Search

Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of Thioploca ingrica in lake sediment.

The relationship between concentrations of chlorophyll a in an open water area and growing areas of the macrophytes Trapa spp. and Nelumbo nucifera was investigated to clarify the role of macrophytes in phytoplankton growth in summer in a eutrophic shallow sand lake, Lake Honsagata, Japan. The chlorophyll a concentrations formed peaks in June 2007 and July 2008 in summer with cyanobacterial blooms composed of mainly Microcystis, Anabaena, Phormidium, and Oscillatoria, which decreased toward August and were maintained at different annual levels. The decline of blooms in August was caused by the rapid growth of macrophytes. The composition of phytoplankton and the level of bloom development in summertime differed characteristically from year to year. Also the total vegetation area of N. nucifera and Trapa spp. showed annually marked changes. A significant negative correlation between the concentrations of chlorophyll a and the growth areas of Trapa spp. in August was detected, indicating that the floating-leaved plants, Trapa spp., produced irregular, clear and turbid water states in this shallow eutrophicated lake. These antagonistic relations are explained based on a likely scenario of allelopathic effects on the development of cyanobacteria by Trapa spp. vegetation and the nutrient absorption competition among them. Our study demonstrated the potential of Trapa spp. to control cyanobacterial blooms producing harmful toxins.

Phylogenetic diversity among filamentous sulfur-oxidizing bacteria of the genus Thioploca inhabiting freshwater/brackish environments was analyzed in detail. The 16S rRNA gene sequence of Thioploca found in a freshwater lake in Japan, Lake Okotanpe, was identical to that of Thioploca from Lake Ogawara, a brackish lake. The samples of two lakes could be differentiated by the sequences of their 23S rRNA genes and 16S-23S rRNA internal transcribed spacer (ITS) regions. The 23S rRNA-based phylogenetic relationships between Thioploca samples from four lakes (Lake Okotanpe, Lake Ogawara, Lake Biwa, and Lake Constance) were similar to those based on the 16S rRNA gene sequences. In addition, multiple types of the ITS sequences were obtained from Thioploca inhabiting Lake Okotanpe and Lake Constance. Variations within respective Thioploca populations were also observed in the analysis of the soxB gene, involved in sulfur oxidation. As major members of the sheath-associated microbial community, bacteria of the phylum Chloroflexi were consistently detected in the samples from different lakes. Fluorescence in situ hybridization (FISH) revealed that they were filamentous and abundantly distributed within the sheaths of Thioploca.

We have previously reported that in cultured rat vascular smooth muscle cells (VSMCs), neurotrophin-3 (NT-3) gene expression was suppressed by TPA (12-O-tetradecanoyl phorbol-13-acetate), which induces an AP-1 transcription factor. In the present study, to clarify the mechanism for TPA-mediated downregulation of NT-3 gene expression, effects of cycloheximide and dexamethasone (Dex) on the TPA-mediated downregulation were examined in VSMCs. Pretreatment with cycloheximide, an inhibitor of protein synthesis, or simultaneous treatment with Dex, an inhibitor of AP-1, suppressed the TPA-mediated downregulation of NT-3 gene expression. Furthermore, co-transfection of c-fos and c-jun expression vectors into VSMCs resulted in decrease in the NT-3 gene expression. The present findings suggest that TPA-induced AP-1 de novo synthesis causes the downregulation of NT-3 gene expression in VSMCs.

Brain-derived neurotrophic factor and its receptor,trkB, are thought to play a crucial role for protection against neuronal death induced by brain ischemia, such as in stroke. In the present study we found a missense mutation in thetrkB gene from all of the five substrains of stroke-prone spontaneously hypertensive rats (SHRSP) that were examined. This mutation was not found in six out of seven hypertensive but stroke-resistant ancestral strains (SHR) of SHRSP, nor in any of seven strains of normotensive, non-stroke-prone strains. Hippocampal neurons, which are particularly vulnerable to damage in stroke, were shown to be more susceptible to ischemic damage in SHRSP than in either SHR or normotensive, stroke-resistant controls. The association of a mutatedtrkB gene with the stroke-prone genotype found in this study suggests that thetrkB gene merits further study as a promising candidate gene for stroke.

A DNA fragment containing 8-hydroxydeoxyguanosine was used to prime second strand synthesis of a recombinant pTZI9 phagemid single stranded DNA. The resultant double stranded closed circular product and also the annealed product of the single stranded DNA and the fragment were found to be resistant to cleavage by the restriction enzyme ApaLI in whose recognition sequence the 8-hydroxydeoxyguanosine had been inserted.

A stable nitrogen isotope analysis was used to clarify the relative importance of denitrification and nitrate uptake by plants in the nitrate reduction in a reed belt of L. Kamisagata (N 37°49′, E 138°53′, alt. 4.5 m, depth 30–80 cm, area 0.025 km2), one of about 20 sand dune lakes in Japan. A very high concentration of NO3 −-N with 19.0 ±5.9 mg N l−1 in spring sources decreased during passage through the reed belt along two set transect lines about 120 m long in any season, whereas progressive enrichment in 15N-NO3 − in flowing water was detected. Loss rate of nitrate ranged from 38.4 to 73.1% with an average of 56.7 ±11.6%. Enrichment factors calculated using a Rayleigh curve method ranged from - 1.03 to -5.12%.. The contribution of denitrification to nitrate loss ranged from 6 to 28%, with a mean of 19.5% (±7.0), whereas that of plant uptake was from 72 to 94%, with a mean of 80.5% (±7.0), indicating the importance of vegetation in a sand dune riparian zone. A technique using the variation of natural abundance of 15N may provide useful information on the nitrate dynamics in artificial or natural wetlands under a nondestructive condition.

The neurotrophin-3 (NT-3) gene has previously been reported to consist of three exons including two 5' short untranslated exons and a 3' long exon encoding the entire protein, and to give rise to two classes of transcripts by alternative splicing of the 5' exons to the 3' coding exon. In the present study, we demonstrated the presence of at least four new classes of transcripts of the NT-3 gene, in addition to the two known transcripts. The present finding proposes the further complexity of the regulational mechanism for NT-3 expression.

Most previous researches on neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have focused on the nervous system, because their receptors are widely distributed in neuronal tissues. Recently, however, the participation of neurotrophins in inflammation and atherosclerosis has been proposed. Therefore, the gene expression of neurotrophins is now an urgent issue is to be investigated in nonneuronal tissues. Here, we evaluated the gene expression of neurotrophins and their receptors in rat cultured vascular smooth muscle cells (VSMCs) by the reverse transcriptase-polymerase chain reaction method. The transcripts of NGF, NT-3, and TrkC (high-affinity receptor for NT-3), and two BDNF alternative spliced transcript variants with exons 3 and 4 were clearly detected in VSMCs cultured under conventional culture conditions. The upregulation of mRNA levels for NGF, two BDNF variants with exons 1 and 2, low-affinity neurotrophin receptor, and high-affinity receptors, TrkA (for NGF) and TrkB (for BDNF), was observed in response to the treatment with serum and phorbol-ester following the serum-starvation. In contrast, the expression of NT-3 and TrkC genes was downregulated under these conditions. Co-expression of these factors and their receptors and the characteristic regulation of their gene transcriptions suggest that these factors play crucial roles in the function of VSMCs through an autocrine mechanism.

Summary The effect of aging on expression of a natural glutamine suppressor tRNA (tRNA Gln(UmUG) ) was studied in different tissues of mice; this tRNA recognizes UAG and inserts glutamine at the site of the termination codon. The level of tRNA Gln(UmUG) was found to be strongly increased in aged mice, compared to newborn and mature animals. An elevated expression of tRNA Gln(UmUG) has also been found in retrovirus-infected cells; in Moloney virus-infected cells the suppressor tRNA allows to read-through the UAG codon within the retroviral protease gene. We suggest that the increase in the level of tRNA Gln(UmUG) may influence retroviral gene expression with age.

The dominant +1 frameshift suppressors sufA6, sufB1 and sufB2, in Salmonella typhimurium act at runs of C and affect tRNA(Pro)1, tRNA(Pro)2 and tRNA(Pro)2, respectively. A recessive +1 frameshift suppressor, sufC, has a similar suppressor specificity (Riddle, D.L., and Roth, J.R., Mol. Biol. 66, 483 and 495, 1972). We show that sufC strains harbour two frameshift suppressors of which one, sufX201, is allelic to sufB. We cloned the sufB+ wild type allele and by recombination in vivo the mutations sufB1, sufB2 and sufX201. Determination of the DNA sequence revealed that the sufB1 and sufB2 mutations result in an extra G in the anticodon loop of the minor tRNA(Pro)2. The sufX201 mutation results in a base substitution (G43 to A43) in the anticodon stem of this tRNA. Although the sufB1 and sufB2 mutations were earlier shown to be dominant, the sufB+ wild type allele on multi copy plasmid inhibited the chromosomal sufB1, sufB2 and sufX201 mediated frameshift suppression but not that mediated by the dominant sufA6 mutation. These results are discussed in view of the possible coding specificity of these mutated tRNAs. The DNA sequence showed a potential consensus promoter sequence upstream of the structural gene for tRNA(Pro)2 and downstream a dyad symmetrical structure followed by a T cluster, a possible rho-independent termination signal. The Salmonella tRNA(Pro)2 gene is identical to the Escherichia coli counterpart reported by Komine, Y. et al. (J. Mol. Biol. 212, 579-598, 1990). While the 5' flanking sequence similarity between the two species is about 83%, the similarity of the 3' flanking sequence is only 42%. Still, the Salmonella tRNA(Pro)2 gene has a rho-independent transcriptional termination signal similar to the one present in E. coli tRNA(Pro)2 gene.

The purpose of this study is to investigate the relationship between gingival discoloration and Ag2S. Minute granules of dental alloys were embedded in the gingiva during a 5-11 months span, and their histological and colorimetric analyses were undertaken. Results showed the following; 1. Cases of low melting silver alloy, 12% Au-Ag-Pd alloy, and Ni-Cr alloy all resulted in gingival discoloration, but it did not occur during the specified experimental period. 2. Regardless of the size of the granules or time, the electron microanalyser showed no signs of sulfer detection in cases of low melting silver alloy. However, in the case of 12% Au-Ag-Pd alloy, sulfer was detected 5 months later for only those granules having the size of 0.2-0.3 microns. No sulfer was detected in cases of Ni-Cr alloy. 3. Although no proliferation of inflammatory cells were observed around those areas surrounding the 12% Au-Ag-Pd alloy granules, they appeared in cases of the low melting silver alloy and Ni-Cr alloy.

The purpose of this study is to analyze, using CIELAB color system, the range ofgingival color variables in healthy gingiva of the maxillary incisor region.Results indicated:1. Color differences (dE) among the bilateral attached andmarginal gingiva of the incisor and lateral incisor regions were under 2.0. However, the attached and marginal gingiva of the canine regions were about 2.88 and2.45 respectively. The papillary gingiva was over 2.0 in each region.2. Compared to the incisor region, an increase in chromaticness and hue a*, b* with a decrease in lightness was observed in the attached, marginal, and papillary gingiva of the lateral incisor and canine region. The range in dE was from 2.28 to 3.36. In the attached and papillary gingiva, dE between the incisor and canine region was significantly bigger than the incisor and lateral incisor region.3.Decrease in chromaticness, lightness, and hue a*, b* was observed from the marginal to papillary gingiva, and dE was from 3.13 to 3.71 with a high being in theincisor region.4. Increase in chromaticness and hue a*, b* with a slight decreasein lightness was observed from the attached to marginal gingiva, and dE was from2.25 to 2.75.

An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA2Ile or tRNAminorIle (ref. 1) has a novel modified nucleo-side, lysidine (L; ref. 2) (Fig. la) in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA (ref. 1). We isolated the gene for tRNA2Ile (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene. Replacement of L(34) of tRNA2Ile molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity. Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.

By carrying out oligonucleotide-directed mutagenesis, in vitro, on a 3.3 kb XhoI-HindIII fragment from Moloney murine leukaemia virus Mo-MuLV proviral DNA, inserted into the phagemid pTZ19R, nine separate fragments have been prepared in which mutations have been inserted at and around the gag-pol gene junction. Using these mutant fragments Mo-MuLV proviral DNA has been reassembled and cloned into pBR322. Examination of the mutant proviral DNAs in mouse culture cells indicates that a terminator codon at the gag-pol junction is essential for function, but any of the three chain terminator codons gives an active virus. Also the region of secondary structure surrounding the terminator codon must be preserved.