Biomedical subjects

Hidenori Hayashi

Publications and source records attributed to Hidenori Hayashi.

At least 19 recordsLinked to original sources

PubMed2006

Histidine kinases play important roles in the perception and signal transduction of hydrogen peroxide in the cyanobacterium, Synechocystis sp. PCC 6803.

Oxidative stress caused by reactive oxygen species and, in particular, to hydrogen peroxide (H(2)O(2)) has a major impact on all biological systems, including plants and microorganisms. We investigated the H(2)O(2)-inducible expression of genes in the cyanobacterium Synechocystis sp. PCC 6803 using genome-wide DNA microarrays. Our systematic screening of a library of mutant lines with defects in histidine kinases (Hiks) by RNA slot-blot hybridization and DNA-microarray analysis suggested that four Hiks, namely, Hik33, Hik34, Hik16 and Hik41, are involved in the perception and transduction of H(2)O(2) signals that regulate the gene expression of 26 of the 77 H(2)O(2)-inducible genes with induction factors higher than 4.0. Among the four Hiks, Hik33 was the main contributor and was responsible for 22 of the 26 H(2)O(2)-inducible genes under the control of the Hiks. By contrast to Hik33, PerR encoding putative peroxide-sensing protein is involved in the regulation of only nine H(2)O(2)-inducible genes.

Bacterial Proteins↗

PubMed2006

Immunogenic variation between multiple HLA-A*0201-restricted, Hepatitis C Virus-derived epitopes for cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the immune control of Hepatitis C Virus (HCV) infection. In the current study, a number of HLA-A*0201-restricted CTL epitopes derived from HCV were evaluated by examining the peptide-binding affinity for major histocompatibility complex (MHC) class I molecules, the stability of peptide-MHC complexes, killing activities of peptide-induced CTLs, and frequencies of intracellular interferon (IFN)-gamma-positive CD8+ T cells. Among 24 peptides tested, 15 peptides induced high or medium killing activities of peptide-specific CTLs. Thirteen of the 15 peptides exhibited high or medium binding affinities for HLA-A*0201 molecules, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. In contrast, the stability of peptide-MHC class I complexes was not correlated with killing activities of peptide-induced CTLs. Furthermore, only a limited number of peptides could induce high or medium frequencies of IFN-gamma-producing CD8+ T cells, which were generally considered to play a crucial role for the clearance of HCV. Analyses of the immunogenicity of CTL epitopes such as in the current study should provide important information about the design of an efficient HCV vaccine that induces vigorous, sustained, and broad HCV-specific CTL responses.

Amino Acid Sequence↗

PubMed2006

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.

Bifidobacterium↗

PubMed2006

Diversity of the Clostridium coccoides group in human fecal microbiota as determined by 16S rRNA gene library.

Fecal microbiota were analyzed in seven healthy individuals by 16S rRNA gene libraries (universal library) using universal primers, and the Clostridium coccoides group libraries using the universal primer 27F and the C. coccoides group-specific primer Erec482. The universal libraries were used in our previous studies. The 972 clones obtained from two different primer set libraries belonged to the C. coccoides group and were classified into 139 operational taxonomic units (OTU) (at least 98% sequence similarity). Of these, 41 OTU were detected commonly from universal libraries and C. coccoides group libraries. One hundred and ten OTU were detected from the C. coccoides group libraries. Fifteen new OTU were isolated from the C. coccoides group libraries in human gut. Most of the OTU did not correspond to known species, thus representing as-yet-uncultured bacteria. We also detected OTU that related to the butyrate-producing bacteria. The C. coccoides group consisted of an average of 35 OTU, although there were differences in the number and the type of species in each individual. When fecal microbiota were analyzed using universal libraries, the OTU belonging to the C. coccoides group detected in elderly individuals were fewer than those detected in adult individuals. When C. coccoides group libraries were used, the numbers of OTU in elderly and adult individuals were not different. Interindividual differences in the composition of OTU belonging to the C. coccoides group were also observed in fecal microbiota.

Adult↗

PubMed2006

Increase in terminal restriction fragments of Bacteroidetes-derived 16S rRNA genes after administration of short-chain fructooligosaccharides.

It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.

Animals↗

PubMed2005

The rpoH gene encoding heat shock sigma factor sigma32 of psychrophilic bacterium Colwellia maris.

The rpoH gene encoding a heat shock sigma factor, sigma(32), was cloned from the psychrophilic bacterium Colwellia maris. The deduced amino acid sequence of sigma(32) from C. maris is more than 60% homologous to that of sigma(32) from mesophilic bacteria. The RpoH box, a 9-amino-acid sequence region (QRKLFFNLR) specific to sigma(32), and two downstream box sequences complementary to a part of 16S rRNA were identified. Primer extension analysis showed that the C. maris rpoH is expressed from only one sigma(70)-type promoter. Northern blot analysis showed that the level of rpoH mRNA was clearly increased at 20 degrees C, a temperature that induces heat shock in this organism. In the presence of an inhibitor of transcriptional initiation, the degradation of rpoH mRNA was much slower at 20 degrees C than at 10 degrees C. Thus, increased stability of the rpoH mRNA might be responsible for the rpoH mRNA accumulation. The predicted secondary structure of the 5'-region of C. maris rpoH mRNA was different from the conserved patterns reported for most mesophilic bacteria, and the base pairing of the downstream boxes appeared to be less stable than that of Escherichia coli rpoH mRNA. Thus, essential features that ensure the HSP expression at a relatively low temperature are embedded in the rpoH gene of psychrophiles.

Amino Acid Sequence↗

PubMed2005

The histidine kinase Hik34 is involved in thermotolerance by regulating the expression of heat shock genes in synechocystis.

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (DeltaHik34 cells) survived incubation at 48 degrees C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in DeltaHik34 cells after incubation of cells at 42 degrees C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44 degrees C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

Bacterial Proteins↗

PubMed2005

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota.

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.

Adult↗

PubMed2005

Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism.

Microbiota in gut contents of jejunum, ileum, caecum and recto-sigmoid colon obtained from three elderly individuals at autopsy were compared using 16S rRNA gene libraries and terminal restriction fragment length polymorphism (T-RFLP). Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets of total genomic DNA extracted from each sample of gut contents. An average of 90 randomly selected clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using the 16S rRNA gene amplified from each sample. The lengths of the terminal restriction fragments were analysed after digestion with HhaI and MspI. The jejunal and ileal microbiota consisted of simple microbial communities of streptococci, lactobacilli, 'Gammaproteobacteria', the Enterococcus group and the Bacteroides group. Most of the species were facultative anaerobes or aerobes. The Clostridium coccoides group and the Clostridium leptum subgroup, which are the most predominant groups in human faeces, were not detected in samples from the upper gastrointestinal tract. The caecal microbiota was more complex than the jejunal and ileal microbiota. The C. coccoides group, the C. leptum subgroup and the Bacteroides group were detected in the caecum. The recto-sigmoidal colonic microbiota consisted of complex microbial communities, with numerous species that belonged to the C. coccoides group, the C. leptum subgroup, the Bacteroides group, 'Gammaproteobacteria', the Bifidobacterium group, streptococci and lactobacilli, and included more than 26 operational taxonomic units. The results showed marked individual differences in the composition of microbiota in each region.

Aged↗

PubMed2005

Detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling.

Although the micro-organisms forming the cutaneous microbiota are considered to play important roles in the modification and prevention of skin diseases, a comprehensive analysis of their composition has not yet been carried out because of difficulties in determining yet-to-be-cultured micro-organisms in the samples. Swab-scrubbed forehead skin samples of five healthy volunteers were analysed by profiling 16S rRNA genes, as well as by conventional culture methods, to provide a profile of the cutaneous microbiota that included yet-to-be-cultured bacteria from normal human skin. Cluster analyses of the 16S rRNA gene sequences indicated a marked increase in diversity compared with that derived from the culture methods. Nineteen previously recognized species and 13 novel phylotypes were obtained from the analysis of 416 clones. In addition to well-known bacteria such as Staphylococcus epidermidis and Propionibacterium acnes, phylotype A, the 16S rRNA gene of which is 97 % similar to that of Methylophilus methylotrophus, was detected in three of the five samples, in one of which it was the predominant clone. Culture-independent genetic profiling of 16S rRNA genes for detecting human cutaneous microbiota has allowed us to detect potentially novel components of the cutaneous microbiota in humans.

Bacteria↗

PubMed2005

Systematic characterization of the ADP-ribose pyrophosphatase family in the Cyanobacterium Synechocystis sp. strain PCC 6803.

We have characterized four putative ADP-ribose pyrophosphatases Sll1054, Slr0920, Slr1134, and Slr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. Sll1054 and Slr0920 hydrolyzed ADP-ribose specifically, while Slr1134 hydrolyzed not only ADP-ribose but also NADH and flavin adenine dinucleotide. By contrast, Slr1690 showed very low activity for ADP-ribose and had four substitutions of amino acids in the Nudix motif, indicating that Slr1690 is not an active ADP-ribose pyrophosphatase. However, the quadruple mutation of Slr1690, T73G/I88E/K92E/A94G, which replaced the mutated amino acids with those conserved in the Nudix motif, resulted in a significant (6.1 x 10(2)-fold) increase in the k(cat) value. These results suggest that Slr1690 might have evolved from an active ADP-ribose pyrophosphatase. Functional and clustering analyses suggested that Sll1054 is a bacterial type, while the other three and Slr0787, which was characterized previously (Raffaelli et al., FEBS Lett. 444:222-226, 1999), are phylogenetically diverse types that originated from an archaeal Nudix protein via molecular evolutionary mechanisms, such as domain fusion and amino acid substitution.

Amino Acid Sequence↗

PubMed2005

Direct cloning of genes encoding novel xylanases from the human gut.

The aim of this study was to identify a novel 1,4-beta-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43,552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 degrees C and stable up to 50 degrees C at pH 6.5 and over the pH range 4.0-11.0 at 25 degrees C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.

Algorithms↗

PubMed2005

Culture-independent analysis of fecal microbiota in cattle.

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.

Animals↗

PubMed2004

Five histidine kinases perceive osmotic stress and regulate distinct sets of genes in Synechocystis.

Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.

Bacterial Proteins↗

PubMed2004

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803.

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.

Cyanobacteria↗

PubMed2004

PPARalpha agonists clofibrate and gemfibrozil inhibit cell growth, down-regulate hCG and up-regulate progesterone secretions in immortalized human trophoblast cells.

We studied effects of PPARalpha agonists clofibric acid and gemfibrozil on cell growth and functions of immortalized human extravillous trophoblast cells. Levels of DNA and protein gradually increased during incubation for 4 days. Gemfibrozil (>0.25mM) and clofibric acid (2.5mM) suppressed the rate of increase in DNA and protein. Specific activities of fatty acyl-CoA oxidase and catalase were increased to about 1.2-2.0 times the control value by 0.05mM gemfibrozil and 1.0 and 2.5mM clofibric acid after incubation for 3 days. Acid phosphatase activity showed a small increase in response to both agents, but esterase activity changed little. The secretion of progesterone from the cells into the medium was increased by 0.25mM gemfibrozil and 1.0 and 2.5mM clofibric acid after incubation for 3 days, but that of human chorionic gonadotropin (hCG) was decreased by 0.35mM gemfibrozil and 2.5mM clofibric acid. The specific activity of lactate dehydrogenase in the cells was hardly changed at all after incubation for 3 days. These results suggest that gemfibrozil and clofibric acid inhibit the proliferation of trophoblast cells. Cell metabolism is probably affected by both agents. The two agents may down-regulate hCG and up-regulate progesterone secretions.

Anticholesteremic Agents↗

PubMed2004

Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp. PCC 7002.

We cloned the gene for a novel Nudix hydrolase in the cyanobacterium Synechococcus sp. PCC 7002 and termed it nuhA. The deduced amino acid sequence of NuhA included the Nudix motif, GX(5)EX(7)RELXEEXGV, which is common to Nudix hydrolases, and in addition, a proline at the 15th amino acid from the C-terminus of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases. The recombinant NuhA with a hexahistidine tag was overexpressed in Escherichia coli and purified. The recombinant NuhA hydrolyzed ADP-ribose specifically among various nucleoside diphosphate derivatives. The hydrolytic activity for ADP-ribose required Mg(2+) and was optimal at pH 9.5. The V(max) and K(m) values of hydrolysis were 23.6 units mg(-1) and 0.094 mM, respectively. NuhA contained an uncharacterized domain in the C-terminal region, termed Pfam-B-3116, which is conserved in several hypothetical proteins. The mutated NuhA deficient in the Pfam-B-3116 domain failed to form the hexamers that are characteristic of NuhA, and exhibited a significantly higher K(m) value for ADP-ribose, suggesting that the Pfam-B-3116 domain might be responsible for oligomerization of NuhA and full binding affinity for ADP-ribose. These unique features suggest that NuhA is a novel type of ADP-ribose pyrophosphatase.

Adenosine Diphosphate Ribose↗

PubMed2004

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries.

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.

Age Factors↗