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H Okamoto

Publications and source records attributed to H Okamoto.

At least 361 records · Page 20Linked to original sources
PubMed1997

Muscarinic receptor-mediated dual regulation of ADP-ribosyl cyclase in NG108-15 neuronal cell membranes.

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma x glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30-52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

ADP-ribosyl Cyclase↗
PubMed1997

A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology.

By means of representational difference analysis, a viral clone (N22) of 500 nucleotides was isolated from serum of a patient (TT) with posttransfusion hepatitis of unknown etiology. The N22 clone showed a poor homology to any reported sequences. Oligonucleotide primers were deduced from the N22 sequence for detecting it by polymerase chain reaction. N22 sequence in serum banded at a sucrose density of 1.26 g/cm3, indicating its association with a viral particle which was designated TT virus (TTV). Since nucleic acids of TTV were sensitive to DNase I, it would be a DNA virus. TTV DNA was detected in sera from three of the five patients with posttransfusion non-A to G hepatitis, including the index case (TT). TTV DNA titers closely correlated with aminotransferase levels in the three patients. These results indicate that TTV would be a novel DNA virus with a possible capacity to induce posttransfusion non-A to G hepatitis.

Aged↗
PubMed1997

Purification, characterization, and localization of an ADP-ribosylactin hydrolase that uses ADP-ribosylated actin from rat brains as a substrate.

Mammalian ADP-ribosylation is poorly understood. An ADP-ribosylprotein hydrolase that acted on ADP-ribosylated actin was purified from rat brain. The molecular weight of this enzyme was 62, 000 as determined by SDS-polyacrylamide gel electrophoresis and gel filtration. Enzyme activity with ADP-ribosylated actin as a substrate was inhibited by NAD, ATP, ADP, and ADP-ribose, but not by AMP. Mg2+ increased Vmax. Purified ADP-ribosylactin hydrolase catalyzed the hydrolysis of ADP-ribosylated subunits Gsalpha, Gialpha, and Goalpha and elongation factor-2. After de-ADP-ribosylation by the purified ADP-ribosylactin hydrolase, the proteins were re-ADP-ribosylated by brain mono-ADP-ribosyltransferases and bacterial toxins. The actin that was de-modified by ADP-ribosylactin hydrolase could form actin filaments. Two kinds of monoclonal antibodies against ADP-ribosylactin hydrolase were prepared and characterized. In an immunohistochemical study, the plasma membranes and cytoplasmic regions of the nerve cells in the rat brain were immunoreactive. In subcellular fractionation of the brains, most of the ADP-ribosylactin hydrolase activity was found in the cytosol and synaptosome fractions. When the synaptosomes were treated with a hypotonic solution, ADP-ribosylactin hydrolase activity was found in the supernatant. Our findings suggest that brain ADP-ribosylactin hydrolase has the important function of polymerizing actin for signal transduction in the cytosol of nerve cells and synaptosomes.

Actins↗
PubMed1997

The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II.

The human immunodeficiency virus encodes the transcriptional transactivator Tat, which binds to the transactivation response (TAR) RNA stem-loop in the viral long terminal repeat (LTR) and increases rates of elongation rather than initiation of transcription by RNA polymerase II (Pol II). In this study, we demonstrate that Tat binds directly to the cyclin-dependent kinase 7 (CDK7), which leads to productive interactions between Tat and the CDK-activating kinase (CAK) complex and between Tat and TFIIH. Tat activates the phosphorylation of the carboxy-terminal domain (CTD) of Pol II by CAK in vitro. The ability of CAK to phosphorylate the CTD can be inhibited specifically by a CDK7 pseudosubstrate peptide that also inhibits transcriptional activation by Tat in vitro and in vivo. We conclude that the phosphorylation of the CTD by CAK is essential for Tat transactivation. Our data identify a cellular protein that interacts with the activation domain of Tat, demonstrate that this interaction is critical for the function of Tat, and provide a mechanism by which Tat increases the processivity of Pol II.

Animals↗
PubMed1997

Mutation rate of GB virus C/hepatitis G virus over the entire genome and in subgenomic regions.

A patient on maintenance hemodialysis was infected with a recently discovered putative non-A to -E hepatitis virus designated GB virus C (GBV-C) or hepatitis G virus (HGV) by transfusion. The viral isolate was recovered from the patient soon after she turned positive for GBV-C/HGV RNA in serum (GS185) and 8.4 years thereafter (GS193), and the entire nucleotide sequences were determined. They both had a genomic length of 9391 nucleotides with a defective C gene made of only 42 nucleotides. Between GS185 and GS193, 31 (0.33%) nucleotides were different, which changed 5 (0.18%) of the encoded 2842 amino acids. Thus, GBV-C/HGV was estimated to have a mutation rate of 3.9 x 10(-4) base substitutions per site per year. Nucleotide conversions were distributed over subgenomic regions, except in the 5' untranslated region of 552 nucleotides and a defective short C gene, which were conserved in sequence. The change in the putative envelope genes (E1 and E2) was no different from that in the entire genome with only 6 (0.35%) nucleotide substitutions among the 1730, just 1 of which induced an amino acid conversion. Taken along with the comparison of the two isolates with the reported five GBV-C or HGV isolates, these results indicate that GBV-C/HGV would not have hypervariable regions and would use a strategy for viral persistence that is different from immune escape.

Amino Acid Sequence↗
PubMed1997

Role of cyclic ADP-ribose in ATP-activated potassium currents in alveolar macrophages.

There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells. In the present study, using the nystatin perforated patch recording configuration, we found that ATP (0.3-30 microM) induced a transient outward current in a concentration-dependent manner and that the reversal potential of the ATP-induced outward current was close to the K+ equilibrium potential, indicating that the membrane behaves like a K+ electrode in the presence of ATP. The first application of ATP to alveolar macrophages perfused with Ca2+-free external solution could induce the outward current, but the response to ATP was diminished with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid, also diminished the response. When cyclic ADP-ribose (cADPR) was applied to the macrophage cytoplasm, a transient outward current was elicited. Thereafter, the successive outward current was inhibited, suggesting the involvement of cADPR in the response. Intracellular perfusion with inositol 1,4, 5-trisphosphate also induced a transient outward current, but the successive current was not inhibited. The ATP-induced outward current was abolished when 8-amino-cADPR (as a blocker of cADPR, 10(-6)-10(-5) M) was introduced into the cytoplasm. Homogenates of alveolar macrophages showed both ADP-ribosyl cyclase and cADPR hydrolase activities, and CD38 (ADP-ribosyl cyclase/cADPR hydrolase) expression was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These results indicate that ATP activates K+ currents by releasing Ca2+ from cADPR-sensitive internal Ca2+ stores.

Adenosine Diphosphate Ribose↗
PubMed1997

Attenuation of nitric oxide synthase induction in IRF-1-deficient glial cells.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) exerts inhibitory and cytotoxic effects on various cells including neuronal cells. Glial NO production, mediated via induction of iNOS, is thought to facilitate neuronal damage during cerebral ischemia. Recently, interferon regulatory factor-1 (IRF-1) has been reported to be an essential transcription factor for iNOS mRNA induction in murine macrophages. However, expression of IRF-1 and its role in the central nervous system have not been examined. In the present study, by using primary glial cell cultures from mice with targeted disruption of the IRF-1 gene, we investigated whether IRF-1 is involved in iNOS mRNA induction in glial cells. After stimulation with lipopolysaccharide and interferon-gamma, IRF-1 mRNA was strongly induced in wild-type (IRF-1 +/+) glial cells. iNOS mRNA induction and nitrite production in IRF-1 -/- glial cells were reduced as compared with those observed in IRF-1 +/+ glial cells. Diethyldithiocarbamate, a selective inhibitor of nuclear transcription factor kappa B (NF-kappa B), completely inhibited iNOS mRNA induction. These results suggest that not only NF-kappa B but also IRF-1 play important roles in iNOS mRNA induction in the central nervous system.

Animals↗
PubMed1997

Molecular cloning of cDNAs for mouse low-molecular-weight and high-molecular-weight prekininogens.

We isolated cDNAs encoding low-molecular-weight (L-) and high-molecular-weight (H-) prekininogens from a mouse liver cDNA library using rat T-kininogen cDNA and rat H-kininogen cDNA respectively, as probes. The signal peptide, the heavy chain, and the bradykinin moiety, which are common between the two prekininogens, consist of 20, 359, and 9 amino acids, respectively, while the light chains of the L- and H-prekininogens are composed of 44 and 273 amino acids, respectively. All 19 cysteine residues present in both mouse prekininogens are located at the same positions relative to those of human origin. The light chain of H-prekininogen contains a characteristic 15-repeated His-Gly sequence and a conserved sequence for binding prekallikrein or factor XI. Northern blotting or reverse transcription-polymerase chain reaction followed by Southern blotting using mouse L- and H-kininogen cDNAs demonstrated that both L- and H-kininogens are predominantly expressed in the liver and kidney. L-Kininogen mRNA was also expressed in other tissues, such as the adrenal gland, brain, spinal cord, testis, lung, heart, and skin, while levels of H-kininogen mRNA in these tissues were too low to detect, suggesting that L-kininogen is synthesized in various tissues of mouse, while H-kininogen is exclusively synthesized in the liver and kidney. A genomic Southern blot using H-prekininogen cDNA revealed that the L- and H-prekininogen mRNAs in mouse are probably encoded by a single gene, as is the case in both human and bovine.

Amino Acid Sequence↗
PubMed1997

Human gene encoding CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing.

We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucose-induced insulin secretion [Takasawa et al. (1993a) Science 259, 370-373] and that CD38 has both ADP-ribosyl cyclase (ADRC) and cADPR hydrolase activities [Takasawa et al. (1993b) J. Biol. Chem. 268, 26052-26054]. In this study, we determined the structure of the human CD38 gene, and showed that two mRNA forms originated by alternative splicing from the CD38 gene. The human CD38 gene consists of 8 exons that extend more than 77 kb on the human genome. Exon 1 encoded the 5'-untranslated region of the mRNA, the N-terminal end of CD38 and the putative transmembrane domain, and exon 2-8 encoded the remainder of CD38: the exon-intron organization of the human CD38 gene is similar to that of the Aplysia ADRC gene [Nata et al. (1995) Gene 158, 213-218]. This structural conservation between human and Aplysia genes suggests that both genes may have evolved from a common ancestral gene.

ADP-ribosyl Cyclase↗
PubMed1997

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase.

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.

ADP-ribosyl Cyclase↗
PubMed1997

Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIII alpha, RegIII beta, RegIII gamma.

Reg (regenerating gene), first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, Reg family, which consists of three subtypes (type I, II, III) based on the primary structures of the encoded proteins of the genes. In mouse, type I and type II Reg genes (i.e. RegI and RegII gene) have so far been isolated. In the present study, the complete nucleotide (nt) sequences of the cDNAs and genes encoding murine type III Reg (regenerating gene product), RegIII alpha, RegIII beta and RegIII gamma were determined. RegIII alpha, RegIII beta and RegIII gamma encode 175-, 175- and 174-amino acid (aa) proteins, respectively, with 60-70% homology. All three genes are composed of six exons and five introns spanning approx. 3 kb, and exhibit distinctive structural features unique for members of the Reg gene family. All the mouse Reg genes, RegIII alpha, RegIII beta, RegIII gamma, RegI and RegII, are assigned to the adjacent site of chromosome 6C by fluorescence in situ hybridization (FISH). RegIII alpha, RegIII beta and RegIII gamma were expressed weakly in pancreas, strongly in intestinal tract, but not in hyperplastic islets, whereas both RegI and RegII were expressed in hyperplastic islets. These results suggest that genes of the mouse Reg family are derived from a common ancestor gene by several gene duplications, and have obtained divergency in expression and function in the process of genetic evolution.

Amino Acid Sequence↗
PubMed1997

Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes.

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

Adenosine Diphosphate Ribose↗
PubMed1997

Sequence-independent inhibition of RNA transcription by DNA dumbbells and other decoys.

DNA dumbbells are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, conferring resistance to exonucleases. Dumbbells may be designed to interact with transcription factors in a sequence-specific manner. The internal based paired sequence of DNA dumbbells in this study contains the X-box, a positive regulatory motif found in all MHC class II DRA promoters. In electrophoretic mobility shift assays (EMSAs), dumbbells and other oligonucleotides ('decoys') with the core X-box sequence were found to compete with the native strand for binding to X-box binding proteins (including RFX1). However, only the X-box dumbbell was capable of forming detectable complexes with such proteins using EMSA. In a model cell system, dumbbells were tested for their ability to block RFX1VP16 activation of a plasmid containing multiple repeats of the X-box linked to the CAT gene. While it appeared that dumbbells could block this activation, the effect was non-specific. This and further evidence suggests an inhibition of transcription, most likely via an interaction with the general transcriptional machinery.

Animals↗
PubMed1997

Concurrent daily carboplatin and accelerated hyperfractionated thoracic radiotherapy in locally advanced nonsmall cell lung cancer.

PURPOSE: This study was designed to evaluate the feasibility and efficacy of accelerated hyperfractionated thoracic radiotherapy concurrently combined with daily carboplatin in patients with locally advanced, unresectable nonsmall cell lung cancer. METHODS AND MATERIALS: Thirty-one patients with locally advanced nonsmall cell lung cancer were treated with continuous course, twice daily thoracic radiotherapy (1.5 Gy each) to a total of 60 Gy over 4 weeks. Carboplatin (25 mg/m2) i.v. was given immediately before each morning thoracic radiotherapy. Blood samples were taken to measure the blood free platinum pharmacokinetics on day 1. RESULTS: All 31 patients completed the protocol treatment without delay. The median age was 73 years, and the majority had Stage IIIA (32%) or IIIB (48%) disease. Major acute toxicity (> or = Grade 3) included 17 patients (55%) with leukopenia, 5 patients (16%) with thrombocytopenia, and 7 patients (23%) with esophagitis. One possible treatment-related death due to diffuse pneumonitis was observed. There were three complete responses (CRs) and 23 partial responses (PRs) in the radiation field, for a response rate of 84%. The relapse pattern was predominantly loco-regional, and the median survival time was 9.8 months. The area under the plasma level-time curve (AUC) of free platinum correlated significantly (r = -0.41, p = 0.04) with the surviving fraction of leukocytes, but not with the severity of the esophagitis. Responders had significantly (p = 0.04 by Welch's t-test) higher AUCs than nonresponders. CONCLUSIONS: This combination therapy was feasible and efficacious against locally advanced nonsmall cell lung cancer. Although long-term local control still remains unsatisfactory, pharmacokinetic data are suggestive of a role for platinum in enhancing the radiation effect.

Aged↗
PubMed1997

Infection with GB virus C and hepatitis C virus in hemodialysis patients and blood donors in Beijing.

RNAs of GB virus C (GBV-C) and hepatitis C virus (HCV) were sought by reverse-transcription polymerase chain reaction with nested primers deduced from the 5' untranslated region: 79 patients on maintenance hemodialysis, 205 commercial blood donors, and 205 voluntary donors in Beijing were studied. GBV-C RNA was detected in 43 (54%) patients and 17 (8%) commercial donors, and HCV RNA in 43 (54%) patients and 13 (6%) commercial donors, respectively. By contrast, GBV-C RNA was detected only in 2 (1%) and HCV RNA in none among 205 volunteer blood donors serving as controls. Thus both patients and commercial blood donors were at higher risk for infection with GBV-C (P < 0.001) than controls. HCV RNA was detected more often in patients with GBV-C RNA than without (29/43 or 67%, vs. 14/36 or 39%, P < 0.05) as well as in commercial donors with GBV-C RNA than without (5/17 or 29% vs. 8/188 or 4%, P < 0.01). A phylogenetic tree constructed on a sequence of 100 base pairs in the helicase region indicated that GBV-C isolates from Beijing are more similar to Japanese isolates than to isolates from the United States and Africa. Sequences from certain hemodialysis patients and those from some commercial donors were similar, suggesting nosocomial infection and spread among restricted groups.

Adult↗
PubMed1997

Infection with GB virus C and hepatitis C virus in drug addicts, patients on maintenance hemodialysis, or with chronic liver disease in Nepal.

Infection with GB virus C (GBV-C) and hepatitis C virus (HCV) was surveyed in various populations in Kathmandu, Nepal. GBV-C RNA and HCV RNA were detected in four (2%) and none, respectively, of 181 normal controls. Viral RNAs were detected significantly more frequently (P < 0.001) in 32 (44%) and 43 (60%), respectively, of 72 users of illicit intravenous drug, and in three (14%) and one (5%) of 22 patients on maintenance hemodialysis. The three hemodialysis patients with GBV-C RNA had been transfused with more blood units than the 19 without GBV-C RNA (51 +/- 21 vs. 5 +/- 3 units, P < 0.01), and one was co-infected with HCV. Of 145 patients with chronic liver disease, GBV-C RNA was detected in four (3%) and HCV RNA in 12 (8%); only one patient with GBV-C RNA was without markers of HCV or hepatitis B virus infection. In the 32 drug addicts infected with GBV-C, genotypes were G1 in two (6%), G2 in 26 (81%), G3 in three (9%), and the remaining one (3%) was coinfected with G2 and G3. GBV-C genotypes in the 13 individuals in the populations other than drug addicts were G2 in 11 (85%) and G3 in two (15%). HCV genotypes in the 43 drug addicts with viremia were l/1a in 21 (49%), V/3a in 19 (44%) and l/1a plus V/3a in two (5%); these genotypes were not prevalent in normal controls and patients with chronic liver disease in Nepal. These results indicate that GBV-C infection is prevalent in healthy subjects in Nepal at a frequency (2%) comparable with those in the other countries and that GBV-C transmits efficiently by intravenous drug abuse among drug addicts and by transfusion in hemodialysis patients.

Adult↗