Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol.

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol.

Catechol, a naturally occurring and an important industrial chemical, has been shown to have strong promotion activity and induce glandular stomach tumors in rodents. In addition, catechol is a major metabolite of carcinogenic benzene. To clarify the carcinogenic mechanism of catechol, we investigated DNA damage using human cultured cell lines and 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. Catechol increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is known to be correlated with the incidence of cancer, in a human leukemia cell line HL-60, whereas the amount of 8-oxodG in its hydrogen peroxide (H2O2)-resistant clone HP100 was not increased. The formation of 8-oxodG in calf thymus DNA was increased by catechol in the presence of Cu(2+). Catechol caused damage to 32P-labeled DNA fragments in the presence of Cu(2+). When NADH was added, DNA damage was markedly enhanced and clearly observed at relatively low concentrations of catechol (<1 microM). DNA cleavage was enhanced by piperidine treatment, suggesting that catechol plus NADH caused not only deoxyribose phosphate backbone breakage but also base modification. Catechol plus NADH frequently modified thymine residues. Bathocuproine, a specific Cu(+) chelator and catalase inhibited the DNA damage, indicating the participation of Cu(+) and H2O2 in DNA damage. Typical hydroxyl radical scavengers did not inhibit catechol plus Cu(2+)-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H2O2 with Cu(+) participates in catechol-induced DNA damage. Therefore, we conclude that oxidative DNA damage by catechol through the generation of H2O2 plays an important role in the carcinogenic process of catechol and benzene.

Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification.

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specific polymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant allele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproate in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2-1250S. A set of oligonucleotide primers was designed to contain different nucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By testing with control strains, we established PCR conditions for specific amplification. Using these conditions and a simple template preparation procedure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identification of specific yeast strains.

Effects of room temperature, seasonal condition and food intake on finger skin temperature during cold exposure test for diagnosing hand-arm vibration syndrome.

For diagnosing the hand-arm vibration syndrome, peripheral circulation and sensory tests immersing one hand in cold water at 10 degrees C for 10 min have been performed widely in Japan. The authors investigated the effects of room temperature, seasonal condition and food intake on the test results, especially finger skin temperature. Six healthy males were examined repeatedly under six different room temperatures at 10 degrees C, 15 degrees C, 20 degrees C, 22.5 degrees C, 25 degrees C and 30 degrees C. Eight healthy males were examined under room temperatures at 10 degrees C, 20 degrees C and 30 degrees C, repeatedly in winter, spring, summer and autumn. Six healthy males were examined in summer under room temperature at 22.5 degrees C repeatedly 1 hr after, 3 hr after meal and after fasting for 13 hr. The finger skin temperature was strongly affected by room temperature. The finger skin temperature was also affected by seasonal condition. No remarkable effect of food intake was observed. For estimating circulatory function of the upper extremities using the finger skin temperature, the room temperature should be strictly controlled and the effect of seasonal condition must be taken into consideration.

An endemic condition of biochemical hypoglycemia among male volunteers.

Biochemical hypoglycemia has attracted attention because it triggers accidents through errors in judgment by impairing cognitive function. Its prevalence and associated risk factors have remained unexplored in the area of occupational health. The present study was designed to investigate its epidemiological aspects in male volunteers. The study population of 76 male volunteers, 63 of whom were firefighters and 13 had other jobs, filled out questionnaires by interview covering 37 factors concerning age, physical parameters, past and current health problems, habits (smoking, drinking, and exercise), food preferences and life style. The participants were asked to measure their blood glucose levels 9 times over a representative workday and holiday and record any symptoms of hypoglycemia at the time of measurement. The body fat ratio of each participant was measured by the bioelectrical impedance analysis method. 22.4% of the participants (17/76) experienced biochemical hypoglycemia (less than 60 mg/dl) at the time of blood glucose monitoring. Those who experienced biochemical hypoglycemia had significantly lower peak, average and nadir blood glucose levels (p < 0.05; 115.1 +/- 18.7 mg/dl, 82.2 +/- 7.0 mg/dl, 52.2 +/- 5.6 mg/dl, respectively) than those who did not (129.9 +/- 26.7 mg/dl, 97.0 +/- 9.9 mg/dl, 75.9 +/- 9.7 mg/dl, respectively). No symptom other than sudden hunger (p < 0.05) was correlated with biochemical hypoglycemia. Lower lean body mass index (lean body mass/height2) was the only statistically significant contributory risk factor (p < 0.05, Odds ratio: 2.91, 95% confidence interval 1.29-6.65) by multiple logistic analysis. Mean blood glucose levels had a positive correlation with lean body mass index (P < 0.0001, r2 = 0.372). The present results suggest that biochemical hypoglycemia is a potential risk problem in occupational settings. Low L.B.M.I. males should be educated about the possibility of biochemical hypoglycemia whoever have experienced a feeling of sudden hunger. Such precautions should be highly recommended to workers under dangerous and/or tense conditions in order to prevent error accidents in occupational settings.

Epidemiological study on hypoglycemia endemic to female nurses and other workers.

Hypoglycemia has been reported to occur frequently among individuals with health-related occupations, especially hospital nurses. In this study, we investigated the risk factors of hypoglycemia. Both female hospital nurses and other female workers participated in this study, which included questionnaires, and daily blood glucose monitoring. Responses from 3,415 workers were included for analysis. 2,489 workers reported that they had never had hypoglycemic episodes (symptom-free group), while 926 workers reported that they had experienced hypoglycemic symptoms more than once. Of these workers, 207 had had episodes within one year of the study and had experienced them more than once a month (symptomatic group). Thirty-one volunteers were recruited from the above two groups-21 from the latter group and 10 from the former group-and daily blood glucose levels were determined: 6 days for nurses to cover three shifts (dayshift, nightshift and midnightshift) and 2 days for other workers. Nine of 21 volunteers from the symptomatic group had biochemical hypoglycemic reading(s) (< 60 mg/dl) during the monitoring; however, none of 10 volunteers from the symptom-free group had such readings. These nine volunteers were found to have lower mean blood glucose levels (p < 0.05) and lower minimum blood glucose levels (p < 0.05) than other volunteers. Simple logistic analyses revealed multiple risk factors in the symptomatic group. By adjusting the confounding effects, these multiple risk factors were reduced into a small number of risk factors: occupation as a nurse, histories of hypotension and glucosuria, eating snacks, eating deserts, and insomnia. The present report clearly demonstrated that there are hypoglycemic-prone individuals among symptomatic female workers, who were shown to have multiple risk factors for hypoglycemic symptoms.

Response to psychological stressors in hand-arm vibration syndrome patients.

We investigated urinary catecholamines' response to acute psychological stress test in hand-arm vibration syndrome patients. Thirteen patients with vibration-induced white finger (VWF) in higher frequency of attack, 7 patients with VWF in lower frequency, 6 patients without VWF and 17 healthy subjects were examined. All subjects were male and their average age (SD) was 59.2 (6.4), 56.3 (2.9), 58.2 (4.7) and 56.8 (4.9), respectively. After an initial rest for 1 hour, acute psychological stress test with stressors--mirror drawing, watching horror video and arithmetic under intermittent noise was performed for 1 hour. Subjective complaints to the stress test were greater in patients with hand-arm vibration syndrome than in the healthy controls. The patient group with VWF in higher frequency indicated significant increases of urinary catecholamines (p < 0.05); average values (SD) at rest period and at stress test were 2.42 (1.17) and 3.71 (1.82) micrograms/h for norepinephrine, and 1.47 (0.73) and 2.66 (1.79) micrograms/h for epinephrine, respectively. Increasing tendency of urinary catecholamines was observed in other three groups, however, they were not statistically significant. The sympathoadrenal medullary response to psychological stressors increased especially in hand-arm vibration syndrome patients with VWF in higher frequency.

Orientational Disorder of C60 in Li2CsC60.

The x-ray diffraction of the nonsuperconducting ternary fulleride Li(2)CsC(60) reveals at room temperature a face-centered-cubic (Fm3m) disordered structure that persists to a temperature of 13 Kelvin. The crystal structure is best modeled as containing quasispherical [radius of 3.556(4) angstroms] C(60)(3-) ions, in sharp contrast to their orientational state in superconducting face-centered-cubic K(3)C(60) (merohedral disorder) and primitive cubic Na(2)CsC(60) (orientational order). The orientational disorder of the carbon atoms on the C(60)(3-) sphere was analyzed with symmetry-adapted spherical-harmonic functions. Excess atomic density is evident in the <111> directions, indicating strong bonding Li(+)-C interactions, not encountered before in any of the superconducting alkali fullerides. The intercalate-carbon interactions and the orientational state of the fullerenes have evidently affected the superconducting pair-binding mechanism in this material.

Crystal Structure, Bonding, and Phase Transition of the Superconducting Na2CsC60 Fulleride.

The crystal structure of superconducting Na(2)CsC(60) was studied by high-resolution powder neutron diffraction between 1.6 and 425 K. Contrary to the literature, the structure at low temperatures is primitive cubic [See equation in the PDF file], isostructural with pristine C(60). Anticlockwise rotation of the C(60) units by 98 degrees about [111] allows simultaneous optimization of C(60)-C(60) and alkali-fulleride interactions. Optimal Na(+)-C(60)(3-) coordination is achieved with each sodium ion located above one hexagon face and three hexagon-hexagon fusions of neighboring fulleride ions (coordination number 12). Reduction of the C(60) molecule lengthens the hexagon-hexagon fusions and shortens the pentagon-hexagon fusions (to approximately 1.43 angstroms). On heating, Na(2)CsC(60) undergoes a phase transition to a face-centered-cubic [See equation in the PDF file] phase, best modeled as containing quasi-spherical C(60)(3-) ions. The modified structure and intermolecular potential provide an additional dimension to the behavior of superconducting fullerides and should sensitively affect their electronic and conducting properties.

Temporary threshold shift of temperature sensation caused by vibration exposure.

The temporary threshold shift of temperature sensation due to vibration exposure was studied to clarify the significance of frequency and acceleration on it. The discrete frequencies of the vibrations tested were 32, 63, 125, 250 and 500 Hz, and the accelerations were at the level of 2, 4, 8 and 16 g. The threshold shift of warm sensation (TTSw) was markedly, but that of cool sensation was small and not significantly different from the control value. TTSw increased with a rise in the level of acceleration and was largest at 125 Hz among the frequencies examined. The most effective frequency among the vibrations tested for warm sensation was inferred to be lower than that for vibratory sensation. After exposure, TTSw or the increment of the neutral zone decayed exponentially but bounced slightly in the later period. These patterns of TTSw are similar to those of the vibration-induced TTS of vibratory sensation.

Physiological variations of warm and cool sense with shift of environmental temperature.

In order to obtain control data on the temperature sense (warm and cool threshold values) of fingertips, the relationships between room temperature and either skin temperature, warm threshold or cool threshold of the middle fingertips were investigated in healthy subjects (6 males), using our thermo-esthesiometer. The skin temperature changes in a sigmoidal response with the variation of room temperature. A point of inflection for this response was observed at the room temperature of 15 degrees C, at which the greatest standard deviation of skin temperature occurred. The warm and cool thresholds, on the other hand, were also affected by variations of room temperature. Warm threshold and skin temperature or cool threshold and skin temperature bore a linear relation to each other, and the correlation coefficient was 0.854 in the former, and 0.925 in the latter, respectively. The disorder of temperature sensitivity (warm and cool thresholds) must always be considered together with the room temperature or skin temperature. On the other hand, the width of the neutral zone between warm and cool thresholds was affected by neither the changes of room temperature nor the changes of skin temperature. Hence, the width of the neutral zone was approximately constant, especially, at the room temperatures in the vicinity of 15 degrees C to 25 degrees C.