Effect of vitamin A treatment on superoxide dismutase-deficient yeast strains

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Δ) and CuZnSOD/MnSOD (sod1Δsod2Δ). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.     

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.     

Flavonoid-induced reduction of ENaC expression in the kidney of Dahl salt-sensitive hypertensive rat

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium,Paenibacillus polymyxa A-1

The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K(m) values for FMN and NADH were 2.1 microM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.     

Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patient's family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Identification of effective pollinators of Primula sieboldii E. Morren in a wild habitat in Hiroshima,Japan

Primula sieboldii E. Morren is a heterostylous clonal herb that is widely distributed in Japan but in danger of extinction in the wild. The existence of pollinators in each habitat is imperative for its long-term survival, because seeds can be produced only by insect cross-pollination between different flower morphs. In this study, we identified the pollinators of P. sieboldii in a wild habitat in Hiroshima as those insects that we observed to (a) put the proboscis into a corolla tube, (b) deposit pollen grains on the proboscis, and (c) have a proboscis of appropriate length and width. Effective pollinators were identified from their contribution to pollination. In 2015 and 2016, the flower visitations of 232 and 558 insects, respectively, were recorded and 85 and 13 insects were captured. Two Bombylius species, B. major L. and B. shibakawae Matsumura, accounted for 90% of flower-visiting insects in both years. All 14 species that we captured were considered pollinators of P. sieboldii, because they had proboscises that were long enough to reach pollen and that had pollen grains deposited on them. The total visitation rate of “Bombyliidae” was the highest among all pollinator categories. The results of potential pollen transport per flower per hour, which was based on total pollen number and total visitation rate of each pollinator category, indicated that “Bombyliidae” species were the most effective pollinators of P. sieboldii in this habitat.