PubMed Health⌕ Search

Biomedical subjects

H Inokuchi

Publications and source records attributed to H Inokuchi.

At least 37 records · Page 2Linked to original sources
PubMed1996

10Sa RNA complements the temperature-sensitive phenotype caused by a mutation in the phosphoribosyl pyrophosphate synthetase (prs) gene in Escherichia coli.

From Escherichia coli cells with a deletion in the ssrA gene that encodes 10Sa RNA after treatment with a mutagen, we isolated two temperature-sensitive mutants, which we designated TS15 and TS101. The temperature-sensitive (ts) phenotype of the mutants could be overcome by introduction of the wild-type ssrA gene but not by the mutants of ssrA. By a complementation test using Kohara's mini-set of clones and by subcloning of a fragment from the phage clone 246, we found that both mutations were in the prs gene that encodes phosphoribosyl pyrophosphate synthetase. Sequencing of the mutant prs gene of TS101 showed that residues 215, cysteine, in the encoded protein had been changed to tyrosine. That such a mutant exists suggests that 10Sa RNA associate with the prs gene product in a functional way.

Escherichia coli↗
PubMed1996

Reevaluation of clinical features of ischemic colitis. Analysis of 68 consecutive cases diagnosed by early colonoscopy.

BACKGROUND: Ischemic colitis (IC) is generally considered a disease of elderly patients who have associated diseases. The aim of the present study was to reevaluate the clinical features of IC. METHODS: We retrospectively analyzed the clinical characteristics, background, and endoscopic and histologic changes in 68 consecutive patients (16 men and 52 women) with this disease diagnosed by early colonoscopy. RESULTS: The patients' age ranged from 22 to 98 years (mean, 55 years). Twenty-three patients (34%, including 19 women) were less than 50 years of age. The classical predisposing factors were not discernible in patients younger than 50. Chronic constipation and prior history of abdominal surgery were common in both young and old patients. Early colonoscopy (especially by the 3rd day from onset) showed endoscopic and histologic findings consistent with the characteristics of IC. CONCLUSIONS: IC is not limited only to the elderly, and it should be considered in the differential diagnosis of colitis with melena in younger patients, especially females, who do not have any predisposing factors. Chronic constipation and prior history of abdominal surgery were commonly associated in both young and old patients. Early colonoscopy, especially by the 3rd day from the clinical onset, is essential for the accurate diagnosis of IC.

Abdomen↗
PubMed1996

[A preliminary study of percutaneous ethanol injection therapy for hepatocellular carcinoma: evaluation of the ethanol diffusion area by the ethanol mixed with gadolinium].

To evaluate the ethanol diffusion area after Lipoidalization in 3 patients with advanced HCC treated by Lipoidalization-PEIT combination therapy, 99.9% ethanol mixed with Gadolinium was used for PEIT (Gd-PEIT). T1-weighted MR images wear obtained 1 hr after Gd-PEIT. The area of homogeneous hyperintense change on T1-weighted MR images was taken to be the ethanol diffusion area. In all 3 patients, homogeneous hyperintensity throughout the tumor over the capsule was recognized on T1-weighted MR images after treatment. The results suggests that T1-weighted MR images after Gd-PEIT provide a valuable tool by which to directly evaluate the ethanol diffusion area for advanced HCC treated by Lipoidalization followed by PEIT.

Carcinoma, Hepatocellular↗
PubMed1996

[Measurement of gastric emptying by magnetic resonance imaging].

The purpose of the study was to establish a new method of measuring gastric emptying using MR Imaging in human. Gastric emptying was measured in 6 healthy male volunteers aged from 28 to 43 years, using MR imaging and RI. The measurements were performed after the oral administration of liquid meal containing glucose, protein and fat. The MR imaging was performed with 0.5T superconducting magnet machine, and consecutive 12 transaxial T1 weighted spin echo images (TR/TE = 300/17) of the upper abdomen were recorded every 10 minutes for more than 1 hour. Gastric emptying curves and their T1/2 values obtained by MR imaging and RI method were correlated well in 5 of 6 cases. We concluded that a non invasive and radiation free method using MR imaging was proved to be a useful tool for measuring gastric emptying.

Adult↗
PubMed1996

[Validation of the hospital anxiety and depression scale in a gastro-intestinal clinic].

This study examined the validity of the Japanese version of the hospital anxiety and depression scale (HAD) in a gastro-intestinal outpatient clinic. One hundred and twenty-three men and 142 women consulting a gastro-intestinal outpatient clinic at a primary care hospital in Kyoto during 1995 were surveyed. Item-remainder correlation and internal consistency were examined for reliability. Concurrent validities were examined using the stait-trait anxiety inventory (STAI) and Zung's self-rating depression scale (SDS). The prevalence of psychiatric disorder in this population ranged from 27% to 39%. Cronbach's coefficients were greater than 0.8 for the anxiety subscale and more than 0.7 for the depression subscale. Spearman's correlation of the anxiety subscale scores and the STAI were r = 0.678 for men, and r = 0.717 for women. The correlation of depression subscale scores and SDS were r = 0.457 for men, and r = 0.565 for women. It is suggested that the use of the HAD to general hospital outpatients clinic would facilitate detecting emotional disorders in outpatients.

Adult↗
PubMed1995

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli.

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of glutamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.

Aldehyde Oxidoreductases↗
PubMed1995

The human protoporphyrinogen oxidase gene (PPOX): organization and location to chromosome 1.

We determined the structure of the human protoporphyrinogen oxidase (PPOX) gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. Southern blotting of human genomic DNA showed that there is a single copy of the PPOX gene, and fluorescence in situ hybridization to metaphase chromosomes mapped the gene to region 1q22. The gene has 13 exons and about 8 kb. The exon/intron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and exons in the gene appear to encode functional protein domains. Primer extension analysis revealed two major transcriptional initiation sites in a region with sequence motifs characteristic of a promoter. The promoter region contains multiple Sp1 elements, CCAAT boxes, and potential GATA-1 binding sites. Mapping of the 5' end PPOX mRNA by polymerase chain reaction indicated that there are the same transcripts in erythroid and nonerythroid cells.

Bacteriophage lambda↗
PubMed1995

Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX.

The hemH gene of Escherichia coli encodes ferrochelatase (EC 4.99.1.1), the enzyme that catalyzes the last step in the production of heme, namely the synthesis of heme from protoporphyrin IX plus Fe2+. The behavioral responses to light were studied in E. coli carrying a hemH mutation. It was shown that the hemH mutant displayed a tumbling response upon illumination and a running response upon removal of the light. The most effect light to induce a tumbling response in the hemH mutant was blue light (396-450 nm). The chemotaxis machinery was needed for the light-induced tumbling response in the hemH mutant. The bacterial defect is an analog of the human inherited disease erythropoietic protoporphyria.

Anaerobiosis↗
PubMed1995

Induction of terminal enzymes for heme biosynthesis during differentiation of mouse erythroleukemia cells.

To examine the induction of terminal enzymes of the heme-biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned. The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria. Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former. The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively. When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau. mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h. The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8-fold within 12 h and remained unchanged thereafter. Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities. The magnitude of PPO induction was less than that of CPO and FEC. Thus, induction of three terminal enzymes of the heme-biosynthetic pathway is an early event in MEL cell differentiation. The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.

Amino Acid Sequence↗
PubMed1995

Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli.

Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway. Hemes are essential components of redox enzymes, such as cytochromes. Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows poorly even in rich medium. By complementation with a human placental cDNA library, we were able to isolate a clone that enhanced the poor growth of such a hemG mutant strain. The clone encoded the gene for human PPO. Sequence analysis revealed that PPO consists of 477 amino acids with a calculated molecular mass of 50.8 kilodaltons. The deduced protein exhibited a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis. The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins. Northern blot analysis revealed the synthesis of a 1.8-kilobase pair mRNA for PPO. A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Furthermore, the cDNA was expressed in vitro as 51-kilodalton protein, and after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of a previously unidentified gene that is involved in the biosynthesis of heme in Escherichia coli.

We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell. Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA. This new gene, designated hemK, was located at 27 min on the linkage map of the E. coli chromosome. By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases. The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA. An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities. H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.

Aldehyde Oxidoreductases↗
PubMed1995

Lack of evidence for P2X-purinoceptor involvement in fast synaptic responses in intact sympathetic ganglia isolated from guinea-pigs.

Recordings were made from neurons in intact pre- and paravertebral guinea-pig sympathetic ganglia using intracellular microelectrodes. Fast excitatory synaptic responses were evoked by stimulation of preganglionic and peripheral nerve trunks. Suramin (0.1-1 mM) did not affect passive or active membrane properties, nor the amplitude or decay time-course of either synaptic potentials or synaptic currents. Synaptic responses were reversibly reduced in amplitude by hexamethonium (98.7 +/- 0.8%, 50-1000 microM) and d-tubocurarine (95.3 +/- 2.6%, 10-280 microM). ATP (0.5-1 mM) and alpha,beta-methylene ATP (1-40 microM) applied in the bathing solution produced no significant changes in resting membrane potential or input resistance. Prolonged application (up to 25 min) of either compound was also without effect on synaptic responses. These substances also did not affect ganglion cells axotomized one to five days in vivo. These data suggest that activation of P2X-purinoceptors is not involved in the generation of fast excitatory synaptic responses in intact guinea-pig sympathetic ganglia. It appears that dissociation of these neurons must markedly increase their sensitivity to purine nucleotides.

Animals↗
PubMed1995

Cloning and identification of the hemG gene encoding protoporphyrinogen oxidase (PPO) of Escherichia coli K-12.

Cells of the VSR751 strain, which was previously isolated as a photoresistant revertant of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, accumulated uroporphyrin (uro), coproporphyrin (copro) and protoporphyrin IX (proto), but did not accumulate as much protoporphyrin as cells of the parental strain (hemH-deleted). Therefore, we concluded that strain VSR751 must be defective in protoporphyrinogen oxidase (PPO), the product of the hemG gene. By complementation analysis using VSR751, we isolated and identified this gene. The hemG gene is located at 86 mim on the E. coli chromosome, just upstream of the rrnA operon, and is transcribed clockwise in the same direction as the rrnA operon. This gene encodes a 181-amino acid protein with a calculated molecular mass of about 21 kDa. Sequence analysis revealed the presence of flavodoxin motif, suggesting tha a cofactor of this enzyme is flavin mononucleotide, which is consistent with the previous report that the mammalian PPO had the flavin cofactor.

Amino Acid Sequence↗
PubMed1995

The significance of membrane lipids in exocytosis: control of liposome-evoked amylase release from secretory granules isolated from the rat parotid gland.

We investigated the significance of the plasma membrane lipid composition in exocytosis in an in vitro interaction system using an intact secretory granular fraction (SG) isolated from the rat parotid gland. When various liposomes (as a model of plasma membranes) were added to this assay system, rapid and transient amylase release from the SG was evoked and increased by Ca2+ in a concentration-dependent manner. The extent depended upon not only the amount of liposomes but also their lipid composition. The addition of 1,2-diacylglycerol and phosphatidic acid to egg yolk phosphatidylcholine-liposomes remarkably facilitated the release. On the other hand, that of various free fatty acids had different effects depending upon their molecular species. Furthermore, a fluorescence de-quenching study demonstrated that membrane fusion actually occurred in this interaction system, and appeared to correlate with the amylase release. These results suggest that the transient alteration of the membrane lipid composition upon cell activation is a modulator of the exocytotic membrane interaction.

Amylases↗
PubMed1994

A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli.

We have determined that 10Sa RNA (one of the small stable RNAs found in Escherichia coli) has an interesting structural feature: the 5' end and the 3' end of 10Sa RNA can be arranged in a structure that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10Sa RNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding 10Sa RNA (ssrA) caused a reduction in the rate of cell growth, which was especially apparent at 45 degrees C, and a reduction in motility on semisolid agar. These phenotypic characteristics of the deletion strain (delta ssrA) allowed us to investigate the effects of some mutations in 10Sa RNA in vivo, although the exact function of 10Sa RNA still remains unclear. When the G.U pair (G3.U357) in 10Sa RNA, which may be equivalent to the determinant G.U pair of alanine tRNA, was changed to a G.A or G.C pair, the ability to complement the phenotypic mutations of the delta ssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a G.A pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (alaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of 10Sa RNA are indeed manifested in vivo.

Base Sequence↗
PubMed1994

Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase.

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.

Amino Acid Sequence↗
PubMed1994

Characterization of microsomal long-chain acyl-CoA hydrolase activity in the rat submandibular gland.

1. Long-chain fatty acyl-CoA hydrolase in submandibular gland microsomes was characterized and compared to that in liver ones. 2. In rat submandibular gland, the microsomal long-chain acyl-CoA hydrolase showed a higher relative activity for polyunsaturated fatty acyl-CoAs than that of liver microsomes. 3. It was suggested that the hydrolase in rat submandibular gland microsomes may play a role in modulation in the acyl-CoA pool size.

Animals↗