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H Inokuchi

Publications and source records attributed to H Inokuchi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of anticodon nuclease residues involved in tRNALys cleavage specificity.

The tRNALys-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional prr locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNALys 5' to the wobble base, yielding 5'-OH and 2', 3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNALys were searched for at the C-half. Random mutagenesis of the low-G+C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNALys. Taken together, the data suggest that the anticodon of tRNALys harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.

Amino Acid Sequence↗
PubMed1999

Oxidation of protoporphyrinogen IX in Escherichia coli is mediated by the aerobic coproporphyrinogen oxidase.

Protoporphyrinogen oxidase, the penultimate enzyme involved in the biosynthetic pathway for heme, catalyzes the removal of six electrons from protoporphyrinogen IX to generate protoporphyrin IX. In Escherichia coli, this enzyme is encoded by the hemG gene. In this study we examined possible alternate pathways for the oxidation of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E. coli mutants that can still grow normally when the hemG gene is disrupted. One of these mutants was characterized in detail and had a mutation in the promoter region of the hemF gene, which encodes aerobic coproporphyrinogen oxidase, the enzyme involved in the step immediately before protoporphyrinogen oxidase. Measurement of the promoter activity of the hemF gene showed that the level of transcription was elevated by the mutation. Overexpression of a wild-type hemF gene cloned in a multicopy plasmid also restored the growth of deltahemG strain. Extracts from cells that overexpress hemF exhibited an increased ability to oxidize protoporphyrinogen IX to protoporphyrin IX. These findings suggest that the E. coli aerobic coproporphyrinogen oxidase has an intrinsic capacity to oxidize not only coproporphyrinogen III but also protoporphyrinogen IX.

Base Sequence↗
PubMed1999

Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan.

A seroepidemiologic, nested case-control study was conducted to evaluate the risk for atrophic gastritis associated with Helicobacter pylori infection. Atrophic gastritis was diagnosed on the basis of serum pepsinogen levels: pepsinogen I level < or = 70 ng/ml and pepsinogen I/pepsinogen II ratio < or = 3.0. Cases were 23 men and 39 women who were not diagnosed with atrophic gastritis in 1987, but who were diagnosed with the condition in 1992. Controls were the 120 men and 282 women who did not meet the serologic criteria for atrophic gastritis in either time period. Neither cases nor controls had a history of upper gastrointestinal operations. Helicobacter pylori infection at the initial survey was associated with a significantly increased risk of atrophic gastritis incidence for both sexes combined (odds ratio = 3.72; 95% confidence interval, 1.78-7.79; P = 0.0005). Cigarette smoking and consumption of alcohol and green-yellow vegetables were not associated with incidence of atrophic gastritis.

Adult↗
PubMed1999

Down-regulation of TGF-beta receptors in human colorectal cancer: implications for cancer development.

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-beta (TGF-beta). TGF-beta also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-beta and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-beta1, type I and type II receptor mRNA and immunohistochemical staining of TGF-beta1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-beta receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-beta receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-beta1 mRNA and the protein were detected in all colorectal cancers. TGF-beta receptor mRNAs and TGF-beta1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-beta stimulation. The escape of human colon cancer from TGF-beta-mediated growth inhibition by down-regulation of TGF-beta receptors as well as the effects of TGF-beta on stroma formation and angiogenesis indicate a possible role for TGF-beta in the progression of colon cancer in an intact host.

Adenoma↗
PubMed1999

Membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 pyramidal neurons.

Intracellular and single-electrode voltage-clamp recordings were made to investigate the process of membrane dysfunction induced by superfusion with oxygen and glucose-deprived (ischemia-simulating) medium in hippocampal CA1 pyramidal neurons of rat tissue slices. To assess correlation between potential change and membrane dysfunction, the recorded neurons were stained intracellularly with biocytin. A rapid depolarization was produced approximately 6 min after starting superfusion with ischemia-simulating medium. When oxygen and glucose were reintroduced to the bathing medium immediately after generating the rapid depolarization, the membrane did not repolarize but depolarized further, the potential reaching 0 mV approximately 5 min after the reintroduction. In single-electrode voltage-clamp recording, a corresponding rapid inward current was observed when the membrane potential was held at -70 mV. After the reintroduction of oxygen and glucose, the current induced by ischemia-simulating medium partially returned to preexposure levels. These results suggest that the membrane depolarization is involved with the membrane dysfunction. The morphological aspects of biocytin-stained neurons during ischemic exposure were not significantly different from control neurons before the rapid depolarization. On the other hand, small blebs were observed on the surface of the neuron within 0.5 min of generating the rapid depolarization, and blebs increased in size after 1 min. After 3 min, neurons became larger and swollen. The long and transverse axes and area of the cross-sectional cell body were increased significantly 1 and 3 min after the rapid depolarization. When Ca2+-free (0 mM) with Co2+ (2.5 mM)-containing medium including oxygen and glucose was applied within 1 min after the rapid depolarization, the membrane potential was restored completely to the preexposure level in the majority of neurons. In these neurons, the long axis was lengthened without any blebs being apparent on the membrane surface. These results suggest that the membrane dysfunction induced by in vitro ischemia may be due to a Ca2+-dependent process that commences approximately 1.5 min after and is completed 3 min after the onset of the rapid depolarization. Because small blebs occurred immediately after the rapid depolarization and large blebs appeared 1.5-3 min after, it is likely that the transformation from small to large blebs may result in the observed irreversible membrane dysfunction.

Animals↗
PubMed1999

[Evaluation of the efficacy and the cost-effectiveness of proton pump inhibitor-based dual and triple therapy regimens for H. pylori eradication in peptic ulcer disease].

To evaluate the efficacy and the cost-effectiveness of H. pylori eradication for the treatment of peptic ulcer disease, a randomized controlled trial to compare the efficacy of proton pump inhibitor-based dual and triple regimens was conducted. A decision analysis was also performed to assess H. pylori eradication compared to the conventional maintenance strategy. Two hundred and thirty-four peptic ulcer patients suffering from H. pylori infection were randomly treated with either omeprazole 20 mg bid + amoxicillin 500 mg qid + clarithromycin 400 mg bid (OAC) or with omeprazole 20 mg bid + clarithromycin 400 mg bid (OC) for 14 days. The eradication rate with OAC was 87.6% (92/105) (95% Confidence Interval (CI): 81-94%), which was significantly higher than that with OC (61.9% (60/97) (95% CI: 52-72%)) (p < 0.001, chi 2 test). Both regimens were safe and well tolerated. H. pylori eradication was more effective and less costly than conventional strategy in a long term perspective. OAC was more cost-effective than OC. In conclusion, H. pylori eradication is a cost-effective alternative to conventional treatment. We recommend 2 weeks triple regimen with omeprazole 20 mg bid + amoxicillin 500 mg qid + clarithromycin 400 mg bid (OAC) as a first-line treatment in all patients with peptic ulcers associated with H. pylori infection.

Amoxicillin↗
PubMed1999

Comparison of 1-week and 2-week triple therapy with omeprazole, amoxicillin, and clarithromycin in peptic ulcer patients with Helicobacter pylori infection: results of a randomized controlled trial.

This study was a comparison of 1-week and 2-week triple therapies with omeprazole, amoxicillin, and clarithromycin (OAC) in patients with peptic ulcer disease and Helicobacter pylori infection. A total of 147 peptic ulcer patients with H. pylori infection assessed by histology and culture were randomly treated with omeprazole 20mg bid + amoxicillin 1000mg bid + clarithromycin 400mg bid for either 1 week (OAC1w) or 2 weeks (OAC2w). Both groups then received omeprazole 20mg daily for 2 weeks followed by ranitidine 300mg daily for 4 weeks. Eradication of H. pylori was assessed by histology, culture, and the 13C-urea breath test (13C-UBT) at least 6 weeks after cessation of antimicrobial therapy. Intention-to-treat eradication rates were 78.2% (95%CI 69%-87%) with OAC1w and 88.4% (95%CI 81%-96%) with OAC2w. Per-protocol eradication rates were 86.0% (95%CI 78%-94%) with OAC1w, 97.0% (95%CI 93%-100%) with OAC2w. There was no significant difference in the eradication rates between OAC1w and OAC2w. Side effects were mild and self-limiting in both groups. In conclusion, both 1- and 2-week triple therapy with OAC are well tolerated and provide good eradication rates in peptic ulcer patients in Japan. The eradication rate of the 2-week regimen was higher than that of the 1-week regimen, but the difference was not statistically significant. Further studies including long-term economic considerations are required to determine the optimal duration of treatment.

Adolescent↗
PubMed1998

Co-expression of yeast amber suppressor tRNATyr and tyrosyl-tRNA synthetase in Escherichia coli: possibility to expand the genetic code.

An efficient system was developed for the co-expression of a yeast tRNATyr/tyrosyl-tRNA synthetase (TyrRS) pair in Escherichia coli. Analysis of suppression patterns using several sets of E. coli and lambda phage mutants indicated that the expressed yeast suppressor tRNATyr was aminoacylated only with tyrosine by its cognate yeast TyrRS and not by E. coli TyrRS or other aminoacyl-tRNA synthetases. This extra tRNA/TyrRS pair is expected to be a key bridgehead for developing an in vivo system for the site-directed incorporation of unnatural amino acids into proteins.

Bacteriophage lambda↗
PubMed1998

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12.

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42 degrees C but not at 32 degrees C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32 degrees C but not at 42 degrees C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant.

Cell Division↗
PubMed1998

Mild hypothermia protects rat hippocampal CA1 neurons from irreversible membrane dysfunction induced by experimental ischemia.

In order to examine the effects of hypothermia on the changes in membrane potential induced by experimental ischemia (deprivation of oxygen and glucose), intracellular recordings were made from single CA1 pyramidal neurons in slice preparations of rat hippocampus. Application of ischemic medium caused irreversible changes in membrane potential consisting of an initial hyperpolarization, then a slow depolarization and a rapid depolarization. At temperatures of 35 degrees C and 37 degrees C, once the rapid depolarization occurred, readministration of oxygen and glucose failed to restore the membrane potential, a state referred to as irreversible membrane dysfunction. When the temperature was lowered to between 27 degrees C and 33 degrees C, the membrane potential returned to the control resting membrane potential in 75% of the neurons. The temperature coefficients (Q10) of the latency, the amplitude, and the maximal slope of the rapid depolarization were 2.5, 1.4 and 2.9, respectively. It is concluded that the critical neuroprotective temperature in ischemia-induced membrane dysfunction is found to be 33 degrees C in single CA1 neurons in vitro.

Animals↗
PubMed1998

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Cell Nucleus↗
PubMed1998

Economic evaluation of Helicobacter pylori eradication for the treatment of duodenal ulcer disease in Japan: a decision analysis to assess eradication strategy in comparison with a conventional strategy.

To evaluate the socio-economic effects of Helicobacter pylori eradication in the treatment of duodenal ulcer disease in Japan, a clinical decision analysis was performed to assess H. pylori eradication therapy compared with the conventional strategy of maintenance with histamine-2 receptor antagonists. A decision tree-based state transition model (Markov chain approach) implemented to simulate a 5 year period of follow up was constructed. The H. pylori eradication strategy was found to be superior to the conventional maintenance strategy with regard to clinical effectiveness and other dimensions of a patient's outcome. Furthermore, in a long-term perspective, the eradication strategy was less costly than the maintenance strategy. Helicobacter pylori eradication should be recommended as the first choice treatment of H. pylori-positive duodenal ulcer patients. The clinical implication of H. pylori eradication entails an improvement in clinical effectiveness and other dimensions of a patient's outcome and a significant reduction in the costs of duodenal ulcer treatment. The long-term total costs do not depend on the initial drug cost of an eradication regimen. Pursuing a high eradication rate of H. pylori is essential in improving the patient's outcome and the cost-effectiveness of treatment.

Anti-Bacterial Agents↗
PubMed1998

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu.

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.

Alkyl and Aryl Transferases↗
PubMed1997

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells.

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30-Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA-DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of beta-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Aerobiosis↗
PubMed1997

The effects of catechol on various membrane conductances in lumbar sympathetic postganglionic neurones of the guinea-pig.

The effects of catechol on membrane properties in lumbar sympathetic postganglionic neurones isolated from guinea-pigs were studied in vitro in current and voltage clamp using single intracellular microelectrodes. Neurones with properties characteristic of two previously described classes of neurone (phasic and tonic) were studied. Catechol (3-12 mM) produced a few mV depolarization and a dose-dependent increase in membrane resistance which were both larger in tonic than in phasic neurones. In the presence of catechol, both phasic and tonic neurones fired only a single action potential at the beginning of a maintained depolarizing current step. In both neurone types, catechol reduced action potential amplitude and slowed its time course. The peak of the afterhyperpolarization became delayed and reduced in amplitude, particularly in tonic neurones. The time constant of inactivation of IA was reduced by catechol without change in the voltage sensitivity of activation or inactivation: IC50 was 3 mM in phasic and 4 mM in tonic neurones. Catechol also blocked a slow voltage-activated K+ current (resembling ID) that was present in many tonic neurones. Catechol did not modify the slow calcium-activated potassium current (gKCa1) or the anomalous rectifier; neither did it appear to affect the fast calcium-activated potassium current (IC) or the delayed rectifier. Catechol did not change the overall rate of spontaneous synaptic activity nor enhance the release of quanta of ACh from preganglionic terminals evoked by nerve stimulation. We conclude that, in addition to blocking IA, catechol blocks the slow ID-like current in sympathetic neurones. It also has a profound effect on the action potential probably by increasing inactivation of voltage-dependent Na+ channels. The change from tonic to phasic discharge in tonic neurones cannot be attributed solely to its effects on IA.

Action Potentials↗
PubMed1997

Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli.

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins.

Aldehyde Oxidoreductases↗
PubMed1997

Helicobacter pylori infection and gastric cancer. A nested case-control study in a rural area of Japan.

We conducted a seroepidemiological nested case-control study to determine the association of gastric cancer with Helicobacter pylori infection and atrophic gastritis. A cohort of 2858 participants in an annual multiphasic health check-up were followed for eight years. Data for 45 gastric cancer cases and 225 sex-, age-, and address-matched control subjects were analyzed. Helicobacter pylori infection was determined by IgG antibodies, and atrophic gastritis was diagnosed by both serum pepsinogen I level (< or = 70 ng/ml) and the pepsinogen I/II ratio (< or = 3.0). Univariate analysis showed that Helicobacter pylori and atrophic gastritis were significantly associated with gastric cancer. In a multivariate analysis, atrophic gastritis was associated with significantly increased risk of cancer (odds ratio, 3.38; 95% confidence interval, 1.54-7.42); however, Helicobacter pylori was not associated with cancer (odds ratio, 1.84; 95% confidence interval, 0.59-5.72). These results suggest that Helicobacter pylori infection alone is not directly associated with gastric carcinogenesis but has an indirect relation to gastric cancer through the development of atrophic gastritis.

Adult↗