Isolation of Aspergillus oryzae mutants producing low levels of 2,4,6-trichloroanisole.

Musty or moldy off-odor in sake severely reduces its quality. Such off-odor is caused by 2,4,6-trichloroanisole (TCA), a compound that is produced by Aspergillus oryzae during sake production by O-methylating the precursor 2,4,6-trichlorophenol (TCP). TCP suppresses the growth of fungi, including A. oryzae, although TCA does not. Therefore, strains that are unable to convert TCP to TCA should be sensitive to TCP in the medium. Nevertheless, A. oryzae with a disrupted O-methyltransferase gene (ΔomtT) grew in a medium containing TCP. In agar medium, we observed no growth difference between the ΔomtT strain and a non-disrupted transformant; however, a significant growth delay was observed with the ΔomtT strain grown in liquid medium containing 0.5 µg/mL of TCP. This strain was more sensitive to low concentrations of TCP, suggesting that omtT contributes to the conversion (detoxification) of TCP in liquid culture. We generated A. oryzae RIB 40 mutants by ultraviolet irradiation and then cultured them in liquid medium containing TCP to obtain strains that did not produce moldy odor. The slow-growing strains were cultured in agar plates and then used to make koji with added TCP. We obtained three strains with lower TCA-producing ability and with sufficient hydrolase activities for sake brewing.

Growth characteristics of Aspergillus oryzae in the presence of 2,4,6-trichlorophenol.

During the making of rice-koji for sake production, 2,4,6-trichlorophenol (TCP) is O-methylated to 2,4,6-trichloroanisole (TCA) by the koji-mold, Aspergillus oryzae, resulting in a musty/moldy off-odor, which significantly reduces the quality of sake. Thus, we aim to develop A. oryzae strains with a less-efficient ability to produce TCA. TCP is a fungicide that suppresses the growth of fungi, whereas TCA does not. The exact effects of TCP on the growth of A. oryzae are unknown. However, it is assumed that a strain with low TCP conversion ability will be sensitive to TCP concentration. In this study, we investigated the effects of the different concentrations of TCP on the growth suppression of A. oryzae. As the TCP concentration in the media increased, the growth rate, and conidia formation of A. oryzae slowed down. No growth was observed in liquid culture (for 1 day at 30°C) containing more than 30 µg/mL of TCP and in agar culture (for 7 days at 30°C) containing more than 50 µg/mL of TCP. However, A. oryzae was able to grow on alpha rice containing higher concentrations of TCP. The results in agar culture are consistent with the effects of TCP on other Aspergillus species.

Passive cigarette smoking changes the circadian rhythm of clock genes in rat intervertebral discs.

We aimed to elucidate the molecular changes in intervertebral discs (IVDs) caused by passive smoking. Rats were subjected to 8 weeks of passive smoking; thereafter, their lumbar vertebrae were harvested. The annulus fibrosus and cartilage endplate (AF/CEP) were harvested together, and the nucleus pulposus (NP) was isolated separately. The expression of 27,342 rat genes was analyzed. In 3 "nonsmoking" rats, 96 of 112 genes whose expression varied ≥10-fold between the AF/CEP and NP were more highly expressed in the AF/CEP. With these differentially expressed genes, we uncovered novel AF/CEP and NP marker genes and indicated their possible novel functions. Although passive smoking induced less marked alteration in the gene expression profiles of both the AF/CEP and NP, multiple clock-related genes showed altered expression. These genes were expressed with a circadian rhythm in IVD cells, and most genes showed a phase shift of -6 to -9 h induced by passive smoking. Some clock-related genes showed abolished oscillation in the NP. Passive smoking also changed the expression levels of proteases and protease inhibitors and reduced the expression of NP marker genes. Thus, passive smoking induces changes in the circadian rhythm of a peripheral clock (IVD clock) that might be involved in molecular events related to IVD degeneration.

The transfer of radioactive cesium and potassium from rice to sake.

Using rice grains contaminated with radioactive cesium ((134)Cs and (137)Cs) that was released by the Fukushima Daiichi Nuclear Power Plant Accident in March of 2011, we investigated the behaviors of the radioactive cesium and potassium (total K and (40)K) during sake brewing. Cesiumis a congener of K, and yeast cells have the ability to take up Cs using known K transporters. During rice polishing, the concentrations of radioactive Cs and K in the polished rice grains decreased gradually until a milling ratio (polished rice weight/brown rice weight) of 70% was reached. No significant changes were observed below this milling ratio. Sake was brewed on a small scale using the 70% polished rice. The transfer ratio of radioactive Cs to sake and to the sake cake was significantly different than the ratio of K. Approximately 36% and 23% of radioactive Cs in the polished rice was transferred to the sake and sake cake, respectively; however, 40% was removed by washing and steeping the rice grains. On the other hand, 25% and 40% of K in the polished rice was recovered in the sake and sake cake, respectively, and 35% was removed by washing and steeping the rice grains. From the present results, the concentration of radioactive Cs in sake would be 4 Bq/kg fresh weight, which is well below the regulation values (100 Bq/kg), even using brown rice containing 100 Bq/kg of radioactive Cs.

The relation between insulin resistance and lifestyle in Japanese female university students.

Using the homeostasis model assessment (HOMA) index, we investigated the link between insulin resistance and lifestyle in Japanese female university students. We used data for 57 Japanese female university students (21.0±0.8 years) who were enrolled in a cross-sectional investigation study. We performed full blood examinations, and anthropometric parameters, nutrient oral intake and daily step counts were measured. The mean HOMA index for the subjects was 1.3±0.6, and 12 subjects were over the level of 1.6, which is considered to indicate insulin resistance in Japan. The HOMA index was positively correlated with abdominal circumference (r＝0.542, p＜0.0001), triglycerides, low density lipoprotein (LDL) cholesterol and systolic blood pressure. In addition, the HOMA index was negatively correlated with n-3 fatty acid and positively correlated with the n-6/n-3 fatty acid ratio (r＝0.304, p＝0.0216). Daily step count was negatively correlated with the HOMA index, but not at a significant level (r＝－0.237, p＝0.0809). Higher HOMA index in some Japanese female university students was noted, and that was associated with lifestyle, especially n-6/n-3 fatty acid ratio of nutrient oral intake.

Prevalence and risk factors for renal artery stenosis and chronic kidney disease in Japanese patients with peripheral arterial disease.

The prevalence and risk factors for renal artery stenosis (RAS) and chronic kidney disease (CKD) are unclear in Japanese patients with peripheral arterial disease (PAD). To examine these issues, we performed renal angiography in 410 patients with PAD. Renal function and damage were assessed using the estimated glomerular filtration rate (eGFR) and urinary level of microalbumin (MA). Multiple logistic and multiple regression analyses were used to examine the relationships of potential risk factors with RAS and CKD. In all, 94 subjects (22.9%) had RAS >50% and 45 subjects (11.0%) had RAS >75%. The incidences of an abnormal level of MA and renal insufficiency (eGFR <60 ml min(-1) per 1.73 m(2)) were 37.0 and 60.7%, respectively. RAS > or =50% was associated with critical limb ischemia (CLI; hazard ratio (HR) 2.519; 95% confidence interval (CI) 1.203-5.276, P=0.014), coronary heart disease (CHD; HR 2.143; 95% CI 1.129-4.069; P=0.020) and hypertension (HR 1.907; 95% CI 1.009-3.628; P=0.045). RAS > or =75% had a relationship with hypertension (HR 3.093; 95% CI 1.002-9.548; P=0.048). eGFR was negatively correlated with age, uric acid and CHD (P=0.013), and MA had a significant positive correlation with low-density lipoprotein cholesterol, CLI, age, CHD and diabetes (P<0.001). These results show that the prevalences of RAS and CKD are very high in Japanese patients with PAD; that CLI and CHD are major risk factors for RAS; and that hyperuricemia, hypercholesterolemia and diabetes are risk factors for CKD in PAD. We also found that MA is a simple and noninvasive marker of renal dysfunction and general vascular damage.

Effects of sulfur dioxide on formation of fishy off-odor and undesirable taste in wine consumed with seafood.

In order to evaluate sensory compatibility of alcoholic beverages with food, beverages and dried squid, namely, "surume", a common Japanese accompaniment, were consumed together. White wine and dried squid pairings had a more undesirable taste and more fishy off-odor than sake and dried squid pairings. The undesirable taste and fishy off-odor appeared to be caused by degradation of unsaturated fatty acids (e.g., docosahexaenoic acid (DHA)), which are found in fish and squid. Upon addition of DHA to the beverage, bitterness intensity, measured by instrumental taste sensor analysis, and the concentration of certain aldehydes reported to contribute to fishy flavors, increased in white wines, whereas they remained largely the same in sake. Among the major chemical constituents that distinguish wine from sake, only wine-specific sulfite markedly increased bitterness intensity and aldehyde levels upon addition of DHA. These results suggest that sulfur dioxide in wine participated in degradation of unsaturated fatty acids, causing an increase in undesirable taste and fishy off-odor in wine and seafood pairings.

An antioxidant effect by acyclic retinoid suppresses liver tumor in mice.

The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor alpha (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P<0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2'-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of beta-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.

Increase in serum troponin-I following rotational atherectomy reliably predicts the occurrence of reversible wall motion abnormalities.

BACKGROUND: There is growing evidence that microdebris pulverized by rotational atherectomy (RA) may have a deleterious effect on coronary microcirculation and ventricular contraction. However, the incidence and the predictors of worsening of left ventricular wall motion following RA are unknown. METHODS: Thirty patients without a known previous anterior wall myocardial infarction who underwent RA-facilitated angioplasty for de novo lesions of left anterior descending coronary artery were evaluated. Global and anterior wall regional left ventricular ejection fraction (LVEF and AREF) were analyzed before and after RA. Serum troponin-I, CK, and CK-MB concentrations were assayed. RESULTS: Target lesion length was greater (P=0.03), post-procedural minimal lumen diameter was smaller (P<0.01), and serum troponin-I immediately after RA was higher (P=0.01) in 16 patients with a decrease in the AREF of at least 15% than in those without. Stepwise logistic regression analysis identified one independent predictor of such worsening of AREF, troponin-I> or =0.1 ng/ml immediately after the procedure (P<0.01), with sensitivity of 81%, specificity of 79%, and relative risk of 3.8. CONCLUSIONS: Our results suggest that RA can cause substantial deterioration in left ventricular wall motion. Serum troponin-I, a sensitive indicator of myocardial damage, may be useful in predicting reversible LV wall motion abnormalities following RA.

Peristent tissue proliferation of multilink stents is dependent on preprocedural plaque area: a serial intravascular ultrasound analysis.

It is not known whether any factors are related to tissue proliferation within and surrounding stents in humans. The authors used serial intravascular ultrasound (IVUS) to evaluate the relationship between IVUS parameters and tissue proliferation within and surrounding Multilink stents. They were able to analyze preinterventional and postinterventional and follow-up IVUS studies in 33 native vessel lesions in 33 patients with stable angina pectoris. Quantitative coronary angiography and IVUS measurements were performed before and after intervention and at follow-up. IVUS imaging using an automatic transducer pullback device allowed follow-up analysis of the same lesion site. The vessel area at the lesion site increased from 17.1 +/- 4.5 mm2 after intervention to 18.5 +/- 5.9 mm2 at follow-up (p<0.01). The in-stent tissue growth (after intervention to follow-up) in-stent plaque area (PA) was 1.6 +/- 1.1 mm2, and the peristent tissue growth (after intervention to follow-up) peristent PA was 0.8 +/- 2.3 mm2. In multivariate analysis, the preprocedural PA at the lesion site was the best predictor of the peristent tissue growth, whereas no factors predicted the in-stent tissue growth. Risk factors, clinical characteristics, and quantitative coronary angiographic parameters showed no relation to the peristent tissue growth or the in-stent tissue growth. The peristent tissue growth was closely related to the preprocedural plaque size, while the factors that affect the in-stent tissue growth were not identified.