RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

Deficiency of the cystine-transporter gene, xCT, does not exacerbate the deleterious phenotypic consequences of SOD1 knockout in mice

Both α-lipoic acid (LA) and ascorbic acid (vitamin C) have been shown to improve endothelial dysfunction, a precursor of atherosclerosis. Since oxidant stress can cause endothelial dysfunction, we tested the interaction and efficacy of these antioxidants in preventing oxidant damage to lipids due to both intra- and extracellular oxidant stresses in EA.hy926 endothelial cells. LA spared intracellular ascorbate in culture and in response to an intracellular oxidant stress induced by the redox cycling agent menadione. Extracellular oxidant stress generated by incubating cells for 2 h in with 0.2 mg/ml LDL and 5 μM Cu²⁺ caused a time-dependent increase of the lipid peroxidation product malondialdehyde in both cells and LDL, preceded by rapid disappearance of` α-tocopherol in LDL. α-Lipoic acid at concentrations of 40–80 μM blunted these effects. Similarly, intracellular ascorbate concentrations of 1–2 mM also prevented Cu²⁺-induced lipid peroxidation in LDL and cells. Cu²⁺-dependent oxidation of LDL in the presence of ascorbate-loaded cells decreased intracellular ascorbate by 20%, but this decrease was not reversed by LA. Both LA and ascorbate protect endothelial cells and LDL from either intra- or extracellular oxidant stress, but that LA does not spare ascorbate in oxidatively stressed cells.     

Ingested beta-carotene enhances glutathione level and up-regulates the activity of cysteine cathepsin in murine splenocytes

To elucidate health benefits of beta-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of beta-carotene. In mice supplemented with beta-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of beta-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated beta-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.     

Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an ^L111^F substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS. Nucleotide sequence data reported in this article are available in the DDBJ/EMBL/GenBank database under accession numbers AB292042-292043 and AB294577-294580.     

Neutralization of acidified water by use of Rhizopus delemar immobilized with a cellulose membrane

To solve serious environmental problems caused by the acidification of pond and lake water by acid rain, remediation methods must be used to keep water pH values neutral. In this study, a microbial method to neutralize acidified water was developed. The neutralization activities of 30 strains of bacteria, yeasts and fungi were measured with a medium adjusted to pH 3.0. Because fungi showed high neutralization properties, the Rhizopus delemar fungus was used to study the characteristics of acidified water neutralization. When R. delemar cells were cultured in a media acidified with nitric, sulfuric and hydrochloric acids, the cells neutralized acids by secreting basic compounds including ammonia. The cells also assimilated nitric acid. R. delemar was used to neutralize pond water adjusted to pH 4.0 with nitric acid. R. delemar cells increased the pH value of pond water from 4.0 to around 7.0 within 2 days, although indigenous microorganisms had not been able to neutralize the same pond water. In this study, R. delemar immobilized in a cellulose tube neutralized acidified water repeatedly by the draw-fill method.     

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.     

Sequence and phylogenetic analyses of novel glucosyltransferase genes of mutans streptococci isolated from pig oral cavity

Nucleotide sequences of water-insoluble glucan-producing glucosyltransferase (gtf) genes of new mutans streptococci isolated from pig oral cavity, Streptococcus orisuis JCM14035, and of Streptococcus criceti HS-6 were determined. The gtf gene of S. orisuis JCM14035 consisted of a 4,401 bp ORF encoding for a 1,466 amino acids, and was revealed to belong to the gtfI group. The percent homology of amino acid sequence of the GTF-I from S. orisuis and S. criceti are 95.0%, however, this score ranges from 77.0% to 78.0% when compared to Streptococcus sobrinus 6715. The deduced N-terminal amino acid sequence was considered responsible for the secretion of GTF-I in S. orisuis JCM14035 and S. criceti HS-6 with high similarity to known GTF proteins from other streptococci. In addition, two other conserved regions, i.e., N-terminal putative catalytic-site and C-terminal glucan binding domain, were also found in GTF-Is of S. orisuis JCM14035 and S. criceti HS-6. Phylogenetic analysis suggested that S. orisuis JCM14035 and S. criceti HS-6, closely related to each other, resemble S. sobrinus and S. downei based on the amino acid sequences of the GTFs.     

Genetic Variation and Population Structure of Hair Crab (<Emphasis Type="Italic">Erimacrus isenbeckii</Emphasis> ) in Japan Inferred from Mitochondrial DNA Sequence Analysis

Genetic variation and population structure of hair crab (Erimacrus isenbeckii) were examined using nucleotide sequence analysis of 580 base pairs (bp) in the 3′ portion of the mitochondrial cytochrome c oxidase subunit I gene (COI) of 20 samples collected from 16 locales in Japan (the Hokkaido and Honshu Islands) and one in Korea. A total of 27 haplotypes was defined by 23 variable nucleotide sites in the examined COI region. Pairwise population F _ST estimates and neighbor-joining tree inferred distinct genetic differentiation between the representative samples from the Pacific Ocean off the Eastern Hokkaido Island and the Sea of Japan, while others were intermediate between these two groups. AMOVA also showed a weak but significant differentiation among these three groups. The present results suggest a moderate population structure of hair crab, probably influenced by high gene flow between regional populations due to sea current dependent larval dispersal of this species.