermentation or aerobic respiration depending on the availability of glucose. Glucose-dependent modulation of intracellular metabolic pathways influence on the reproductive potential and lifespan of the cells, what links glucose with calorie restriction studies. At the same time, there is a noticeable lack of data concerning the calorie excess and its consequences at the cellular level. Using the yeast Saccharomyces cerevisiae cells as a research model, a significant relationship between glucose concentration, biosynthetic efficiency, reproductive potential and total lifespan of yeast cells was found. High glucose concentrations, corresponding to the calorie excess conditions, lead to an increase in the level of reactive oxygen species, an increase in cell size and cell biomass, but at the same time, it reduces the reproductive potential and shortens the total lifespan of the yeast cell. The negative impact of glucose excess on the physiological state of the cell as well as the complexity and interrelationships of intracellular metabolic pathways suggest that the issue of glucose metabolism need further investigations.

Reproductive Potential of Yeast Cells Depends on Overall Action of Interconnected Changes in Central Carbon Metabolism, Cellular Biosynthetic Capacity, and Proteostasis.

Carbon metabolism is a crucial aspect of cell life. Glucose, as the primary source of energy and carbon skeleton, determines the type of cell metabolism and biosynthetic capabilities, which, through the regulation of cell size, may affect the reproductive capacity of the yeast cell. Calorie restriction is considered as the most effective way to improve cellular physiological capacity, and its molecular mechanisms are complex and include several nutrient signaling pathways. It is widely assumed that the metabolic shift from fermentation to respiration is treated as a substantial driving force for the mechanism of calorie restriction and its influence on reproductive capabilities of cells. In this paper, we propose another approach to this issue based on analysis the connection between energy-producing and biomass formation pathways which are closed in the metabolic triangle, i.e., the respiration-glycolysis-pentose phosphate pathway. The analyses were based on the use of cells lacking hexokinase 2 (∆hxk2) and conditions of different glucose concentration corresponding to the calorie restriction and the calorie excess. Hexokinase 2 is the key enzyme involved in central carbon metabolism and is also treated as a calorie restriction mimetic. The experimental model used allows us to explain both the role of increased respiration as an effect of calorie restriction but also other aspects of carbon metabolism and the related metabolic flux in regulation of reproductive potential of the cells. The obtained results reveal that increased respiration is not a prerequisite for reproductive potential extension but rather an accompanying effect of the positive role of calorie restriction. More important seems to be the changes connected with fluxes in central carbon metabolic pathways resulting in low biosynthetic capabilities and improved proteostasis.

Linkage between Carbon Metabolism, Redox Status and Cellular Physiology in the Yeast Saccharomyces cerevisiae Devoid of SOD1 or SOD2 Gene.

Saccharomyces cerevisiae yeast cells may generate energy both by fermentation and aerobic respiration, which are dependent on the type and availability of carbon sources. Cells adapt to changes in nutrient availability, which entails the specific costs and benefits of different types of metabolism but also may cause alteration in redox homeostasis, both by changes in reactive oxygen species (ROS) and in cellular reductant molecules contents. In this study, yeast cells devoid of the SOD1 or SOD2 gene and fermentative or respiratory conditions were used to unravel the connection between the type of metabolism and redox status of cells and also how this affects selected parameters of cellular physiology. The performed analysis provides an argument that the source of ROS depends on the type of metabolism and non-mitochondrial sources are an important pool of ROS in yeast cells, especially under fermentative metabolism. There is a strict interconnection between carbon metabolism and redox status, which in turn has an influence on the physiological efficiency of the cells. Furthermore, pyridine nucleotide cofactors play an important role in these relationships.

Less is more or more is less: Implications of glucose metabolism in the regulation of the reproductive potential and total lifespan of the Saccharomyces cerevisiae yeast.

Carbohydrates are dietary nutrients that have an influence on cells physiology, cell reproductive capacity and, consequently, the lifespan of organisms. They are used in cellular processes after conversion to glucose, which is the primary source of energy and carbon skeleton for biosynthetic processes. Studies of the influence of glucose on cellular parameters and lifespan of organisms are primarily concerned with the effect of low glucose concentration defined as calorie restriction conditions. However, the effect of high glucose concentration on cell physiology is also very important. Thus, a comparative analysis of the effects of low and high glucose concentration conditions on cell efficiency was proposed with regard to reproductive capacity and total lifespan of the cell. Glucose concentration determines the type of metabolism and biosynthetic capabilities, which in turn, through the regulation on the cell size, may affect the reproductive capacity of cells. This study was conducted on yeast cells of wild-type and mutant strains Δgpa2 and Δgpr1 with glucose signalling pathway impairment. Such an experimental model enabled testing both the role of glucose concentration in the regulation of metabolic changes and the extent to which these changes depend on the extracellular or intracellular glucose concentrations. It has been shown here that calorie/glucose excess connected with changes in cell metabolic fluxes increases biosynthetic capabilities of yeast cells. This leads to an increase in cell dry weight accompanied by the increase in cell size and a simultaneous decrease in the reproductive potential and the overall length of cell life.

Disorders in NADPH generation via pentose phosphate pathway influence the reproductive potential of the Saccharomyces cerevisiae yeast due to changes in redox status.

Intermediary metabolites have a crucial impact on basic cell functions. There is a relationship between cellular metabolism and redox balance. To maintain redox homoeostasis, the cooperation of both glutathione and nicotine adenine dinucleotides is necessary. Availability of nicotinamide adenine dinucleotide phosphate (NADPH) as a major electron donor is critical for many intracellular redox reactions. The activity of glucose-6-phosphate dehydrogenase (Zwf1p) and 6-phosphogluconate dehydrogenase (Gnd1p and Gnd2p) is responsible for NADPH formation in a pentose phosphate (PP) pathway. In this study, we examine the impact of redox homoeostasis on cellular physiology and proliferation. We have noted that the Δzwf1 mutant lacking the rate-limiting enzyme of the PP pathway shows changes in the cellular redox status caused by disorders in NADPH generation. This leads to a decrease in reproductive potential but without affecting the total lifespan of the cell. The results presented in this paper show that nicotine adenine dinucleotides play a central role in cellular physiology.

Autofluorescence of yeast Saccharomyces cerevisiae cells caused by glucose metabolism products and its methodological implications.

Autofluorescence is the natural fluorescence emitted by cellular compounds which have light emission properties. The main examples of these compounds, identified as an endogenous fluorophores, include aromatic amino acids, vitamins, coenzymes and electron acceptors. As many of them play a critical role in cell metabolism, changes in their content may provide important information on the physiological status of the cell. Nevertheless, the simultaneous occurrence of different endogenous fluorophores in cells makes it difficult to interpret the autofluorescence signal. Autofluorescence values may also be imposed on values obtained through exogenous fluorescent dyes. This study evaluates the origin and the methodological implications of autofluorescence observed in yeast cells. The results show that the level of autofluorescence may differ between yeast cells, which are a result of different concentrations of endogenous fluorophores, including tryptophan, pyridoxine and riboflavin. The study also shows an important influence of autofluorescence on the results obtained by methods based on external fluorescent dyes.

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

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

[Duplication of DNA--a mechanism for the development of new functionality of genes]. / Duplikacja DNA--mechanizm rozwoju nowej funkcjonalnosci genów.

The amplification of DNA is considered as a mechanism for rapid evolution of organisms. Duplication can be especially advantageous in the case of changing environmental conditions. Whole genome duplication maintains the proper balance between gene expression. This seems to be the main reason why WGD is more favorable than duplication of the fragments of DNA. The polyploidy status disappear as a result of the loss of the majority of duplicated genes. The preservation of duplicated genes is associated with the development of their new functions. Polyploidization is often noted for plants. However due to sequencing technique, the duplications episodes are more frequently reports also for the other systematic taxa, including animals. The occurrence of ancient genome duplication is also considered for yeast Saccharomyces cerevisiae. The existence of two active copies of ribosomal protein genes can be a confirmation of this process. Development of the fermentation process might be one of the probable causes of the yeast genome duplication.