Overexpression of PMA1 enhances tolerance to various types of stress and constitutively activates the SAPK pathways in Saccharomyces cerevisiae.

PMA1 encodes a transmembrane polypeptide that functions to pump protons out of the cell. Ectopic PMA1 overexpression in Saccharomyces cerevisiae enhances tolerance to weak acids, reactive oxygen species (ROS) and ethanol, and changes the following physiological properties: better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide production. The enhanced stress tolerance was dependent on the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway, but not the MAPK Slt2 of the cell wall integrity (CWI) pathway; however, a PMA1 overexpression constitutively activated both Hog1 and Slt2.

Overexpression of OLE1 enhances stress tolerance and constitutively activates the MAPK HOG pathway in Saccharomyces cerevisiae.

OLE1 of Saccharomyces cerevisiae encodes the sole and essential Δ-9 desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Upon ectopic overexpression of OLE1 in S. cerevisiae, significant increases in the membrane oleic acid content were observed.

Dissection of the HOG pathway activated by hydrogen peroxide in Saccharomyces cerevisiae.

Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has long been implicated in transducing the oxidative stress-induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1, we reveal that the signal from hydrogen peroxide (H O ) flows through Ssk1, the response regulator of the two-component system of the HOG pathway.

Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry.

Loss of Dfg5 glycosylphosphatidylinositol-anchored membrane protein confers enhanced heat tolerance in Saccharomyces cerevisiae.

The protein product of Saccharomyces cerevisiae DFG5 gene is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein and a putative glycosidase/glycosyltransferase that links other GPI-anchored proteins to β-glucans in the cell wall. Upon exposure to heat (41°C), DFG5 deletion mutant dfg5Δ displayed significantly enhanced heat tolerance as well as lowered level of reactive oxygen species and decreased membrane permeability compared with those in the control (BY4741). Comparative transcriptome profiles of BY4741 and dfg5Δ revealed that 38 and 23 genes were up- and down-regulated in dfg5Δ respectively.

Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252.

Mutations of the TATA-binding protein confer enhanced tolerance to hyperosmotic stress in Saccharomyces cerevisiae.

Previously, it was shown that overexpression of either of two SPT15 mutant alleles, SPT15-M2 and SPT15-M3, which encode mutant TATA-binding proteins, confer enhanced ethanol tolerance in Saccharomyces cerevisiae. In this study, we demonstrated that strains overexpressing SPT15-M2 or SPT15-M3 were tolerant to hyperosmotic stress caused by high concentrations of glucose, salt, and sorbitol. The enhanced tolerance to high glucose concentrations in particular improved ethanol production from very high gravity (VHG) ethanol fermentations.

Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae.

Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance.

Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans.

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H(2)O(2) scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H(2)O(2), menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH.

A MAP kinase pathway is implicated in the pseudohyphal induction by hydrogen peroxide in Candica albicans.

Hydrogen peroxide (H(2)O(2)) functions as a ubiquitous intracellular messenger besides as an oxidative stress molecule. This dual role is based on the distinct cellular responses against different concentrations of H(2)O(2). Previously, we demonstrated that both low (> 1 mM) and high (4-10 mM) doses of exogenous H(2)O(2) induce filamentous growth with distinct cell morphology and growth rate in Candida albicans, suggesting the different transcription response.

Construction of Saccharomyces cerevisiae strains with enhanced ethanol tolerance by mutagenesis of the TATA-binding protein gene and identification of novel genes associated with ethanol tolerance.

Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565-1568), seems to a powerful tool for creating ethanol-tolerant strains.

Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B.

The human genome contains genes encoding for over 40 different types of kinesin and kinesin-like proteins. Of these, the functions of 13 kinesins remain uncharacterized. In this study, we constructed a plasmid containing the ORF of KIF18B and revealed that the KIF18B message of approximately 3kb is expressed in a tissue- and cell type-specific manners.

Thermodynamics of DNA condensation induced by poly(ethylene glycol)-block-polylysine through polyion complex micelle formation.

Isothermal titration calorimetry (ITC) was carried out to explore the condensation process of plasmid DNA (pDNA) molecules induced by poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) as a condensing agent. The ITC curves measured can be divided into two distinctive endothermic binding processes: the first was the binding of PEG-PLL to the elongated pDNA, and the second was the binding that accompanied the pDNA conformational transition. The thermodynamic parameters were obtained by fitting each ITC curve using our recently developed fitting method.

Hydrogen peroxide induces hyphal differentiation in Candida albicans.

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H(2)O(2) in Candida albicans. This finding is confirmed by the changing intracellular concentration of H(2)O(2). In order to induce the same level of differentiation, low concentrations of exogenous H(2)O(2) are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.

A novel role of the chromokinesin Kif4A in DNA damage response.

Chromokinesins are microtubule-motor molecules that possess chromatin binding activity and are important for mitotic and meiotic regulation. The chromokinesin-member Kif4A is unique in that it localizes to nucleus during interphase of the cell cycle. Kif4 deletion by gene targeting in mouse embryonic cells was known to associate with DNA damage response.

Reactivation of p53 in cells expressing hepatitis B virus X-protein involves p53 phosphorylation and a reduction of Hdm2.

Multifunctional activities of the hepatitis B virus X-protein (HBx) in cells have been largely implicated in the development of liver cancer; one of these activities is the loss of p53 function by sequestering p53 in the cytoplasm. We have previously found that doxorubicin increased the p53 levels in cells containing p53-binding HBx protein and restored the p53-mediated transcriptional activity that was suppressed by HBx. Here, we investigated the mechanism underlying p53 reactivation.

Development of a fitting model suitable for the isothermal titration calorimetric curve of DNA with cationic ligands.

A novel curve fitting model was developed for the isothermal titration calorimetry (ITC) of a cationic ligand binding to DNA. The ligand binding often generates a DNA conformational change from an elongated random coil into a compact collapsed form that is referred to as "DNA condensation". The ligand binding can be classified into two regimes having different binding constants Ki, i.

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination.

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP.

Complete sequence of a gene encoding KAR3-related kinesin-like protein in Candida albicans.

In contrast to Saccharomyces cerevisiae, little is known about the kinesin-like protein (KLP) in Candida albicans. The motor domain of kinesin, or KLP, contains a subregion, which is well conserved from yeast to humans. A similarity search, with the murine ubiquitous kinesin heavy chain region as a query, revealed 6 contigs that contain putative KLPs in the genome of C.

Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans.

We previously identified several proteins that are differentially expressed in pathogenic hyphae by comparing protein profiles of yeast and hyphae of Candida albicans. One of these, thiol-specific antioxidant 1 (TSA1), attracted our attention because it may play some roles in surviving an unfavourable oxidative environment created by host cells. Two alleles of the C.

Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology.

The aging process is known to be regulated by specific genes in various organisms, including yeast, the nematode C. elegans, fruitflies and mice. To explore the novel genes involved in aging process, we applied cDNA microarray technology to a replicative senescence model of human dermal fibroblasts (HDF).

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery.

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells.

Kinesin superfamily protein member 4 (KIF4) is localized to midzone and midbody in dividing cells.

In association with microtubules, a variety of kinesins play important roles in cellular functions such as intracellular transport of organelles or vesicles, signal transduction, and cell division. In a previous study we revealed that human kinesin superfamily protein member 4 (KIF4) is a chromokinesin that binds to chromosomes. Since localization of several kinds of kinesin at midzone called central spindle, or midbody that connects two daughter cells, or both, suggests their implication in cell division, we investigated KIF4 localization of during mitosis and cytokinesis in Hela cells.

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production.

Background: The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release.

Results: A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis.

Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35.

A large portion of human kinesin superfamily protein member 4 (KIF4) is associated with the nuclear matrix during the interphase, while a small portion is found in the cytoplasm. During mitosis, it is associated with chromosomes throughout the entire process. In the present study, we identified a protein that interacts with KIF4 using a yeast two-hybrid system, co-immunoprecipitation and co-fractionation.