Biomedical subjects

H Inokuchi

Publications and source records attributed to H Inokuchi.

At least 19 recordsLinked to original sources

PubMed1996

Molecular cloning and characterization of a cDNA that encodes protoporphyrinogen oxidase of Arabidopsis thaliana.

A cDNA encoding protoporphyrinogen oxidase (PPOX), the last enzyme common to the biosynthetic pathways for chlorophylls and hemes, was obtained from a library of Arabidopsis thaliana cDNA constructed in a lambda vector by screening for complementation of a hemG mutant of Escherichia coli. Extracts of E. coli cells transformed with the Arabidopsis PPOX cDNA had high PPOX activity, and this activity was markedly inhibited by acifluorfen, a specific inhibitor of PPOX. Sequence analysis revealed that the cDNA for Arabidopsis PPOX encodes a protein of 537 amino acids (aa) with a calculated molecular mass of 57.7 kDa. The deduced aa sequence exhibited similarity to sequences of PPOX from Bacillus subtilis, mouse, and human. However, the PPOX of Arabidopsis contained a putative leader peptide for import into mitochondria (mt). southern analysis indicated that the PPOX whose cDNA we cloned is encoded by a single gene in Arabidopsis. Northern blot analysis showed that the level of expression of the gene in Arabidopsis leaves was high. whereas it was low in roots and floral buds. To our knowledge, this is the first report for the cloning of a cDNA for a plant PPOX.

Amino Acid Sequence↗

PubMed1996

Genetic analysis of functional connectivity between substrate recognition domains of Escherichia coli glutaminyl-tRNA synthetase.

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype on Escherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA(Gln): GlnRS: ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3' end of tRNA(Gln). The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischarge supF tRNA(Tyr) considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguish supE tRNA(CUA)(Gln) from tRNA2(Gln) considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K(m) for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.

Adenosine Triphosphate↗

PubMed1996

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification.

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Amino Acid Sequence↗

PubMed1996

Three electrophysiological classes of guinea pig sympathetic postganglionic neurone have distinct morphologies.

Sympathetic postganglionic neurones can be differentiated electrophysiologically into three classes (phasic, Ph; tonic, T; and long-afterhyperpolarising, LAH) based on their potassium channel expression and consequent differences in excitability. We tested whether neuronal morphology differs between these classes. Neurones in coeliac, inferior mesenteric, and lower lumbar paravertebral ganglia of guinea pigs were filled with biocytin during in vitro experiments in which electrical properties were recorded. The dimensions of somata and dendrites were measured in approximately equal numbers of stained neurones of each class. The three electrophysiological classes were distinct in terms of soma shape, soma size (Ph < T = LAH), total dendritic length (LAH < Ph < T) and average length of dendrites (LAH < Ph < T) (P < 0.0001, multivariate analysis of variance). The mean number of primary dendrites also differed (LAH 13, Ph 16, T 20). The majority of dendrites did not branch, the ratios of terminations to primary dendrites being 1.36 (LAH), 1.63 (Ph) and 1.81 (T). Overall, LAH neurones, with medium-sized somata but the smallest dendritic trees, were more distinct morphologically than Ph and T neurones. The morphological differences between classes were not dependent on differences in location. Further, there was no apparent relation between morphology and the pattern of synaptic input each class receives. The results indicate that three distinct groups of sympathetic postganglionic neurone exist in adult guinea pigs, although more than three functions are subserved by these neurones.

Animals↗

PubMed1996

Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.

Escherichia coli↗

PubMed1996

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

Base Sequence↗

PubMed1996

10Sa RNA is associated with 70S ribosome particles in Escherichia coli.

The intracellular distribution of 10Sa RNA in Escherichia coli was investigated in cell extracts. Northern hybridization revealed that a large fraction of 10Sa RNA cosediments with 70S ribosomes. When 70S ribosomes were dissociated into 50S and 30S subunits in the presence of low levels of Mg2+ ions, almost all of the 10Sa RNA disappeared from both subunits. The extent of the association of the 10Sa RNA with ribosomes was much enhanced during the growth phase of the cells. These results suggest the possibility that 10Sa RNA might function on the ribosomes in E. coli cells.

Autoradiography↗

PubMed1996

Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

Aldehyde Oxidoreductases↗

PubMed1996

Control of the availability of exogenous 5-aminolevulinic acid in Escherichia coli.

We observed novel phenomena on the growth of a hemA deletion mutant (H500) of Escherichia coli: First, H500 cells grown in rich medium required an increased amount of exogenous 5-aminolevulinic acid (ALA) for normal growth, as compared with cells grown in poor medium. Second, H500 cells grown with an insufficient supply of ALA had a prolonged lag phase but relatively normal growth during the logarithmic phase. An increased requirement of exogenous ALA in rich media was due to an impermeability of the cells to ALA, resulting in a reduced ability to synthesize porphyrins. The latter could be also explained by change in permeability that was repressed under heme-deficient conditions.

Aldehyde Oxidoreductases↗

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↗