Trehalose accumulation and radiation resistance due to prior heat stress in Saccharomyces cerevisiae.

In this study, we examined the accumulation of trehalose, a stress-responsive substance, upon gamma-ray irradiation by evaluating the cause of trehalose accumulation and the development of gamma-ray resistance through intracellular trehalose accumulation. Saccharomyces cerevisiae cells cultured to the logarithmic growth phase were irradiated with gamma rays, and the intracellular trehalose content was measured. However, trehalose was not detectable. The yeast cells with trehalose accumulation caused by pre-treatment at 40 °C were irradiated with gamma rays, and the resistance of these cells to gamma radiation was compared with that of cells without heat treatment. Trehalose accumulation resulted in gamma-ray resistance and suppressed the increase in reactive oxygen species, lipid peroxidation, and DNA double-strand break production in yeast cells. The tests were also performed with a trehalose-6-phosphate-synthase (TPS1)-deficient mutant strain (Δtps1) unable to synthesize trehalose, and the results revealed that TPS1 was involved in protection against oxidative stress.

Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

Distribution characteristics and source identification of polychlorinated dibenzo-p-dioxin and dibenzofurans, and dioxin-like polychlorinated biphenyls in the waters from River Kanzaki, running through Osaka urban area, Japan.

This is the first to elucidate the distribution and sources of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs), and dioxin-like polychlorinated biphenyls in the waters from Kanzaki River, which is one of the most heavily polluted rivers in Japan. The World Health Organization (WHO)-toxic equivalent quantities (TEQs) in waters from the mainstream exceeded the Japanese environmental standard (1.0 pg-TEQ/L). The PCDD/PCDFs were dominated by highly chlorinated DFs, which predominantly contributed to the WHO-TEQs, suggesting that the main causes would be the incineration-related wastes. To find the sources, the dioxin congener concentrations in water and sediment samples from its tributary small waterways were determined. Abnormally high WHO-TEQs were detected in a water (50 pg-TEQ/L) and a sediment sample (41,000 ng-TEQ/kg dry weight) near the industrial solid waste incinerators (ISWIs). The PCDD/PCDF characteristics agreed well with those of the incinerator-related wastes as seen in the mainstream. These facts indicate that the dioxin pollution in the mainstream could be largely related to the industrial wastes from the ISWIs. Here, a TEQ apportionment method was used to understand the contribution of the pyrogenic sources to the WHO-TEQs. The average contribution ratios of the pyrogenic sources to WHO-TEQs were more than 80% for river waters from the mainstream, indicating that the elevated WHO-TEQs in the mainstream had been largely caused by the ISWIs.

Thermotolerant bacteriocin-producing lactic acid bacteria isolated from thai local fermented foods and their bacteriocin productivity.

Twenty-one samples of Thai local fermented foods were screened for thermotolerant bacteriocin-producing lactic acid bacteria. From 529 isolates of lactic acid bacteria, 121 isolates were able to inhibit the growth of certain bacterial strains. Of these 121 isolates, only 11 produced antibacterial agents that were capable of inhibiting the growth of multiple bacterial strains in a liquid medium. One strain (KKU 170) of these 11 isolates produced an antibacterial agent that could strongly inhibit the growth of selected strains of gram-positive bacteria including Listeria sp. The antibacterial agent produced by the strain KKU 170 was identified as a bacteriocin since it was inactivated by proteinase K treatment. The strain KKU 170 was identified as Pediococcus acidilactici by both biochemical tests and molecular biological techniques. Optimal production of bacteriocin by the strain KKU 170 was found in culture medium containing 0.2% glucose, at an initial culture pH of 6.5, and temperature of 45 ºC. The maximum bacteriocin activity (1600 AU ml(-1)) was reached at the late exponential phase of growth and displayed primary metabolite production. The partially purified bacteriocin of the strain KKU 170 was tolerant to heat treatment at 121 ºC for 30 min.

Isolation of Aluminum-Tolerant and -Absorbing Yeast.

Aluminum ions are toxic to bacteria and are thus frequently used for preservation in the food industry. However, at higher concentrations, aluminum is toxic to animals. The extraction of aluminum from aluminum-contaminated foods would therefore be beneficial. Based on the discovery of yeast strains that can tolerate and absorb toxic metals, we aimed to identify strains that could tolerate and absorb aluminum. In this study, yeast were isolated from soil samples and cultured in medium containing the toxic concentration of aluminum chloride (5 mM) for Saccharomyces cerevisiae BY4741. Among aluminum-tolerant strains, two strains, Alt-OF2 and Alt-OF5, were identified as aluminum-absorbing. D1/D2 sequencing revealed that both strains belonged to the genus Schizoblastosporion (syn. Nadsonia).

Seasonal change in the atmospheric concentration of particulate polycyclic aromatic hydrocarbons in Ho Chi Minh City, Vietnam.

We analyzed atmospheric particulate polycyclic aromatic hydrocarbons (PAHs) in Ho Chi Minh City, Vietnam, for 19 months. The average concentrations of total PAHs at dry and rainy seasons were 4.28 +/- 2.83 and 15.71 +/- 8.21 ng m(-3), respectively. The use of motorcycles without catalytic converters, estimated to be main emission sources of PAHs, would be higher during the dry season. PAH concentrations show a negative correlation with sunshine duration (r = -0.51). Furthermore, the ratio of average PAH concentration in the dry season to that in the rainy season shows a positive correlation with photolytic half-life (r = 0.94). Thus, seasonal changes in PAH concentrations are attributable to their photolytic degradation.

Identification and characterization of a psychrophilic yeast strain newly isolated from the fermentative starter (Loog-pang) of a traditional drink in Thailand.

One psychrophilic yeast strain, that grew well in a cold environment such as in a refrigerator, was isolated from the yeast starter (Loog-pang) of a traditional alcohol drink in Thailand. The isolated strain OPU-FC11 was identified as Cryptococcus diffluens by the assay for 26S ribosomal DNA and the test for carbon source assimilation. OPU-FC11 showed a good amount of growth at 4 degrees 0 at which a commonly found yeast like Saccharomyces cerevisiae did not grow, and produced cold-adapted enzymes that showed a relatively high activity at lower temperatures.

Thermotolerant Yeast Kluyveromyces marxianus Reveals More Tolerance to Heat Shock than the Brewery Yeast Saccharomyces cerevisiae.

The thermotolerant yeast Kluyveromyces marxianus, growing at high temperature (45℃) , showed stronger survival under heat shock at 50℃ than the brewing yeast Saccharomyces cerevisiae, which was unable to grow at 45℃. The survival rate of K. marxianus decreased to 10% during heat shock at 50℃ for 20 min, and to less than 0.01% at 60℃ for 20 min. Cells with damaged cellular membranes were infrequently observed at 50℃ and had decreased significantly from heat shock at 60℃. The metabolic activity of K. marxianus was retained at 50℃, whereas that of S. cerevisiae was not. The trehalose content of K. marxianus was approximately two times that of S. cerevisiae. These results suggest that K. marxianus protects itself from heat shock-induced damage through the use of trehalose (a protective molecule in S. cerevisiae) as well as other different factors.

Growth characteristics of fusants by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis.

Intergeneric fusants were obtained by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis. Two thermotolerant fusants growing at 40 degrees C were screened on the medium containing soluble starch. These fusants showed weak growth in a soluble starch medium and the production of a little amylase. The carbon source assimilation and the chromosome composition of the fusants were similar to those of the K. marxianus parent. However, a chromosomal difference from K. marxianus was recognized in the fusants. These results show the possibility that the fusants are amylase-producing strains rearranged from K. marxianus.

Fermentative production of l-galactonate by using recombinant Saccharomyces cerevisiae containing the endogenous galacturonate reductase gene from Cryptococcus diffluens.

The GAR1 gene, encoding d-galacturonate reductase in Cryptococcus diffluens, was isolated, and the GAR1-expression plasmid was constructed by insertion of GAR1 downstream of the yeast constitutive promoter in the yeast-integrating vector. Recombinant Saccharomyces cerevisiae expressing C. diffluensd-galacturonate reductase from a genome integrated copy of the gene was cultured for use the conversion of d-galacturonic acid to l-galactonic acid. The optimum conditions for l-galactonic acid production were determined in terms of the initial concentration of d-galacturonic acid, fermentation pH, and mixed sugars. The following conditions yielded high efficiency in the conversion of d-galacturonic acid to l-galactonic acid in large-scale cultures: 0.1% initial d-galacturonic acid concentration, pH 3.5, and glucose as additional sugar. The aerobic condition was necessary for the conversion of d-galacturonic acid. Subculture of that recombinant was not showing to decrease of the d-galacturonic acid conversion rate even though it was repeated in ten generations. Culturing in scale-up, the conversion rate of d-galacturonic acid to l-galactonic acid was increased.

Isolation and Characterization of a Variant Manganese Resistant Strain of Saccharomyces cerevisiae.

　Manganese contamination in water is one of the most serious problems in Southeast Asian countries, including Vietnam. Bioremediation using microorganisms, especially from the brewing yeast Saccharomyces cerevisiae, is expected to be a useful technique to remove manganese from contaminated water. Yeast strain S. cerevisiae BY4741 as the wild-type strain and some manganese-accumulating mutants bred from BY4741 were examined for cell growth and manganese accumulation in YPD liquid medium containing various concentrations of Mn2+.　Variants accumulating larger concentrations of manganese were isolated by the repeated screening of survivors in YPD media containing10mM Mn2+. Manganese was accumulated by the yeast cells during growth, but the growth of BY4741 was retarded with increasing Mn2+ concentrations and almost inhibited at 15mM Mn2+. One variant isolate, named IM3, showed no retardation of growth up to 15 mM Mn2+ and could absorb over 4-fold more manganese than the BY4741 strain. Effects of culture temperature and pH on the growth and manganese accumulation were analyzed for IM3. Maximum accumulation was shown at 30℃, pH 6.0 while the optimal growth was shown at 37℃, pH 5.0 - 7.0. Interestingly, IM3 could grow a little at pH 9.0 when manganese was added to the culture media, while it could not grow without the addition of manganese.

Roles of Catalase and Trehalose in the Protection from Hydrogen Peroxide Toxicity in Saccharomyces cerevisiae.

The roles of catalase and trehalose in Saccharomyces cerevisiae subject to hydrogen peroxide (H2O2) treatment were examined by measuring the catalase activity and intracellular trehalose levels in mutants lacking catalase or trehalose synthetase. Intracellular trehalose was elevated but the survival rate after H2O2 treatment remained low in mutants with deletion of the Catalase T gene. On the other hand, deletion of the trehalose synthetase gene increased the catalase activity in mutated yeast to levels higher than those in the wild-type strain, and these mutants exhibited some degree of tolerance to H2O2 treatment. These results suggest that Catalase T is critical in the yeast response to oxidative damage caused by H2O2 treatment, but trehalose also plays a role in protection against H2O2 treatment.

Improvement of the growth defect in salt- and ethanol-tolerant yeast mutagenized with error-prone DNA polymerization by using backcross cell fusion.

Salt- and ethanol-tolerant mutants of Saccharomyces cerevisiae, isolated from the uracil-requiring mutant derived from Taiken No. 396 by proofreading-deficient DNA polymerization, showed less growth than their parent strain. The fusants, between these tolerant mutants and the lysine-requiring mutant from Taiken No. 396 obtained by the protoplast fusion, indicated improved growth.

Gas-particle concentrations of atmospheric polycyclic aromatic hydrocarbons at an urban and a residential site in Osaka, Japan: effect of the formation of atmospherically stable layer on their temporal change.

A comparative study on atmospheric polycyclic aromatic hydrocarbons (PAHs) in particulate matter and the gaseous phase was performed at an urban and a residential site in Osaka, Japan, during 2005-2006. PAH concentrations at the urban site were found to be approximately twice higher than those at the residential site. At both sites, particulate PAH concentrations increased mainly in winter while the trends of temporal change in gaseous PAH concentrations were not clearly observed. The main sources of PAHs were estimated to be local traffic, e.g., diesel engines with catalytic converter. PAH concentrations did not significantly negatively correlate with ozone concentrations and meteorological parameters. Gas-particle partitioning coefficients of representative PAHs with low molecular weight (LMW) significantly negatively correlated with ambient temperature, showing that temporal change in the LMW PAH concentrations in PM could be attributable to the shift of their gas-particle distribution caused by the change in ambient temperature. For the first time, we studied the effect of the formation of atmospherically stable layer following an increase in PAH concentrations in Japan. At the urban site, PAHs showed a significant positive correlation with potential temperature gradients, indicating that temporal variability in PAH concentrations would be dominantly controlled by the formation of atmospherically stable layer in Osaka area.

Characterization of D-galacturonate reductase purified from the psychrophilic yeast species Cryptococcus diffluens.

D-Galacturonic acid reductase was purified from a psychrophilic yeast strain of Cryptococcus diffluens, which was isolated from Satho, a traditional alcohol drink in Thailand. This enzyme, named Cd-GalUAR, assimilates D-galacturonic acid and requires NADPH as a cofactor. Cd-GalUAR is about 45 kDa and stable from pH 6.5 to 7.5 and up to 35°C. Its optimum pH and temperature are pH 7.0 and 40°C, respectively. However, 80% of its maximum activity remained at 4°C. The reaction of Cd-GalUAR from D-galacturonic acid produces L-galactonic acid, which was identified by (13)C NMR and LC-MS. Three amino acid sequences were determined from trypsin-digested peptides of Cd-GalUAR. Similar sequences are found in many NAD or NADP oxidoreductases, including some D-galacturonate reductases. Our results suggest that Cd-GalUAR is the first D-galacturonate reductase identified in yeast.

Effect of extraction temperature on pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and dioxin-like polychlorinated biphenyls from a sediment sample using polar and non-polar solvents.

This study is the first to investigate the effect of extraction temperature (150, 175, and 200 degrees C) on the simultaneous pressurized liquid extraction (PLE) of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) from a sediment sample (certified reference material: JSAC 0431). To provide basic data that will help to determine the optimum conditions for PLE, six single solvents (toluene, dichloromethane [DCM], benzene, acetonitrile, acetone, and methanol) were used as an extraction solvent. The results obtained by PLE with each solvent were compared with those obtained by Soxhlet extraction using toluene. The efficiency of PLE for 2,3,7,8-substituted PCDDs, 2,3,7,8-substituted PCDFs, and DL-PCBs, as obtained by PLE using the solvents toluene, DCM, benzene, and acetonitrile, showed an increase with increasing extraction temperature. The recoveries of PCDDs, PCDFs, and DL-PCBs obtained by PLE using toluene were similar to or inferior to those obtained using the Soxhlet method. In contrast, their recoveries obtained by PLE using DCM, benzene, and acetonitrile were similar to or higher than those obtained using the Soxhlet method. Notably, the recoveries of PCDDs, PCDFs, and DL-PCBs using benzene at 200 degrees C were 117+/-15, 123+/-13, and 115+/-12%, respectively. When using acetone and toluene, the recoveries of tetra-hexaCDDs and tetra-hexaCDFs also increased with extraction temperature. In particular, the recoveries of 2,3,7,8-tetraCDD and 2,3,7,8-tetraCDF obtained with acetone at 200 degrees C were 181+/-21 and 167+/-14%, respectively. However, the efficiency of PLE for heptaCDDs and/or heptaCDFs was insufficient at all the tested temperatures. Furthermore, the efficiency of PLE for octaCDD and/or octaCDF showed a pronounced decrease with increasing extraction temperature, yielding recoveries of approximately 40% at 200 degrees C. We found that heptaCDF isomers were produced from authentic octaCDF during the PLE procedure with acetone at 200 degrees C, indicating that the lower recoveries of highly chlorinated DDs and/or DFs are attributable to their degradation.

Characteristics of the abundance of polychlorinated dibenzo-p-dioxin and dibenzofurans, and dioxin-like polychlorinated biphenyls in sediment samples from selected Asian regions in Can Gio, Southern Vietnam and Osaka, Japan.

The levels of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) were determined in sediment samples from Can Gio, South Vietnam, and Osaka, Japan. Can Gio is known for the defoliation of its mangrove forests by aerial spraying with Agent Orange during the Vietnam War, whereas Osaka is renowned for a PCDD/PCDF pollution accident at a municipal solid-waste incinerator. For comparison, we also analyzed PCDD/PCDFs and DL-PCBs in sediment samples from Hue and Hanoi, Vietnam. The toxic equivalent quantity (TEQ) values in Can Gio were as high as those in Hue, Hanoi, and suburban areas of Osaka, but much lower than those in urban areas of Osaka. The proportion of the World Health Organization (WHO)-TEQ value contributed by 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD) in Can Gio was approximately 30%, higher than the values in the other sample areas. These data suggest that residual sedimentary TCDD that originated from aerial spraying of Agent Orange occur in only low concentrations in Can Gio. The main contributors to WHO-TEQ values in Can Gio are natural sources, as in Hue. In contrast, commercial PCBs are the dominant contributors to WHO-TEQ values in Hanoi. In Osaka, agrochemicals used in rice cultivation, the incineration of solid waste, and commercial PCBs equally contributed to WHO-TEQ values at suburban locations. The dumping of incinerator-related materials and/or the inadequate management of commercial PCBs have resulted in significantly elevated WHO-TEQ values of 240-370 ng kg(-1)dw at urban locations in Osaka.

Concentrations of atmospheric polycyclic aromatic hydrocarbons in particulate matter and the gaseous phase at roadside sites in Hanoi, Vietnam.

We analyzed the concentrations of polycyclic aromatic hydrocarbons (PAHs) in both particulate matter (PM) and the gaseous phase at 10 roadside sites in Hanoi, Vietnam. The average concentrations of 47 PAHs ( summation 47PAHs) were 63 +/- 82 ng m(-3) in PM and 480 +/- 300 ng m(-3) in the gaseous phase. The PAHs mainly originated from motorcycles without catalytic converters. The highest concentrations of summation 47PAHs in both PM and the gaseous phase were observed at a terminal for buses and trucks. The operation of large commercial vehicles led to increased PAH pollution at the terminal site.

Characteristics of wines made by Saccharomyces mutants which produce a polygalacturonase under wine-making conditions.

Wines by yeast mutants producing polygalacturonase in high glucose concentration, from Saccharomyces wine-making strains, had higher filterability and more concentrated anthocyanin contents than that of their parent strains. These results suggest that the clarification process was improved at a lower cost by the low viscosity and that high-quality wines result from the increase in the anthocyanin contents.

Endo-polygalacturonase in Saccharomyces wine yeasts: effect of carbon source on enzyme production.

Eight wine yeast strains of Saccharomyces sp. were tested for polygalacturonase (PGase) activity, after cultivation on various carbon sources. No strain showed any activity when grown on glucose, while five strains produced PGase in the presence of galactose and polygalacturonate. These data suggest that the PGase of wine strains is repressed by glucose and induced by galactose and polygalacturonate. The existence of the PGase gene in the wine strains and its similarity with that of the laboratory strains was proved by Southern hybridization and PCR amplification. The promoter region of the PGase gene in the wine strains was slightly different from that of the laboratory strains. This possibly explains the different pattern of gene expression in wine and laboratory strains. The PGase of wine strains produced di- or tri-galacturonic acid from polygalacturonic acid, different from the fungal PGase.