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H Ohkawa

Publications and source records attributed to H Ohkawa.

At least 127 records · Page 7Linked to original sources
PubMed1989

Effects of calcitonin gene-related peptide on the non-adrenergic inhibitory potentials in the intestinal smooth muscle cells of the guinea pig.

Effects of calcitonin gene-related peptide (CGRP) on the spontaneous and evoked contractions in the duodenal and ileal preparations, the spontaneous action potentials and the non-adrenergic non-cholinergic inhibitory potentials (NANC i.p.s) in the longitudinal smooth muscle cells of the guinea-pig duodenum and ileum were examined. Preparations were pretreated with atropine (1 microM) and guanethidine (5 microM). CGRP (26 nM) was found to inhibit spontaneous and evoked contractions. The resting membrane potential of the longitudinal smooth muscle was not altered but the frequency of spontaneous action potentials was decreased by the treatment with CGRP (6-104 nM). Field stimulation evoked the NANC i.p.s and the pretreatment with atropine (1 microM) and guanethidine (5 microM) caused an increase in the amplitude of the NANC i.p.s in the longitudinal smooth muscle. However, CGRP (6-104 nM) did not change the amplitude of the NANC i.p.s. The rebound excitation in the longitudinal muscle membrane was inhibited by CGRP. The single spike activity of the myenteric neurons in the duodenum was not affected by CGRP (26-104 nM). The results suggest that CGRP inhibits the intestinal motility by non-neurogenic NANC manner and does not activate the NANC inhibitory neurons in intestine.

Animals↗
PubMed1989

Effect of the proton pump inhibitor, NC-1300, on gastric mucus secretion in rats.

The effects of NC-1300 on gastric acid and mucus glycoprotein secretion were investigated in pylorus-ligated (4 h) rats. NC-1300 (30 mg/kg, intraduodenally) significantly inhibited gastric acid concentration more than 90%. The agent did not affect the total mucus glycoprotein output in the gastric juice, but caused by about 1.5 times denser mucus glycoprotein secretion than that in the control group. The amount of mucus glycoprotein in the mucous gel layer increased by about 1.4 times with this agent. Thus, NC-1300 not only inhibits acid secretion but also promotes mucus secretion, and this may explain its ability to protect the stomach against necrotizing agents.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1989

Novel functions and cellular interaction of human lymphoid stromal cells with lymphoid cell lines in vitro.

Lymphoid stromal (SG) cells have been isolated from the lymph node of a patient with malignant lymphoma, and characterized by positive reaction with a monoclonal antibody against the T-zone stromal cells in human lymph nodes. B-acute lymphoblastic leukemia (BALL) cells showed prominent emperipolesis toward SG cells when they were cocultured, whereas T-acute lymphoblastic leukemia (TALL) cells attached firmly to the surface of SG cells. Autologous peripheral B and T cells behaved, respectively, in the same way as BALL and TALL cells. Both BALL and TALL cells while directly interacting with the SG cells were completely inhibited from incorporating [3H]thymidine, although radioactive grains were observed in 16.4%-12.4% of supernatant BALL and 13.8%-13.0% of supernatant TALL cells in each coculture. Furthermore the media conditioned by SG cells significantly increased the incorporation of [3H]thymidine into the TALL cells as much as 190% of the control. These results indicate that SG cells undergo tissue-specific cellular interactions with B- and T-lymphoid cell lines but not with a myeloid cell line, and they can modify their growth by two distinct mechanisms. SG cells proved to be very useful in studying the effect of the lymphoid microenvironment on the proliferation of lymphocytes in vivo.

Burkitt Lymphoma↗
PubMed1988

Organization and characterization of the virCD genes from Agrobacterium rhizogenes.

We have precisely localized virulent (vir) genes of the hairy root-inducing plasmid pRiA4b on the basis of sequence similarity with the tumor-inducing plasmid pTiA6NC, and shown that the overall organizations of vir genes in both plasmids are fairly analogous, although sizes and spacer lengths in some genes differ from each other. Among the vir genes thus mapped, the virC and virD loci were characterized in detail. Transposon insertions in virD led to loss of tumorigenicity on Kalanchoe stems and carrot discs, and one within virC exhibited an attenuated pathogenicity. The avirulent phenotype of the virD2 strain among these mutants was due to the lack of ability to recombine T-DNA border repeats in Agrobacterium cells. The nucleotide sequence of most parts of the virCD loci were similar in both plasmids. The virCD genes of these two plasmids, therefore, seem comparable both functionally and structurally. Phylogeny of pRi and pTi has also been discussed from the sequence data.

Amino Acid Sequence↗
PubMed1988

Genetically engineered modification of P450 monooxygenases: functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme.

Modified constructions of a microsomal cytochrome P450, of NADPH-cytochrome P450 reductase, and of a P450/reductase fused enzyme were prepared to analyze the function of the amino-terminal hydrophobic regions of these enzymes and the hinge region of the fused enzyme. Expression plasmids for delta P450c, delta reductase, and the delta P450/reductase fused enzyme, all of which lacked their amino-terminal hydrophobic regions, were constructed by inserting each of the corresponding cDNAs between the yeast alcohol dehydrogenase I promoter and the terminator of the expression vector pAAH5. Yeast transformed with plasmids encoding delta P450 and the delta P450/reductase fused enzyme produced smaller amounts of the respective enzymes and showed lower monooxygenase activity toward 7-ethoxycoumarin than did yeast transformed with plasmids encoding the complete enzymes. Both delta P450 and delta P450/reductase were found in the microsomal fraction of the yeast cells. Yeast transformed with the expression plasmid for delta reductase produced 20 times more enzyme than did yeast transformed with the plasmid for the complete enzyme. delta Reductase was present in the soluble fraction and was 33 times more active in reducing cytochrome c than was the complete enzyme. The results suggest that the amino-terminal hydrophobic regions of P450c and the P450/reductase fused enzyme play an important role in their stability and function in the yeast microsomes. By contrast, the amino-terminal-containing P450 reductase appears to be unstable in yeast cells. Altering the size of the hinge regions does not affect the activity of the P450/reductase fused enzyme significantly, but some amino acid changes in this region increase the stability of the fused enzyme slightly.

Animals↗
PubMed1988

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene.

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.

Amino Acid Sequence↗
PubMed1988

Mucus glycoprotein and mucosal protection.

For assessing the participation of mucus glycoproteins in the cytoprotective process, mucus glycoprotein content in the rat gastric mucosa was measured after treatment with 70% ethanol with or without prostaglandin (PG) E derivatives or 20% ethanol pretreatment. Oral administration of two synthetic PGE derivatives did not cause any significant changes in mucus glycoprotein content. Seventy percent ethanol administration caused marked reduction in mucus glycoprotein content (about 50% of control) as well as severe gastric mucosal damage. Treatment with two PGE derivatives (10-100 micrograms/kg) prior to 70% ethanol administration markedly inhibited the gross mucosal lesion, whereas the glycoprotein content under these conditions was significantly less than that in the untreated control group (ranging from 67-88% of control). Pretreatment with 20% ethanol markedly inhibited the gross mucosal damage caused by 70% ethanol dosing but the inhibition in the reduction of mucus glycoprotein content was restricted to about 80% of the untreated controls. In summary, cytoprotection induced by PG was not accompanied by entire conservation of intramucosal mucus glycoprotein in the gastric mucosa.

Alprostadil↗
PubMed1988

Immunohistochemical studies on B cell lymphomas with special reference to T cell infiltration and its significance as a prognostic factor.

Twenty-eight cases of B cell lymphoma were studied immunohistochemically utilizing a panel of monoclonal antibodies. Of 27 cases tested, 26 were B-1-positive and 22 of 24 cases were HLA-DR positive. There were eight BA-1-positive cases, including one follicular lymphoma, and 10 J-5-positive cases including five diffuse large cell lymphomas. Some of the J-5-positive diffuse large cell lymphomas were considered to be of follicular center cell origin. Infiltration and distribution of non-neoplastic T cell subsets, BA-1-positive B cells, dendritic reticulum cells, Langerhans cells and HNK cells showed close similarity between the structure of follicular lymphomas and that of the reactive follicles. Such similarity to the normal counterpart structure was less apparent in diffuse lymphomas, especially in the large cell type. These findings were interpreted to be an expression of different degrees of neoplastic deviation. There was evidence to suggest that, regardless of the histological classification, a large number of infiltrating non-neoplastic T cells was related to good prognosis.

Adult↗
PubMed1987

Effect of direct cell-to-cell interaction between the KM-102 clonal human marrow stromal cell line and the HL-60 myeloid leukemic cell line on the differentiation and proliferation of the HL-60 line.

Hematopoietic cellular interaction was investigated in a coculture of the human clonal marrow stromal line, KM-102, and the myeloid leukemia cell line, HL-60. In the coculture, a large number of HL-60 cells remained in the supernatant but some of them became firmly attached to KM-102 cells. The attached HL-60 cells showed little positive reaction in the NBT test when the culture was supplemented with 10(-9) to 10(-7) M 1 alpha, 25-dihydroxyvitamin D3. In contrast, differentiation in the supernatant HL-60 cells was strikingly responsive to the agent in a dose-dependent way. Furthermore, the complete inhibition was observed in the incorporation of [3H]thymidine into the attached HL-60 cells with autoradiography, but 23.6% of the supernatant cells moderately incorporated [3H] thymidine into their nuclei. There was no attachment between HL-60 cells and stromal cells from human thymus and lymph node, or between lymphocytic leukemia cells and marrow stromal cells. These results indicate that there is direct cellular interaction between myeloid leukemic cells and marrow stromal cells which modulates the proliferation and differentiation of the myeloid leukemic cells. This modulation by marrow stromal cells is more strongly affected by this interaction than by exogenously added differentiation-inducing agents. Apparently marrow stromal cells produce a definitive milieu for the proliferation and differentiation of myeloid leukemic cells.

Bone Marrow Cells↗
PubMed1987

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli.

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.

Amino Acid Sequence↗
PubMed1987

A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase.

A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and rat NADPH-cytochrome P450 reductase was constructed by combining the cytochrome P450c cDNA with the cDNA fragment encoding the protease-solubilized moiety of the NADPH-cytochrome P450 reductase. The hybrid cDNA was inserted between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 to yield expression plasmid pAMP19. Saccharomyces cerevisiae AH22 cells transformed with the expression plasmid pAMP19 produced a 130-kD protein reactive with both anti-cytochrome P450c Ig and antireductase Ig. The yeast cells containing the fused enzyme exhibited about four times higher monooxygenase activity toward 7-ethoxycoumarin than those containing rat cytochrome P450c alone. The fused enzyme was purified from the yeast microsomal fraction by sequential chromatography with DEAE-cellulose and 2',5'-ADP Sepharose 4B columns. The preparation had an apparent molecular weight of 130 kD and the same sequence of the 10 amino-terminal amino acids as that of rat cytochrome P450c. Spectral properties of the fused enzyme indicated the presence of a protoheme, flavin adenine dinucleotide, and flavin mononucleotide in the molecule. The reaction mechanism of the fused enzyme followed first-order kinetics. These results clearly indicate that the fused enzyme is a new self-catalytic P450 monooxygenase. Trypsin treatment of yeast microsomes containing the fused enzyme suggested that the P450 moiety is embedded in the microsomal membrane with the reductase moiety lying on the cytoplasmic side.

Animals↗
PubMed1987

Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d.

Three chimeric cytochrome P450 cDNAs were constructed by replacing the central region, carboxy-terminal region, or both central and carboxy-terminal regions of cytochrome P450c cDNA with the corresponding regions of cytochrome P450d cDNA. These were inserted between the alcohol dehydrogenase I promoter and terminator of yeast expression vector pAAH5 to form expression plasmids pACDC2, pACCD1, and pACDD2. On introduction of each of these plasmids into Saccharomyces cerevisiae AH22 cells, chimeric cytochrome P450 proteins were expressed in AH22/pACDC2, AH22/pACCD1, and AH22/pACDD2 cells at the level of at least 10(5), 4 X 10(5) molecules per cell, respectively. The reduced CO-difference spectra showed that AH22/pACCD1 and AH22/pACDD2 cells contained 4 X 10(5) and 10(5) molecules per cell of the corresponding chimeric cytochrome P450 hemoproteins, designated as cytochrome P450ccd and cytochrome P450cdd, respectively. Cytochrome P450ccd exhibited higher monooxygenase activities toward 7-ethoxycoumarin, acetanilide, and benzo[alpha]pyrene than cytochrome P450c, although the substrate specificity of cytochrome P450ccd seemed to be the same as that of cytochrome P450c. Cytochrome P450cdd exhibited lower activities toward 7-ethoxycoumarin and benzo[alpha]pyrene, and a higher activity toward acetanilide as compared with those of cytochrome P450c and cytochrome P450ccd. Therefore, the substrate specificity of cytochrome P450cdd seemed to be the same as that of cytochrome P450d. These results suggest that the central one-third region of cytochrome P450c and cytochrome P450d is responsible for substrate-binding, and that the carboxy-terminal third of both cytochromes P450 plays an important role in electron transport.

Animals↗
PubMed1987

Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi.

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).

Aspergillus↗
PubMed1986

Expression of rat NADPH-cytochrome P-450 reductase cDNA in Saccharomyces cerevisiae.

A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S. cerevisiae cells.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA.

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.

Amino Acid Sequence↗
PubMed1986

Monooxygenase activity of Saccharomyces cerevisiae cells transformed with expression plasmids carrying rat cytochrome P-450MC cDNA.

The recombinant plasmids pAMC1 and pJMC1 were constructed; the former contained the cytochrome P-450MC (P-450MC) cDNA expression unit consisting of yeast alcohol dehydrogenase I (ADH) promoter, rat P-450MC cDNA and ADH terminator, and the Leu 2 marker gene, and the latter contained the same expression unit and the leu 2-d gene. Saccharomyces cerevisiae AH22 cells transformed with each of the recombinant plasmids were examined for plasmid copy number, P-450MC mRNA level, P-450MC content, and monooxygenase activity. The S. cerevisiae AH22/pJMC1 cells contained about 2-fold higher levels of the plasmid, P-450MC mRNA, and P-450MC than the AH22/pAMC1 cells. Monooxygenase activity towards 7-ethoxycoumarin and acetanilide of the AH22/pJMC1 cells was 1.7-fold and 1.5-fold higher than that of the AH22/pAMC1 cells, respectively, whereas the activity of the AH22/pAMC1 cells towards 7-ethoxycoumarin and acetanilide was more than 1,000-fold 10-fold higher than that of the control AH22/pAAH5 cells which contain no P-450MC cDNA, respectively. Therefore, it is likely that monooxygenase activity of the AH22 cells carrying rat P-450MC cDNA was approximately proportional to the expression level of P-450MC cDNA.

Acetanilides↗
PubMed1986

Distribution of 8 alpha-hydroxyriboflavin in rat organs.

From a survey of flavin compounds using a high performance liquid chromatograph equipped with a fluorescence detector, 8 alpha-hydroxyriboflavin, its derivative, riboflavin, FMN, and FAD were found in rat organs. The derivative of 8 alpha-hydroxyriboflavin was purified from the kidney sequentially by DEAE column chromatography, paper chromatography, and high performance liquid chromatography, and was determined to be 8 alpha-hydroxyriboflavin 5'-phosphate. The amount of 8 alpha-hydroxyriboflavin in the brain, heart, liver, and kidney of the rat was calculated to be 0.010, 0.026, 0.172, and 0.103 nmol per g of wet tissue, respectively. The amount of 8 alpha-hydroxyriboflavin 5'-phosphate in the brain, heart, and liver was less than 0.002 nmol, while that in the kidney was 0.330 nmol, per g of wet tissue. On the other hand, we could not detect 7 alpha-hydroxyriboflavin in the organ extracts, even though it was excreted into human urine along with 8 alpha-hydroxyriboflavin. This is the first reported occurrence of 8 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin 5'-phosphate in higher animal organs.

Animals↗