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. The constitutive Hog1 activation required the MAPK kinase kinase (MAP3K) Ssk2 of the HOG pathway, but not Ste11 and Ssk22, two other MAP3Ks of the same pathway. The constitutive Slt2 activation did not require Rom2 and the membrane sensors of the CWI pathway, whereas Bck1 was indispensable. The PMA1 overexpression activated the stress response element but not the cyclic AMP response element and the Rlm1 transcription factor. PMA1 overexpression may facilitate the construction of industrial strains with simultaneous tolerance to weak acids, ROS, and ethanol.

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 (H2 O2 ) flows through Ssk1, the response regulator of the two-component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAP3K) Ssk2 but not its paralog Ssk22 or another MAP3K Ste11 of the pathway, culminating in Hog1 phosphorylation via the MAP2K Pbs2. When overexpressed, Ssk2 is also activated in an Ssk1-independent manner. Unlike in mammals, H2 O2 does not cause endoplasmic reticulum stress, which can activate Hog1 through the conventional unfolded protein response. Hog1 activated by H2 O2 is retained in the cytoplasm, but is still able to activate the cAMP- or stress-responsive elements by unknown mechanisms.

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. OLE1-overexpressing strains displayed enhanced tolerance to various stresses, better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide content. The OLE1-mediated enhanced stress tolerance was considerably diminished upon deletion of HOG1, which encodes the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway. Furthermore, OLE1 overexpression constitutively activated Hog1, which remained in the cytoplasm. Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets, including CTT1, HSP12, and STL1, and further, the cAMP and stress response elements present in the promoter. OLE1 overexpression neither caused nor relieved endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute to enhanced tolerance to various types of stress. Biotechnol. Bioeng. 2017;114: 620-631. © 2016 Wiley Periodicals, Inc.

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. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

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 ß-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. Of the 23 down-regulated genes, 11 of 13 viable deletion mutants were identified to be tolerant to heat, suggesting that the down-regulation of those genes might have contributed to the enhanced heat tolerance in dfg5Δ. Deletion of DFG5 caused slight activation of mitogen-activated protein kinases Hog1 in the high-osmolarity glycerol pathway and Slt2 in the cell wall integrity pathway. Therefore, a model is proposed on the signal transduction pathways associated with deletion of DFG5 upon heat stress.

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. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

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. The strains displayed constitutive and sustained activation of Hog1, a central kinase in the high osmolarity glycerol (HOG) signal transduction pathway of S. cerevisiae. However, the cell growth defect known to be caused by constitutive and sustained activation of Hog1 was not observed. We also found that reactive oxygen species (ROS) were accumulated to a less extent upon exposure to high glucose concentration in our osmotolerant strains. We identified six new genes (GPH1, HSP12, AIM17, SSA4, USV1, and IGD1), the individual deletion of which renders cells sensitive to 50 % glucose. In spite of the presence of multiple copies of stress response element in their promoters, it was apparent that those genes were not controlled at the transcriptional level by the HOG pathway under the high glucose conditions. Combined with previously published results, overexpression of SPT15-M2 or SPT15-M3 clearly provides a basis for improved tolerance to ethanol and osmotic stress, which enables construction of strains of any genetic background that need enhanced tolerance to high concentrations of ethanol and glucose, promoting the feasibility for VHG ethanol fermentation.

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. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.

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. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H(2)O(2). These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H(2)O(2), but its cellular function is specified to 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. In this study, we revealed that the sub-toxic and toxic levels of H(2)O(2) indeed induced pseudohyphae, but not true hyphae. Supporting this, several hyphae-specific genes that are expressed in true hyphae induced by serum were not detected in either sub-toxic or toxic H(2)O(2) condition. A DNA microarray analysis was conducted to reveal the transcription profiles in cells treated with sub-toxic and toxic conditions of H(2)O(2). Under the sub-toxic condition, a small number of genes involved in cell proliferation and metabolism were up-regulated, whereas a large number of genes were up-regulated in the toxic condition where the genes required for growth and proliferation were selectively restricted. For pseudohyphal induction by sub-toxic H(2)O(2), Cek1 MAPK activating the transcription factor Cph1 was shown to be important. The absence of expression of several hyphae-specific genes known to be downstream targets of Cph1-signaling pathway for true hyphae formation suggests that the Cek1-mediated signaling pathway is not solely responsible for pseudohyphal formation by subtoxic H(2)O(2) and, but instead, complex networking pathway may exists by the activation of different regulators.

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. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME.

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. A polypeptide of 842 amino acids was deduced from the ORF sequence. We identified another form of 873 amino acids which arises from alternative splicing at the C-terminal end. We also generated an anti-KIF18B antibody which detects a protein band of 120kDa. Western analyses showed that the protein level of KIF18B is elevated at late G(2) through metaphase, very similar to cyclin B1. Immunocytochemical staining revealed that the KIF18B protein is present predominantly in the nucleus and to a lesser extent in the cytoplasm of interphase cells. During mitosis, most KIF18B was found to be closely associated with astral microtubules emanating from the spindle pole during prometaphase and metaphase. Meanwhile, KIF18B was not detected at anaphase and telophase, consistent with the Western blotting data. The nuclear localization signal was roughly determined by using several EGFP-tagged deletion mutants of KIF18B. Together, the expression of KIF18B is regulated in a cell cycle-dependent manner and therefore may play an important role(s) in cell division.

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. The binding of PEG-PLL to the pDNA was accompanied by a small increase in enthalpy, a large increase in entropy, and a large decrease in free energy. The binding stabilized as the polymerization degree of PLL on PEG-PLL increased and the salt concentration decreased. Changes in the thermodynamic parameters are discussed in relation to both the polymerization degree of PLL on PEG-PLL and the salt concentration.

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. However, its precise role in DNA damage or repair pathway is not clear. Here we report that Kif4A associates with BRCA2 in a biochemical identification and that the interaction is mediated by the Kif4A C-terminal cargo-binding domain and BRCA2 C-terminal conserved region. Upon nucleus-specific laser micro-irradiation, Kif4A was rapidly recruited to sites of DNA damage. Significantly, the depletion of Kif4A from cells by shRNA impaired the ionizing-radiation induced foci (IRIF) formation of Rad51, both quantitatively and qualitatively. In contrast, the IRIF of gamma-H2AX or NBS1 was largely intact. Moreover, Kif4A knockdown rendered cells hypersensitive to ionizing radiation in a colonogenic survival assay. We further demonstrated that Kif4A deficiency led to significantly decreased homologous recombination in an I-SceI endonuclease induced in vivo recombination assay. Together, our results suggest a novel role for a chromokinesin family member in the DNA damage response by modulating the BRCA2/Rad51 pathway.

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. We found that six phosphorylation sites of the Serine residues of p53 were efficiently phosphorylated in HBx-expressing ChangX-34 cells, suggesting that the binding of HBx to the p53 protein does not interfere with the phosphorylation of p53 by signaling kinases. In addition, doxorubicin caused a dramatic reduction of Hdm2 mRNA and protein levels in cells expressing HBx. Intriguingly, reactivation of p53 was accompanied with a nuclear accumulation of p53 and the phosphorylated p53 at Serine15 was only detected in nuclear fraction, but not in cytosolic fraction of doxorubicin-treated ChangX-34 cells. Functional restoration of the p53 protein in HBx-expressing cells occurs according to the dual effects of doxorubicin: a significant reduction of Hdm2 expression and a nuclear accumulation of the phosphorylated p53 protein. Thus, proper usage of doxorubicin as an effective antitumor agent may be reevaluated and can be extended to tumors primarily caused by infection of DNA tumor viruses.

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.e., the binding to an elongated DNA chain with a binding constant K1 and with K2 that occurred during the conformational transition. The two-variable curve fitting models are usually bound by a strict regulation on the difference in the values of the binding constants K1 > K2. For the DNA condensation, however, the relationships for K1 and K2 are still unclear. The novel curve fitting model developed in this study takes into account this uncertainty on the relationship of the binding constants and is highly flexible for the two-variable binding constant system.

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. albicans. Of these, the length of an open reading (ORF) of 375 amino acids, temporarily designated CaKAR3, was noticeably short compared with the closely related S. cerevisiae KAR3 (ScKAR3) of 729 amino acids. This finding prompted us to isolate a lambda genomic clone containing the complete CaKAR3 ORF, and here the complete sequence of CaKAR3 is reported. CaKAR3 is a C-terminus motor protein, of 687 amino acids, encoded by a non-disrupting gene. When compared with ScKAR3, the amino terminal region of 112 amino acids was unique, with the middle part of the 306 amino acids exhibiting 25% identity and 44% similarity, while the remaining C-terminal motor domain exhibited 64% identity and 78% similarity, and have been submitted to GeneBank under the accession number AY182242.

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. albicans TSA1 (CaTSA1) gene are located in opposite orientation on the same chromosome. Using PCR-directed disruption cassettes and URA-Blaster, a series of deletion mutants that lack one to four copies were constructed to examine the functions of CaTSA1. Northern and Western analyses showed that both the transcript and protein products of CaTSA1 decreased proportionally to the disrupted copy number and were completely absent in the null mutant, indicating that all four TSA1 copies are equally functional at the transcriptional level. Intracellular H2O2 increased by an order of magnitude in deletion mutants lacking three to four copies, suggesting that CaTsa1p is not a redundant H2O2 scavenger. CaTsa1p was indispensable for yeast-to-hyphal transition when C. albicans was cultured under oxidative stress. The level of its oxidized form increased approximately five-fold in hyphal cells, whereas that of the reduced form increased two-fold compared to yeast cells. The ratio of oxidized to reduced form was increased three-fold in hyphal cells. This overall increase was found to be controlled at the post-transcriptional level. Interestingly, CaTsa1p is translocated to the nucleus of hyphal cells. These findings may be of biological significance for differentiation and pathogenicity.

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. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.