Regulatory role of monoamine neurotransmitters in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> cells

Proliferation of Saccharomyces cerevisiae EPF cells on solid maltose-peptone-yeast extract (MPY) medium was stimulated by the addition of monoamine neurotransmitters. Dopamine turned out to be the most efficient among them: it caused ∼8-fold growth stimulation at 1 μM concentration. The dopamine effect was partly mimicked by apomorphine, a dopamine receptor agonist. Serotonin and histamine produced less significant (1.5–2-fold) effects, and norepinephrine virtually failed to stimulate yeast culture growth. These data point to a specific, apparently receptor-dependent mode of action of the tested neurotransmitters on S. cerevisiae cells. Using high performance liquid chromatography, serotonin, catecholamines (dopamine and norepinephrine), catecholamine precursor dioxyphenylamine, and oxidized amine products (homovanilic acid, dihydrophenylacetic acid, and 5-hydroxyindolacetic acid) were established to be accumulated in yeast cells up to (sub)micromolar concentrations without their release into the culture fluid supernatant (CFS). The results obtained suggest that the tested amine neurotransmitters and related compounds do not serve as autoregulators in the yeast population. Nevertheless, they may be involved in the regulation of yeast population development by other ecosystem components.     

<Emphasis Type=Italic>IXR1</Emphasis> and <Emphasis Type=Italic>HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

The <Emphasis Type=Italic>Pseudomonas fluorescens</Emphasis> secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Pseudomonas fluorescens strains are known to produce a wide range of secondary metabolites including phenazines, siderophores, pyoluteorin, and 2,4 diacetylphloroglucinol (DAPG). DAPG is of particular interest because of its antifungal properties and because its production is associated with inhibition of phytopathogenic fungi in natural disease-suppressive soils. This trait has been exploited to develop strains of P. fluorescens that have potential application as biocontrol agents. Although the biochemistry, genetics and regulation of DAPG production have been well-studied, relatively little is known about how DAPG inhibits fungal growth and how fungi respond to DAPG. Employing a yeast model and a combination of phenotypic assays, molecular genetics and molecular physiological probes, we established that inhibition of fungal growth is caused by impairment of mitochondrial function. The effect of DAPG on yeast is largely fungistatic but DAPG also induces the formation of petite cells. Expression of the multidrug export proteins Pdr5p and Snq2p is increased by DAPG-treatment but this appears to be a secondary effect of mitochondrial damage as no role in enhancing DAPG-tolerance was identified for either Pdr5p or Snq2p.     

<Emphasis Type=Italic>RAD18</Emphasis> gene product of yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6–Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

Expression of human melanocortin 4 receptor in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

The melanocortin 4 receptor (MC4R) is involved in the regulation of energy homeostasis and is known as one of the major hypothalamic regulators of food intake. Several studies have shown that replacement of aspartic acid at position 126 of the MC4R abolishes the ligand binding. We used the modified yeast Saccharomyces cerevisiae strain MMY28 to functionally express the MC4R and characterise the importance of this amino acid for ligand based activation of the receptor. The efficiency of the functional expression system was estimated by activation with αMSH, ACTH and THIQ and compared with cAMP response in mammalian cells. We generated the library of MC4R mutants randomised at the amino acid position 126. Recombinant MC4R clones were screened for the αMSH induced activity in yeast. From 9 different amino acids obtained only the natural aspartic acid displayed the ligand dependent activity of MC4R. The MC4R variants with glutamic acid and leucine at position 126, however, displayed higher background activity than other amino acid substitutions. The results suggest that the yeast expression system is suitable for screening of the MC4R receptor ligands and that the substitution of aspartic acid at position 126 of MC4R by different amino acids functionally inactivates the receptor.     

Interactions of the <Emphasis Type=Italic>HSM3</Emphasis> gene with genes initiating homologous recombination repair in yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

It was assumed previously that the mutator phenotype of the hms3 mutant was determined by processes taking place in the D-loop. As a next step, genetic analysis was performed to study the interactions between the hsm3 mutation and mutations of the genes that control the initial steps of the D-loop formation. The mutations of the MMS4 and XRS2 genes, which initiate the double-strand break formation and subsequent repair, were shown to completely block HSM3-dependent UV-induced mutagenesis. Mutations of the RAD51, RAD52, and RAD54 genes, which are also involved in the D-loop formation, only slightly decreased the level of UV-induced mutagenesis in the hsm3 mutant. Similar results were observed for the interaction of hsm3 with the mph1 mutation, which stabilizes the D-loop. In contrast, the shu1 mutation, which destabilizes the D-loop structure, led to an extremely high level of UV-induced mutagenesis and displayed epistatic interactions with the hsm3 mutation. The results made it possible to assume that the hsm3 mutation destabilizes the D-loop, which is a key substrate of both Rad5- and Rad52-dependent postreplicative repair pathways.     

Metabolism of arabinogalactan from siberian larch by <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> yeast

The ability of Saccharomyces cerevisiae yeast to utilize arabinogalactan (AG) from Siberian larch wood was studied, as well as the influence of phenolic impurities on the rate of its utilization. It was shown that arabinogalactan was intensively involved in the yeast metabolism, and the degradation extent of the polymer chain depended on both the method of purification of the AG sample and the amount of phenolic impurities in this sample.     

Expression and Assembly of Recombinant Surface Layer Proteins in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Most bacterial surface layers (SLs) are formed by self-assembly of a single type of protein. Native and recombinant surface layer monomers are capable to self-assemble on solid substrates and in solution to highly regular nanosized arrays which make them attractive for nanobiotechnological applications. In this study, we expressed the surface layer protein SbsC of Bacillus stearothermophilus ATTC 12980, tagged with Enhanced Green Fluorescent Protein, in the yeast Saccharomyces cerevisiae. We observed a network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis, SL structures were formed de novo in the buds. SL assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point.     

Purification and biochemical characterization of recombinant human H-ferritins from <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Recombinant human H-ferritins produced from Saccharomyces cerevisiae were purified and their molecular properties were characterized. Electrophoresis of the recombinant H-ferritins showed revealed bands with mobilities similar to those of the H-ferritins produced by Escherichia coli. The pI of H-ferritins from yeast was more basic than that of H-ferritins produced by E. coli. When the cells were treated with tunicamycin, the pI of H-ferritins was driven to a sharp band with the pI range similar to that of those produced by E. coli, implying that the H-ferritins from yeast are glycosylated. The iron uptake of H-ferritins was rapid in the initial stage, with a slight reduction when compared to ferritins from E. coli. Recombinant ferritins are commonly used during synthesis of inorganic nanoparticles in their cores for chemical and industrial purposes. Transmission electron microscopy revealed that the reconstitution yield and size distribution of the core minerals were affected depending on the protein shells. The H-ferritins with higher reconstitution yields (64.4%) showed slightly larger sizes (mean 6.52 nm) with narrower size distribution.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Constitutive expression of <Emphasis Type=Italic>ZmsHSP</Emphasis> in <Emphasis Type=Italic>Arabidopsis</Emphasis> enhances their cytokinin sensitivity

A small HSP gene, ZmsHSP, was isolated from Zea mays. Sequence analysis revealed that the open reading frame of ZmsHSP was 477 bp and that it encodes a protein composed of 159 amino acid residues with a calculated molecular mass of 18.17 kD and a predicated isoelectric point (pI) of 5.63. ZmsHSP contains a CS domain (p23-like domain) and shares similarity with the HSP90 co-chaperone p23. The expression level of ZmsHSP was different among various tissues with the highest expression in leaves and the lowest in silks. Results also showed that the expression of ZmsHSP in maize was significantly up-regulated by dehydration. Transgenic Arabidopsis plants overexpressing ZmsHSP under the control of the CaMV 35S promoter had lower endogenous cytokinin content and showed more sensitivity to cytokinin during the germination and early seedling stage than wild-type plants, suggesting that ZmsHSP might has a function in cytokinin response in Zea mays.     

Accumulation of inorganic polyphosphates in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

The yeast Saccharomyces cerevisiae was shown to have a high potential as a phosphate-accumulating organism under growth suppression by nitrogen limitation. The cells took up over 40% of phosphate from the medium containing 30 mM glucose and 5 mM potassium phosphate and over 80% of phosphate on addition of 5 mM magnesium sulfate. The major part of accumulated Pi was reserved as polyphosphates. The content of polyphosphates was ∼57 and ∼75% of the phosphate accumulated by the cells in the absence and presence of magnesium ions, respectively. The content of long-chain polyphosphates increased in the presence of magnesium ions, 5-fold for polymers with the average length of ∼45 phosphate residues, 3.7-fold for polymers with the average chain length of ∼75 residues, and more than 10-fold for polymers with the average chain length of ∼200 residues. On the contrary, the content of polyphosphates with the average chain length of ∼15 phosphate residues decreased threefold. According to the data of electron and confocal microscopy and X-ray microanalysis, the accumulated polyphosphates were localized in the cytoplasm and vacuoles. The cytoplasm of the cells accumulating polyphosphates in the presence of magnesium ions had numerous small phosphorus-containing inclusions; some of them were associated with large electron-transparent inclusions and the cytoplasmic membrane.     

Increased ethanol resistance in <Emphasis Type=Italic>Ethanolic Escherichia coli</Emphasis> by Insertion of heat-shock genes BEM1 and SOD2 from <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Ethanol is generally toxic to microorganisms, and intracellular and extracellular accumulation of ethanol inhibits cell growth and metabolism. In this study, pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) were cloned into pET-32a vector and then introduced into E. coli BL21 to produce ethanol. Heat shock genes (BEM1 and SOD2) from Saccharomyces cerevisiae were inserted into recombinant ethanolic E. coli using pET28_a vector to improve ethanol shock resistance. Three different strains were constructed: Ethanolic E. coli (adhB and pdc genes inserted using pET32_a vector), BEM1 gene-inserted E. coli (BEM1 inserted using pET_28a), and SOD2-inserted E. coli (SOD2 inserted using pET28_a). Construction of these three different strains allowed comparison of the functions of these heat shock genes as well as their roles in ethanol tolerance. The toxicity of ethanol in recombinant ethanolic E. coli was tested by measuring cell growth in response to various ethanol concentrations. The results show that SOD2-inserted E. coli showed higher ethanol resistance than ethanolic E. coli.     

Glutathione peroxidase3 of <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3Δ cells during Cd-treatment.     

Targeted protein depletion in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis> by activation of a bidirectional degron

Background  

Tools for in vivo manipulation of protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target protein degradation at restrictive conditions.