Purification and Properties of an S-PI(Pepstatin Ac)-insensitive Carboxyl Proteinase from a Xanthomonas sp. Bacterium

A S-PI(Pepstatin Ac)-insensitive carboxyl proteinase was found in culture filtrate of a Xanthomonas sp. bacterium. The carboxyl proteinase was highly purified and about 100 mg of the enzyme was obtained from 601 of culture filtrate, with a recovery of 25%. The optimum condition for the action of the purified enzyme toward casein was approx. pH 2.7 and its activity was not inhibited by any of such carboxyl proteinase inhibitors as Pepstatin, S-PI, and DAN but EPNP inhibited it. Such behavior of the enzyme against inhibitors resembles that of Pseudomonas sp. carboxyl proteinase, the first found from a bacterium. Some differences were observed, however, in their properties such as optimum pH, isoelectric point, and amino acid composition.     

Chemical Studies on Pyrethroids

Allethrin-(E)-Ol (IV), allethrin-(E)-al (V) and allethrin-(E)-acid (VI), the metabolites of allethrin (III) in the insect body, were synthesized. Their low toxicities to houseflies seem to support the hypothesis that they are products of the detoxication process of allethrin.     

Absolute Configuration of (+)-1-Acetoxy-p-menth-3-one Isolated from Essential Oil of Mentha gentilis (L.)

The most effective electro-energizing fermentation (E-E F) conditions for l-glutamate (l-Glu) production by Brevibacterium flavum No. 2247 were determined. The adding of 0.01 mm neutral red at the beginning of cultivation was found most effective. A 1.5 V direct current was applied to the culture broth at 6~8 hr after inoculation in the cathode compartment, l-Glu was produced at 51.0 mg per ml, and this is about a 15 % increase in yield compared to the yield of the not electro-energizing (E-E) control (44.3 mg/ml).     

Enzymatic fragmentation of carbohydrate moieties of radish arabinogalactan-protein and elucidation of the structures

We investigated the structures of L-arabino-galactooligosaccharides released from the sugar moieties of a radish arabinogalactan-protein (AGP) by the action of exo-β-(1→3)-galactanase. We detected a series of neutral β-(1→6)-linked galactooligosaccharides forming branches of one to up to at least 19 consecutive Gal groups, together with corresponding acidic derivatives terminating in 4-O-methyl-glucuronic acid (4-Me-GlcA) at the non-reducing end. Some oligosaccharide chains of degree of polymerization (dp) higher than 3 for neutral, and 4 for acidic oligomers were modified with L-Araf residues. The acidic tetrasaccharide 4-Me-β-GlcA-(1→6)[α-L-Araf-(1→3)]-β-Gal-(1→6)-Gal was detected as an abundant L-Araf-containing oligosaccharide among these neutral and acidic oligomers. A pentasaccharide containing an additional L-Araf group attached to the L-Ara in the tetrasaccharide through an α-(1→5)-linkage was also found. We observed L-arabino-galactooligosaccharides substituted with single or disaccharide L-Araf units at different Gal residues along these neutral and acidic β-(1→6)-galactooligosaccharide chains, indicating that these side chains are highly variable in length and substituted variously with L-Araf residues.     

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.     

Bio-functional pickles that reduce blood pressure of rats

Addition of γ-aminobutyric acid (GABA) and angiotensin converting enzyme (ACE)-inhibitory peptides to the pickles was studied in order to develop a new type of pickles that reduce blood pressure. Based on the outcome of these studies, a new type of fermentation bed composed of rice bran and white miso has been successfully developed. The advantage of such pickles is that they not only contain both GABA and ACE-inhibitory peptides, but also that their taste and flavor are excellent, with colors close to the original ones. The new type of pickles could temporarily reduce blood pressure in two types of rats, spontaneously hypertensive rats and NaCl-sensitive model rats. Thus, the newly developed pickles appear to be beneficial for pickle business.     

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.     

Kif14 Mutation Causes Severe Brain Malformation and Hypomyelination

We describe a novel spontaneous mouse mutant, laggard (lag), characterized by a flat head, motor impairment and growth retardation. The mutation is inherited as an autosomal recessive trait, and lag/lag mice suffer from cerebellar ataxia and die before weaning. lag/lag mice exhibit a dramatic reduction in brain size and slender optic nerves. By positional cloning, we identify a splice site mutation in Kif14. Transgenic complementation with wild-type Kif14-cDNA alleviates ataxic phenotype in lag/lag mice. To further confirm that the causative gene is Kif14, we generate Kif14 knockout mice and find that all of the phenotypes of Kif14 knockout mice are similar to those of lag/lag mice. The main morphological abnormality of lag/lag mouse is severe hypomyelination in central nervous system. The lag/lag mice express an array of myelin-related genes at significantly reduced levels. The disrupted cytoarchitecture of the cerebellar and cerebral cortices appears to result from apoptotic cell death. Thus, we conclude that Kif14 is essential for the generation and maturation of late-developing structures such as the myelin sheath, cerebellar and cerebral cortices. So far, no Kif14-deficient mice or mutation in Kif14 has ever been reported and we firstly define the biological function of Kif14 in vivo. The discovery of mammalian models, laggard, has opened up horizons for researchers to add more knowledge regarding the etiology and pathology of brain malformation.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.     

Replication Study in a Japanese Population of Six Susceptibility Loci for Type 2 Diabetes Originally Identified by a Transethnic Meta-Analysis of Genome-Wide Association Studies

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and six susceptibility loci (TMEM154, SSR1, FAF1, POU5F1, ARL15, and MPHOSPH9) originally identified by a transethnic meta-analysis of genome-wide association studies (GWAS) in 2014.MethodsWe genotyped 7,620 Japanese participants (5,817 type 2 diabetes patients and 1,803 controls) for each of the single nucleotide polymorphisms (SNPs) using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using logistic regression analysis.ResultsOf the six SNPs examined in this study, four (rs6813195 near TMEM154, rs17106184 in FAF1, rs3130501 in POU5F1 and rs4275659 near MPHOSPH9) had the same direction of effect as in the original reports, but two (rs9505118 in SSR1 and rs702634 in ARL15) had the opposite direction of effect. Among these loci, rs3130501 and rs4275659 were nominally associated with type 2 diabetes (rs3130501; p = 0.017, odds ratio [OR] = 1.113, 95% confidence interval [CI] 1.019–1.215, rs4275659; p = 0.012, OR = 1.127, 95% CI 1.026–1.238, adjusted for sex, age and body mass index), but we did not observe a significant association with type 2 diabetes for any of the six evaluated SNP loci in our Japanese population.ConclusionsOur results indicate that effects of the six SNP loci identified in the transethnic GWAS meta-analysis are not major among the Japanese, although SNPs in POU5F1 and MPHOSPH9 loci may have some effect on susceptibility to type 2 diabetes in this population.