2-Nitropropane Dioxygenase from Hansenula mrakii: Re-characterization of the Enzyme and Oxidation of Anionic Nitroalkanes

2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula mrakii, has a molecular weight of 42,000, and consists of a single polypeptide. The enzyme contains 1 mol of FAD per mol of enzyme. The iron protein associated with previous preparations was removed by the present purification procedures. The enzyme catalyzes the oxygenative denitrification of anionic nitroalkanes much more effectively than that of the neutral ones with the optimum pH of 6.5. The Michaelis constants for the anionic substrates are as follows: 2-nitropropane, 1.61 mM; 1-nitropropane, 3.23 mM; nitroethane, 3.13 mM, and 3-nitro-2-butanol, 0.59 mM.     

Purification and Properties of meso-α,∊-Diaminopimelate Decarboxylase from Bacillus sphaericus

Soybean 7S and 11S globulins were stored at relative humidities (RHs) of 11% and 96% at 50°C. The redispersibility of the proteins at RH 96% decreased in a short time. However, it did not decrease, when stored for 45 days at RH 11%. Gel filtration showed that the proteins polymerized during storage. The effects of urea, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on the redispersibilities of the proteins at RH 96% showed that the hydrogen, hydrophobic and disulfide bonds participate in the polymerization of 7S globulin, and that the disulfide bond is strongly related to the polymerization of 11S globulin. Redispersibility was restored with 2-ME in both the 7S and 11S globulins and some of the proteins in the supernatant redispersed with 2-ME were observed to be similar to the native ones with respect to the gel filtration, electrophoretic behavior and circular dichroism spectrum.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Purification and Characterization of a Thermostable Carboxypeptidase (Carboxypeptidase Taq) from Thermus aquaticus YT-1

Brassinosteroid (BR) and auxin co-regulate plant growth in a process termed cross-talking. Based on the assumption that their signal transductions are partially shared, inhibitory chemicals for both signal transductions were screened from a commercially available library. A chemical designated as NJ15 (ethyl 2-[5-(3,5-dichlorophenyl)-1,2,3,4-tetrazole-2-yl]acetate) diminished the growth promotion of both adzuki bean epicotyls and Arabidopsis seedlings, by the application of either BR or auxin. To understand its target site(s), bioassays with a high dependence on the signal transduction of either BR (BR-signaling) or auxin (AX-signaling) were performed. NJ15 inhibited the photomorphogenesis of Arabidopsis seedlings grown in the dark, which mainly depends on BR-signaling, while NJ15 also inhibited their gravitropic responses mainly depending on AX-signaling. On the study for the structure–activity relationships of NJ15 analogs, they showed strong correlations on the inhibitory profiles between BR- and AX-signalings. These correlations imply that NJ15 targets the downstream pathway after the integration of BR- and AX-signals.     

Nicotine Dependence and Cost-Effectiveness of Individualized Support for Smoking Cessation: Evidence from Practice at a Worksite in Japan

Given the lack of economic studies evaluating the outcomes of smoking cessation programs from the viewpoint of program sponsors, we conducted a case study to provide relevant information for worksites. The present study was carried out between 2006 and 2008 at a manufacturing factory in the Toyama Prefecture of Japan and included subjects who voluntarily entered a smoking cessation program. The program included face-to-face counselling followed by weekly contact to provide encouragement over six months using e-mail or inter-office mail. Nicotine patches were available if required. All 151 participants stopped smoking immediately. Over the 24-month study period, self-report showed 49.7% abstained continuously from smoking. The rate of 24-month consecutive abstinence was higher in participants with lower Fagerström Test scores for Nicotine Dependence at baseline than in those with higher scores (63.6% for 0–2 points vs. 46.5% for 3–6 points vs. 43.8% for 7–10 points; chi-square test p = 0.19). A logistic regression model showed a significant linear trend for the association between the score and abstinence status after adjustment for possible confounding factors (p = 0.03). The crude incremental cost for one individual to successfully quit smoking due to the support program was ¥46,379 (i.e., ¥100 = $1.28, £0.83, or €1.03 at foreign exchange rates). The corresponding costs for the three categories of the Fagerström Test score for Nicotine Dependence were ¥31,953, ¥47,450 and ¥64,956, respectively. When a sensitivity analysis was conducted based on the 95% confidence interval of the success rate, the variance in the corresponding costs was ¥25,514–45,034 for 0–2 points, ¥38,344–61,824 for 3–6 points, and ¥45,698–108,260 for 7–10 points. The degree of nicotine dependence may therefore be an important determinant of the cost-effectiveness of smoking cessation programs.     

Dapagliflozin,a Sodium-Glucose Co-Transporter 2 Inhibitor,Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.     

Construction of a Model Culture System of Human Colonic Microbiota to Detect Decreased Lachnospiraceae Abundance and Butyrogenesis in the Feces of Ulcerative Colitis Patients

Compositional alteration of the gut microbiota is associated with ulcerative colitis (UC). Here, a model culture system is established for the in vitro human colonic microbiota of UC, which will be helpful for determining medical interventions. 16S ribosomal RNA sequencing confirms that UC models are successfully developed from fecal inoculum and retain the bacterial species biodiversity of UC feces. The UC models closely reproduce the microbial components and successfully preserve distinct clusters from the healthy subjects (HS), as observed in the feces. The relative abundance of bacteria belonging to the family Lachnospiraceae significantly decreases in the UC models compared to that in HS, as observed in the feces. The system detects significantly lower butyrogenesis in the UC models than that in HS, correlating with the decreased abundance of Lachnospiraceae. Interestingly, the relative abundance of Lachnospiraceae does not correlate with disease activity (defined as partial Mayo score), suggesting that Lachnospiraceae persists in UC patients at a decreased level, irrespective of the alteration in disease activity. Moreover, the system shows that administration of Clostridium butyricum MIYAIRI restores butyrogenesis in the UC model. Hence, the model detects deregulation in the intestinal environment in UC patients and may be useful for simulating the effect of probiotics.     

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.