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H Ohmori

Publications and source records attributed to H Ohmori.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of N omega-phosphoarginine hydrolase from rat liver.

N omega-Phosphoarginine hydrolase from rat liver hydrolyzed N omega-phosphoarginine into arginine and inorganic phosphate, whereas it did not release inorganic phosphate from 19 other phosphorylated compounds containing a N-P bond, an O-P bond or a C-P bond. In addition, it was not able to transfer the phosphoryl moiety from N omega-phosphoarginine to ADP. These results indicated that this enzyme was distinct from both phosphoamidase and arginine kinase. Its properties were as follows: thiol compounds were essential for its activity; it was stimulated by 1.5-2-fold in the presence of 0.001% Lubrol, Tween 20, poly(oxyethylene) 9-lauryl ether and Nonidet P-40, while 0.004% sodium lauryl sulfate inhibited the activity completely; concentrations of sodium molybdate and sodium vanadate necessary for 50% inhibition were 7 microM and 12 microM, respectively; some proteins stimulated the activity, while lysophosphatidic acid, lysophosphatidylinositol, and phosphatidic acid suppressed the activity even in the presence of poly(oxyethylene) 9-lauryl ether.

Animals↗
PubMed1992

Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1.

To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide. These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities. Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction. Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350. These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase. The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues. The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies. A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase. The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit.

Amino Acid Sequence↗
PubMed1992

Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes.

An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.

Chemical Phenomena↗
PubMed1992

Establishment of an enzyme release assay for cytotoxic T lymphocyte activity.

In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days. When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.

Animals↗
PubMed1992

Enhancement of DNA transfection efficiency by heat treatment of cultured mammalian cells.

The expression of genes introduced into various mammalian cell lines was enhanced by raising the temperature of the cells to 42 degrees C for a few hours after DNA transfection. This heat treatment resulted in an up to 10-fold increase in the frequency of the cells that transiently expressed a foreign gene such as that of beta-galactosidase, whereas it had only a limited enhancing effect on the development of stable transformants. By immunotitration analysis, it was confirmed that the enhanced expression of beta-galactosidase activity correlated well with the increase of the enzyme protein. This procedure may have an applicability for augmenting the frequency of transient gene expression in many cell types.

Animals↗
PubMed1992

Intravesical instillation of adriamycin in the presence or absence of verapamil for the treatment of superficial bladder cancer: preliminary report of a collaborative study.

A case-controlled collaborative study on the intravesical administration of Adriamycin in the presence or absence of verapamil, a calcium-channel blocker, as chemotherapy of superficial bladder cancer was carried out at two universities, Okayama and Kagoshima, and their affiliated hospitals. Although little is known about the expression of P-glycoprotein in superficial bladder cancer, it may be a cause of multidrug resistance (MDR). Verapamil was used as an inhibitor of P-glycoprotein. Arm A consisted of Adriamycin given at 50 mg/50 ml saline, and arm B constituted Adriamycin given at 50 mg/40 ml saline plus 5 ampules (10 ml) of injectable verapamil. The drugs were instilled into the bladder for 3 consecutive days in each of 3 consecutive weeks for a total of 9 instillations. No significant difference in antitumor effects was observed between arm A and arm B. Recurrent tumors responded better than did primary tumors to both arm-A and arm-B treatments (P = 0.012). In both treatment arms, significant differences (P = 0.031) in the response rate were found between tumors with diameters of less than 1 cm and those measuring 1-3 cm in diameter. Although the number of evaluable patients was limited, recurrent subjects who had previously received Adriamycin instillations responded in both treatment arms.

Administration, Intravesical↗
PubMed1992

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes.

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab')2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).

Animals↗
PubMed1992

Differences of L-myc polymorphic patterns of neuroblastoma in patients under 1 year versus older ages: a preliminary report.

The age of the patient at the onset of symptoms or at diagnosis is generally accepted as one of the most important prognostic factors of neuroblastomas (NBs). Children less than 1 year of age have a better survival rate than older patients, but the reason for this is unknown. Forty-eight unselected NB patients were divided into two groups: less than 1 year (younger NB patient) and over 1 year (older NB patient) of age at diagnosis. Two of 12 younger NB patients and 18 of 36 older NB patients had N-myc amplification in their tumors. To elucidate further the possible genetic difference between younger and older NB patients, studies of restriction fragment length polymorphism (RFLP) of the L-myc gene was carried out in these two groups. The L-myc locus showed 2-allele polymorphism, allele L(10 kb) and S (6.6 kb), after digestion with EcoRI. Patients homozygous for L-band have been reported as individuals having less metastatic potential in some cancers. The allele frequencies of L and S in neuroblastomas of younger NB patients were 0.50 and 0.50, while those of older NB patients were 0.35 and 0.65, respectively. Although we did not determine L-myc RFLP in normal tissue of individual patients, we expect that the distribution of allele L and S is partly affected by possible allelic loss involving the L-myc region. However, the L-myc RFLP patterns in younger NB patients were the same as those of normal individuals and significantly differed from those of older NB patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1992

Ion channels for the mechano-electrical transduction and efferent synapse of the hair cell.

Auditory and vestibular information is applied to the hair cell hair bundle as mechanical energy, and is transduced into electrical energy by gating ion channels. The m-e.t. channel has a unitary conductance of 50 pS and a broad selectivity to monovalent cations and to divalent cations. Ca ions are the most permeable through the channel. The angular displacement of the hair bundle is the primary gating factor. Circumstantial evidence indicates the possibility of the direct gating of channels by the membrane deformation itself. The transduction potential activates voltage gated Ca channel and leads to the release of neurotransmitters which activate afferent neurones. Cholinergic muscarinic receptors likely mediate the inhibitory efferent innervation to the hair cell.

Animals↗
PubMed1992

Selective regulation of antigen-specific IgE response by cyclic AMP level in murine lymphocytes.

We have reported that prostaglandin E2 (PGE2) is a selective stimulator of the antigen-specific IgE response [6]. Because PGE2 is known to elevate intracellular cAMP, we investigated the regulatory role of cAMP in the production of antigen-specific IgE. Anti-TNP IgE response was induced by stimulating TNP-KLH-primed BALB/c spleen cells with the same antigen in vitro. Addition of 10-100 microM dibutyryl cAMP (DBcAMP) to the lymphocyte culture resulted in a 2-3-fold increase in anti-TNP IgE response without affecting the production of anti-TNP IgG1 or IgM. Forskolin, a stimulator of adenylate cyclase, also specifically augmented the IgE response. In contrast, 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, suppressed IgE production in an isotype-specific manner. These results suggest that IgE synthesis can be selectively modulated by intracellular cAMP level. Enhancement of IgE production by DBcAMP was observed, particularly in highly primed spleen cells, suggesting that IgE-committed B cells are subjected to regulation by cAMP.

Animals↗
PubMed1992

Suppression of interleukin 4 production from type 2 helper T cell clone by antisense oligodeoxynucleotide.

Type 2 helper T cell (Th2) clone has been reported to secrete interleukin (IL) 4 and IL5 in response to the specific antigen presented by syngeneic antigen-presenting cells. In the present report, we synthesized phosphorothioate analogue of an antisense oligodeoxynucleotide complementary to nucleotide 17-36 of IL4 mRNA (S-oligo), and tested its ability to inhibit IL4 production from a Th2 clone, D10.G4.1. (D10). D10 cells were cultured with mitomycin C-treated C3H spleen cells in the presence of 100 micrograms/ml conalbumin for 48-72 h. Secreted IL4 and IL5 were assayed biologically using HT2 cells and dextran sulfate-stimulated murine B cells, respectively. When 5-10 micrograms/ml S-oligo was added to the culture, IL4 production from D10 was suppressed by 70-90%. The same concentrations of S-oligo inhibited neither the antigen-induced proliferation of D10 nor the secretion of IL5 from the Th2 clone. These results suggest that this S-oligo is useful for inhibiting the production of IL4 preferentially without affecting other functions of Th2 cells.

Animals↗
PubMed1992

Direct proof that the primary site of action of cytochalasin on cell motility processes is actin.

We have previously described the isolation of a mutant KB cell (Cyt 1 mutant) resistant to the cytotoxic effect of cytochalasin B (CB). The Cyt 1 mutant carries an altered form of beta-actin (beta'-actin) and lacks normal beta-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). Increased resistance of the Cyt 1 mutant to CB in vivo is reflected in altered properties of beta'-actin in vitro (Toyama, S., and S. Toyama. 1988. J. Cell Biol. 107:1499-1504). Here, we show that the mutation in beta-actin is solely responsible for the cytochalasin-resistant phenotype of the Cyt mutant. We have isolated a cDNA clone encoding beta'-actin from Cyt 1 cells. Sequence analysis reveals two mutations in the coding region that substitute two amino acid residues (Val139----Met and Ala295----Asp). Expression of the beta'-actin cDNA confers cytochalasin resistance upon transformed cytochalasin-sensitive KB cells. Levels of resistance to CB in the transformed cell clones correlate well with amounts of beta'-actin polypeptide. Both of the two mutations in beta'-actin are necessary for the high level expression of cytochalasin resistance. Overall, we conclude that the primary site of action of cytochalasin on cell motility processes in vivo is actin.

Actin Cytoskeleton↗
PubMed1992

Comparison of Ki-ras gene mutation among simultaneously occurring multiple urethan-induced lung tumors in individual mice.

Mouse lung tumors were induced in C57BL/6J(female) x A/J(male) F1 mice by a single s.c. injection of urethan. About 6 months later, multiple small-sized lung tumors were detectable in almost all mice. After a further 6 months, some of these tumors became larger than the rest. We examined whether there were any mutational differences among multiple lung tumors in a single mouse. Direct DNA sequencing of a separately amplified Ki-ras gene by polymerase chain reaction (PCR) was carried out with 25 DNA samples from multiple tumors in four mice. Twenty-four of 25 tumors (96%) had mutations at the codon 61 of the Ki-ras gene. The major mutations involved were either AT to GC transition (44%) or AT to TA transversion (44%) at the second base of codon 61. We compared the types of these gene mutations among the tumors from each of two mice from two different groups of siblings and then compared the two groups. Interestingly, in the first group of siblings, we detected CTA in 5/6 tumors in the first mouse and again CTA in 4/6 tumors in the second one. In the second group of siblings, we detected CGA in 5/7 tumors in one mouse and CGA again in 3/5 tumors in the second mouse. These results show that the pattern of Ki-ras codon 61 mutations in urethan-induced lung tumors is similar in tumors developing in siblings, suggesting that host factors have an effect on the carcinogen-induced mutational pattern. There was no major mutational difference between small and large tumors. The results suggested that other event(s) in addition to the mutation of the Ki-ras gene might play a role during the development of large-sized tumors.

Amino Acid Sequence↗
PubMed1992

The effect of caged calcium release on the adaptation of the transduction current in chick hair cells.

1. Intracellular Ca2+ concentration ([Ca2+]i) was raised by photolysis of a caged calcium compound, nitr-5, and its effects on the mechano-electrical transduction (MET) current were studied by a whole-cell patch electrode voltage clamp technique in dissociated hair cells of a chick. Nitr-5 was loaded into the hair cell by incubation with the membrane-permeable form of the compound (nitr-5 AM). 2. Photolysis of nitr-5 by ultraviolet (UV) light irradiation induced outward currents at -50 mV when recorded with a KCl-based intracellular medium without Ca2+ chelating compounds. The average amplitude of the photo-activated outward current was 115 +/- 82 pA (mean +/- S.D., n = 5). 3. The MET current generated at -50 mV showed a decay after step displacement of the hair bundle. This adaptation was accelerated after UV exposure of the cell. The adaptation was further accelerated by hyperpolarization of the membrane and was eliminated in 20-100 microM Ca2+ extracellular media. 4. The displacement-response relationship was shifted towards the positive direction after the UV irradiation. 5. The recovery of the transducer current after step displacement of the hair bundle was accelerated after UV irradiation, for both the inward-going MET current recorded at -50 mV and the outward-going MET current at +54 mV. However, the adaptation was not observed at positive membrane potentials even after the photolysis of nitr-5. 6. The extent of MET current decay was reduced or disappeared in 20-100 microM Ca2+ extracellular media and the offset time course was prolonged at the membrane potential of -50 mV. The current decay was not observed even after the photo-release of intracellular Ca2+ in 50-100 microM Ca2+ extracellular media. 7. These results (paragraphs 3-6) suggest that the MET current adaptation is accelerated by the increase of [Ca2+]i, and that Ca2+ ions entering through MET channels are essential in the development of adaptation. 8. The adaptation of the MET current was reversibly reduced in a dihydrostreptomycin (DHSM, 20-50 microM) medium. The time course of the adaptation changes lagged the changes in the MET current amplitude. 9. The adaptation developed or disappeared with a delay of 10-20 s after the introduction of either the normal-Ca2+ (2.5 mM) or the low-Ca2+ (50-100 microM) extracellular medium, respectively. These delays in the development and the subsidence of adaptation suggest a presence of a Ca2+ buffer site intracellularly between the adaptative site and the MET channel.

Adaptation, Physiological↗
PubMed1992

[Prophylactic intravesical instillation therapy in patients with superficial bladder cancer--results of a randomized prospective study].

A randomized prospective study was conducted for the purpose of investigating the efficacy of intravesical chemoprophylaxis of superficial bladder cancers. Eligible patients were randomized into three groups: 1) adriamycin (ADM) group; intravesical instillation with 50 mg of ADM dissolved in 100 ml physiological saline, 2) mitomycin C (MMC) group; intravesical instillation with 30 mg of MMC dissolved in 100 ml of physiological saline, 3) control group; transurethral resection or transurethral coagulation only. The characteristic features of our protocol consisted of frequent (six times) instillations of the drugs within two weeks after transurethral resection, followed by instillations on two consecutive days at four-week intervals for two years. Furthermore, large quantities (100 ml) of instillation fluid containing relatively low concentrations of the drugs (500 micrograms/ml for ADM or 300 micrograms/ml for MMC) were employed. One hundred and forty-four patients have been submitted to the study; 110 patients were fully evaluable for recurrence and 34 patients were eliminated as non-evaluable patients. The cumulative five-year non-recurrence rates of the patients with multiple tumors were 32% in the MMC group, 25% in the ADM group and 7% in the control group. The cumulative non-recurrence rates of the ADM and MMC groups were significantly higher than that of the control group. It is considered that this instillation therapy with ADM and MMC is useful for preventing the recurrence of superficial bladder cancers.

Administration, Intravesical↗
PubMed1992

[Histopathological study of metallothionein in bladder cancer and renal cell carcinoma].

Metallothionein (MT) is a low molecular-metal binding protein with multiple biological functions. Recently, MT has been implicated as a factor involved in resistance to anticancer drugs, which presumably inactivates anticancer drugs, including cisplatin, and doxorubicin. In this report, we investigated the relationship of MT expression with the clinical features in bladder cancer and renal cell carcinoma. In 35 cases of bladder cancer, 10 cases of renal cell carcinoma and 3 cases of normal mucosa of bladder, the expression of MT was immunohistologically examined by avidinebiotin-peroxidase (ABC) staining of paraffin-embedded tissue specimens with anti-MT antibody. Intense MT expression was noted in all cases of normal mucosa of bladder. MT was detected in 10 of 35 cases of bladder cancer, with the incidence of MT expression being significantly higher increases with lower pathological tumor grade. MT was detected in 8 of 10 cases of renal cell carcinoma, and all of the their normal renal tubules showed more intense staining. A number of hypotheses can be proposed from these observations. First, our observation of decreased MT expression in poorly differentiated carcinomas, which are the more proliferating tumors, this suggests correlation of MT expression with proliferative status of cancer. Second, the higher incidence of MT expression in renal cell carcinoma than in bladder cancer may suggest that it is a factor responsible for the lower efficacy of chemo-therapy in renal cell carcinoma than in bladder cancer.

Aged↗
PubMed1991

Cadmium causes increases of N-myc and multidrug-resistance gene mRNA in neuroblastoma cells.

Since cadmium exposure results in neuropathological alterations in central nervous system, we investigated the effects of cadmium on the gene expression of neuroblastoma (GOTO) cells. We observed an increase in mRNA levels of heat-shock protein (hsp) 70, hsp 90, hsp 32 and metallothionein after treatment of GOTO cells with cadmium, although the time courses of the changes of individual mRNA of the heat-shock proteins and metallothionein were somewhat different from each other. An accumulation of N-myc and multidrug-resistance gene (MDR1) mRNA was detected in the presence of cadmium. This is contrary to the previous report, in which an inverse correlation between the expression of MDR1 gene and N-myc oncogene in human neuroblastoma had been described. However, the increase of N-myc and MDR1 mRNA in the present study is not likely due to the loss of regulatory mechanism of these genes by cytotoxic effects of cadmium, because active protective mechanisms such as heat-shock proteins and metallothionein could be induced under these conditions.

Cadmium↗