Adaptive Role of Cell Death in Yeast Communities Stressed with Macrolide Antifungals.

Microorganisms cooperate with each other to protect themselves from environmental stressors. An extreme case of such cooperation is regulated cell death for the benefit of other cells. Dying cells can provide surviving cells with nutrients or induce their stress response by transmitting an alarm signal; however, the role of dead cells in microbial communities is unclear. Here, we searched for types of stressors the protection from which can be achieved by death of a subpopulation of cells. Thus, we compared the survival of Saccharomyces cerevisiae cells upon exposure to various stressors in the presence of additionally supplemented living versus dead cells. We found that dead cells contribute to yeast community resistance against macrolide antifungals (e.g., amphotericin B [AmB] and filipin) to a greater extent than living cells. Dead yeast cells absorbed more macrolide filipin than control cells because they exposed intracellular sterol-rich membranes. We also showed that, upon the addition of lethal concentrations of AmB, supplementation with AmB-sensitive cells but not with AmB-resistant cells enabled the survival of wild-type cells. Together, our data suggest that cell-to-cell heterogeneity in sensitivity to AmB can be an adaptive mechanism helping yeast communities to resist macrolides, which are naturally occurring antifungal agents. IMPORTANCE Eukaryotic microorganisms harbor elements of programmed cell death (PCD) mechanisms that are homologous to the PCD of multicellular metazoa. However, it is still debated whether microbial PCD has an adaptive role or whether the processes of cell death are an aimless operation in self-regulating molecular mechanisms. Here, we demonstrated that dying yeast cells provide an instant benefit for their community by absorbing macrolides, which are bacterium-derived antifungals. Our results illustrate the principle that the death of a microorganism can contribute to the survival of its kin and suggest that early plasma membrane permeabilization improves community-level protection. The latter makes a striking contrast to the manifestations of apoptosis in higher eukaryotes, the process by which plasma membranes maintain integrity.

The Role of LAM Genes in the Pheromone-Induced Cell Death of S. cerevisiae Yeast.

Lam1-4 proteins perform non-vesicular transport of sterols from the plasma membrane to the endoplasmic reticulum. Disruption of their function leads to an increase in the content of sterols in the plasma membrane. In mammals, homologs of Lam proteins are responsible for the internalization of plasma cholesterol. The biological role of Lam proteins in yeast remains unclear, since the strains lacking individual LAM genes do not display any pronounced phenotype. Deletion of LAM1 (YSP1) gene inhibits the regulated death of Saccharomyces cerevisiae yeast cells induced by the mating pheromone. Here, we investigated whether LAM2 also plays a role in the cell death induced by the excess of mating pheromone and assessed genetic interactions between LAM2 and genes responsible for ergosterol biosynthesis. We have shown that LAM2 deletion partially prevents pheromone-induced death of yeast cells of the laboratory strain W303, while deletions of three other LAM genes - LAM1, LAM3, and LAM4 - does not provide any additional rescuing effect. The UPC2-1 mutation in the transcription factor UPC2 gene, which leads to the excessive accumulation of sterols in the cell, promotes cell survival in the presence of the pheromone and shows additivity with the LAM2 deletion. On the contrary, LAM2 deletion stimulates pheromone-induced cell death in the laboratory strain BY4741. We have found that the deletion of ergosterol biosynthesis genes ERG2 and ERG6 reduces the effect of LAM2 deletion. Deletion of LAM2 in the Δerg4 strain lacking the gene of the last step of ergosterol biosynthesis, significantly increased the proportion of dead cells and decreased the growth rate of the yeast suspension culture even in the absence of the pheromone. We suggest that the absence of the effect of LAM2 deletion in the Δerg6 and Δerg2 strains indicates the inability of Lam2p to transport some ergosterol biosynthesis intermediates, such as lanosterol. Taken together, our data suggest that the role of Lam proteins in the regulated death of yeast cells caused by the mating pheromone is due to their effect on the plasma membrane sterol composition.

Manipulating Cellular Energetics to Slow Aging of Tissues and Organs.

Up to now numerous studies in the field of gerontology have been published. Nevertheless, a well-known food restriction remains the most reliable and efficient way of lifespan extension. Physical activity is also a well-documented anti-aging intervention being especially efficient in slowing down the age-associated decline of skeletal muscle mass. In this review we focus on the molecular mechanisms of the effect of physical exercise on muscle tissues. We also discuss the possibilities of pharmacological extension of this effect to the rest of the tissues. During the exercise, the level of ATP decreases triggering activation of AMP-dependent protein kinase (AMPK). This kinase stimulates antioxidant potential of the cells and their mitochondrial respiratory capacity. The exercise also induces mild oxidative stress, which, in turn, mediates the stimulation via hormetic response. Furthermore, during the exercise cells generate activators of mammalian target of rapamycin (mTOR). The intracellular ATP level increases during the rest periods between exercises thus promoting mTOR activation. Therefore, regular exercise intermittently activates anti-oxidant defenses and mitochondrial biogenesis (via AMPK and the hormetic response) of the muscle tissue, as well as its proliferative potential (via mTOR), which, in turn, impedes the age-dependent muscle atrophy. Thus, the intermittent treatment with activators of (i) AMPK combined with the inducers of hormetic response and of (ii) mTOR might partly mimic the effects of physical exercise. Importantly, pharmacological activation of AMPK takes place in the absence of ATP level decrease. The use of uncouplers of respiration and oxidative phosphorylation at the phase of AMPK activation could also prevent negative consequences of the cellular hyper-energization. It is believed that the decline of both antioxidant and proliferative potentials of the cells causes the age-dependent decline of multiple tissues, rather than only the muscular one. We argue that the approach above is applicable for the majority of tissues in an organism.

Ergosterol Turnover in Yeast: An Interplay between Biosynthesis and Transport.

Sterols are important components of biological membranes that determine the physicochemical properties of lipid bilayer and regulate the functioning of membrane proteins. Being insoluble in water, sterols cannot diffuse between the membrane compartments separated by an aqueous phase. For this reason, distribution of sterols across cellular membranes is rather uneven. Membrane-to-membrane transport of sterols occurs mainly in a non-vesicular fashion and is provided by Lam and Osh proteins. In this review, we discuss the consequences of impairments in sterol biosynthesis and transport mostly relying on the studies performed on the model organism Saccharomyces cerevisiae. Despite the fact that molecular mechanisms underlying the functioning of Lam and Osh proteins are well established, the biological roles of these proteins are still unclear, because deletions of corresponding genes do not affect yeast phenotype. At the same time, disruptions in the biosynthesis of ergosterol, the major sterol of S. cerevisiae, lead to either cell death or reduced stress resistance. However, under certain conditions (e.g., mild salt or thermal stresses), a decrease in the ergosterol levels causes an increase in cell resistance. This suggests that the cells possess a mechanism facilitating rapid adjustment of the plasma membrane sterol content. We argue that the biological role of Lam proteins is, in particular, fast optimization of sterol composition of cell membranes.

Triosephosphates as Intermediates of the Crabtree Effect.

An increase in glucose concentration in the medium rapidly decreases respiration rate in many cell types, including tumor cells. The molecular mechanism of this phenomenon, the Crabtree effect, is still unclear. It was shown earlier that adding the intermediate product of glycolysis fructose-1,6-bisphosphate to isolated mitochondria suppresses their respiration. To study possible roles of glycolytic intermediates in the Crabtree effect, we used a model organism, the yeast Saccharomyces cerevisiae. To have the option to rapidly increase intracellular concentrations of certain glycolytic intermediates, we used mutant cells with glycolysis blocked at different stages. We studied fast effects of glucose addition on the respiration rate in such cells. We found that addition of glucose affected cells with deleted phosphoglycerate mutase (strain gpm1-delta) more strongly than ones with inactivated aldolase or phosphofructokinase. In the case of preincubation of gpm1-delta cells with 2-deoxyglucose, which blocks glycolysis at the stage of 2-deoxyglucosephosphate formation, the effect of glucose addition was absent. This suggests that triosephosphates are intermediates of the Crabtree effect. Apart from this, the incubation of gpm1-delta cells in galactose-containing medium appeared to cause a large increase in their size. It was previously shown that galactose addition did not have any short-term effect on respiration rate of gpm1-delta cells and, at the same time, strongly suppressed their growth rate. Apparently, the influence of increasing triosephosphate concentration on yeast physiology is not limited to the activation of the Crabtree effect.

[The clinical picture and specific microbiological features of acute otitis media]. / Klinika i mikrobiologicheskie osobennosti ostrogo srednego otita.

The objective of the present work was to study the spectrum of bacterial pathogenic agents responsible for the development of acute otitis media under present conditions and to elucidate the relationship between the pathogen species and the clinical course of the inflammatory process in the middle ear. A total of 60 patients of either sex at the age varying from 18 to 64 patients were available for the examination. All of them complained of ear pain, purulent discharge from the ears, hearing impairment, and general weakness. The following methods were employed: the analysis of the patients' complaints and their medical histories, visualexamination of the ENT organs, tonal threshold audiometry, tympanometry, and the analysis of secretion from the tympanic cavity using the real-time PCR technique. The study has demonstrated some regular patterns of the clinical manifestations of the disease depending on its causative agent. Specifically, it turned out that acute otitis media associated with the infection by Streptоcoccus pneumoniae is characterized by the more reactive clinical symptoms and the greater amount of complications compared with acute otitis media caused by Haemophilus influenzae that is largely a subclinical pathology. However, the latter condition more frequently leads to chronization of the pathological process.

Negative Feedback of Glycolysis and Oxidative Phosphorylation: Mechanisms of and Reasons for It.

There are two main pathways of ATP biosynthesis: glycolysis and oxidative phosphorylation. As a rule, the two pathways are not fully active in a single cell. In this review, we discuss mechanisms of glycolytic inhibition of respiration (Warburg and Crabtree effects). What are the reasons for the existence of this negative feedback? It is known that maximal activation of both processes can cause generation of reactive oxygen species. Oxidative phosphorylation is more efficient from the energy point of view, while glycolysis is safer and favors biomass synthesis. This might be the reason why quiescent cells are mainly using oxidative phosphorylation, while the quickly proliferating ones - glycolysis.

Physiological scenarios of programmed loss of mitochondrial DNA function and death of yeast.

Recently it was convincingly shown that the yeast Saccharomyces cerevisiae does possess the basic modules of programmed cell death machinery. As programmed cell death is suicide for a unicellular organism, it is reasonable to assume that they trigger the program when the death is beneficial for the rest of the population. Not surprisingly, most of the scenarios of physiological death of S. cerevisiae, i.e. cell death in stationary culture, during meiosis, during mating, and driven by viruses are dependent on quorum sensing, meaning that they depend on the cell density. Here we also discuss possible mechanisms that govern fitness decline during replicative aging of S. cerevisiae cells. We argue that loss of mitochondrial DNA function that occurs during replicative aging is programmed and adaptive. Indeed, yeast cells with nonfunctional mitochondrial DNA are known to be extremely stress-resistant, and also the presence of a subpopulation of such cells might protect the culture from degeneration by preventing the fixation of opportunistic mutations.

Mitochondrial matrix fragmentation as a protection mechanism of yeast Saccharomyces cerevisiae.

It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non-fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Deltaysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.

Spectroradiometer with wedge interference filters (SWIF): measurements of the spectral optical depths at Mauna Loa Observatory.

A spectroradiometer with wedge interference filters (SWIF) (the filters were produced by Carl Zeiss, Jena, Germany) and a CCD matrix (which was of Russian production) that functions as the sensor has been designed and built for use in ground-based optical sensing of the atmosphere and the Earth's surface in the spectral range of 0.35-1.15 µm. Absolute calibration of this instrument was performed through a series of observations of direct solar radiation at Mauna Loa Observatory (MLO) in Hawaii in May and June 1993. Spectral optical depth (SOD) measurements that were made during these field experiments provided detailed spectral information about both aerosol extinction (scattering plus absorption) and molecular absorption in the atmosphere above the site at MLO. The aerosol-SOD measurements were compared with narrow-band radiometer measurements at wavelengths of 380, 500, and 778 nm The SWIF and narrow-band radiometer measurements are in agreement to within the experimental error. At a wavelength of 500 nm, the aerosol SOD was found to be approximately 0.045. Adescription of the SWIF instrument, its absolute calibration, and the determination of atmospheric SOD's at MLO are presented.

[The effect of moderate doses of ethanol on the evoked neuronal activity of the sensorimotor cortex]. / Vliianie umerennykh doz étanola na vyzvannuiu aktivnost' neironov sensomotornoi kory.

Influence of acute administration of ethanol in moderate doses (170-1200 mg/kg, intraperitoneally) on the evoked activity of sensorimotor cortical neurons in response to electrical stimulation of the contralateral forelimb was studied in outbred rats never earlier exposed to ethanol action. During the first 20-25 min of ethanol action whether augmentation or attenuation in the level of neuronal reactivity could occur as well which in both cases could be accompanied by significant variability of latencies in poststimulus discharges and destabilization of their patterns. Expressed modifications in neuronal patterns at the first stage of ethanol action suggest that ethanol destabilizes evoked reactions of sensorimotor cortical neurons to afferent stimulation. This underlines the need for more complete approach to investigation of primary CNS reactions to ethanol paying more attention to temporal succession of origin and flow of these effects. Possible relations between oppositely directed primary reactions of single cortical neurons and individual characteristics of perception of different animals are discussed.

[The effect of moderate doses of ethanol on the nature of the background neuronal impulse activity of the sensorimotor cortex in rats]. / Vliianie umerennykh doz étanola na kharakter fonovoi impul'snoi aktivnosti neironov sensomotornoi kory krys.

The influence of acute administration of moderate doses of ethanol (170-1200 mcg/kg, intraperitoneally) on the spontaneous impulse activity of motor cortex neurons was studied in outbred rats never earlier exposed to alcohol. Initial reactions of the cortical neurons were shown to be characterized by the changes in the frequency of spontaneous activity which could be increased or decreased during the first 20-25 min after the injection. The changes in the mean frequency of spontaneous discharges were accompanied by the changes in their patterns. Both increase and decrease of the mean frequency at the initial stage of ethanol action was accompanied by significant variability of interspike intervals. It is emphasized that examination of the initial reactions of the cortical neurons to ethanol administration demands studying their temporal duration.

[The single administration of ethanol and the amplitude of the action potentials of the neocortical neurons in rats: extracellular studies]. / Odnokratnoe vvedenie étanola i amplituda potentsialov deistviia neironov neokorteksa krys: ékstrakletochnye issledovaniia.

On outbred rats the influence was studied of acute administration of moderate dozes of ethanol (150 mg--1.2 g/kg, intraperitoneally) on the motor cortex neurones. It was shown that ethanol in the range of these dozes led to a change of the amplitude of extracellularly recorded action potentials, which could be directed to an increase or decrease. In the used range minimum as well as maximum dozes in various animals might elicit contrary effects, i.e. no dependence was observed of the direction of reaction of the studied neurones on the administrated doze. Possible mechanisms are discussed which may be the basis of the change of the amplitude of extracellular action potentials. Conclusion is made that use of moderate dozes of ethanol is a useful condition for studies directed to a search of mechanisms of initial reaction to alcohol action.

[Plasmapheresis in the treatment of critical stages of ischemia in diabetic angiopathy of the lower extremities]. / Plazmaferez v lechenii kriticheskikh stepenei ishemii pri diabeticheskoi angiopatii nizhnikh konechnostei.

The authors have used gravitation plasmapheresis in the treatment of 26 patients with diabetic angiopathies of lower extremities. It was established that continuous gravitation plasmapheresis facilitated the immediate correction of most of the altered biochemical and coagulation parameters of blood, in patients with diabetes mellitus. The use of rheopolyglucinum is followed by stable central hemodynamics and stabilization of the general state of the patients during continuous plasmapheresis which allows to recommend rheopolyglucinum as the main plasma-substituting solution for fractionation of blood in patients with diabetic angiopathies. The gravitation plasmapheresis used in the treatment of critical degrees of ischemia in patients with diabetic angiopathies of lower extremities is thought by the authors to allow to preserve the extremity or to make the level of its amputation lower in most patients.

[Physical work capacity studied by measured physical loading in patients with hereditary hemochromatosis]. / Issledovanie fizicheskoi rabotosposobnosti v usloviiakh dozirovannoi fizicheskoi nagruzki u bol'nykh naseldstvennym gemokhromatozom.

A significant decrease of work fitness was revealed in 23 patients with verified diagnosis of hereditary hemochromatosis exposed to graded physical exercise. It is assumed that the decrease of exercise tolerance in these patients may be most possibly accounted for by the lowering of myocardial contractility as a result of its injury due to iron overload. It is not excluded, however, that nonspecific dystrophic processes determined by diabetes mellitus, liver damage and coronary atherosclerosis in some cases may develop.