Biomedical subjects

Y Asano

Publications and source records attributed to Y Asano.

At least 325 records · Page 18Linked to original sources

PubMed1997

Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.

Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and Pllb starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and Pllb, and the carboxy terminal proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.

Amino Acid Sequence↗

PubMed1997

Arginine vasopressin inhibits interleukin-1 beta-stimulated nitric oxide and cyclic guanosine monophosphate production via the V1 receptor in cultured rat vascular smooth muscle cells.

BACKGROUND: It has been reported that various vasoactive substance modulate cytokine stimulated nitric oxide (NO) production in many cell types. OBJECTIVE: To examine the effects of arginine vasopressin (AVP) on the production of NO and cyclic GMP (cGMP), and on inducible nitric oxide synthase (INOS) in cultured rat vascular smooth muscle cells (VSMC). DESIGN: Because VSMC possess the V1 receptor which clauses vascular contraction and respond to various cytokines for producing NO, we used rat VSMC and selected interleukin-1 beta (IL-1 beta) as a potent stimulator of NO production among various cytokines. We also measured cGMP production, which is the final mediator of NO-induced vascular relaxation, in order to evaluate the physiologic meaning of the present study. METHODS: VSMC were incubated with test agents for 24 h except for a time-course study. Nitrite as a stable end product of NO was measured in the medium. Intracellular cGMP contents were assayed by enzyme immunoassay. INOS messenger RNA expression was analyzed by Northern blotting. RESULTS: AVP inhibited IL-1 beta-induced nitrite production in a dose- and time-dependent manner with concomitant changes in intracellular cGMP contents. On the other hand, AVP did not affect nitrite and cGMP production in the absence of IL-1 beta. Inhibition of nitrite and cGMP production by AVP was reversed by administration of the specific V1 receptor antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine] -Arg8-vasopressin) but not by the oxytocin (OXT) receptor antagonist [d(CH2(5)), TyrMe2, Orn8]-Vasotocin. Administration of the V1 receptor antagonist or OXT receptor antagonist alone did not affect IL-1 beta-stimulated nitrite and cGMP production. Although administration of AVP inhibited IL-1 beta-induced INOS messenger RNA expression, administration of the V1 receptor antagonist but not of the OXT receptor antagonist reversed this inhibition. CONCLUSION: It is suggested that AVP inhibits IL-1 beta-induced NO and cGMP production via the V1 receptor but not via the OXT receptor in VSMC. AVP can cause vascular contraction not only through direct action but also through indirect action by inhibiting NO production under some inflammatory conditions.

Animals↗

PubMed1997

Serum transcriptionally regulates Na(+)-K(+)-ATPase gene expression in vascular smooth muscle cells.

The present study was designed to examine the effects of serum on Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit gene expression in cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas. Addition of 10% serum to VSMC for 24 h increased Na(+)-K(+)-ATPase activity 1.5-fold and alpha 1- and beta 1-subunit protein levels 1.9-fold. Serum (10%) caused a 3.5-fold increase in alpha 1-mRNA levels and a 6.7-fold increase in beta 1-mRNA levels, with peak elevations at 12 h. The protein synthesis inhibitor cycloheximide abolished serum-mediated beta 1-mRNA induction but did not affect serum-mediated alpha 1-mRNA induction. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) significantly reduced serum-mediated beta 1-mRNA induction but had no effect on serum-mediated alpha 1-mRNA induction. Transfection experiments with the 5'-flanking sequences of the alpha 1- or beta 1-subunit genes linked to the luciferase reporter gene revealed that 10% serum caused 2.8- and 6.5-fold increases in luciferase activity, respectively. Among growth factors, only basic fibroblast growth factor (FGF) enhanced luciferase activities for the alpha 1- and beta 1-subunit genes. We conclude that 1) serum stimulates alpha 1- and beta 1-mRNA expression, alpha 1- and beta 1-subunit protein accumulation, and Na(+)-K(+)-ATPase activity; 2) serum-mediated beta 1-mRNA induction partly requires de novo synthesis of intermediate regulatory proteins and activation of PKC and TK, whereas serum-mediated alpha 1-mRNA induction occurs through PKC- and TK-independent mechanisms; 3) the 5'-flanking regions of the alpha 1- and beta 1-subunit genes are serum responsive; and 4) FGF mimics stimulatory effects of serum on promoter activities for the alpha 1- and beta 1-subunit genes.

Animals↗

PubMed1997

Pial arteriolar constriction to alpha 2-adrenergic agonist dexmedetomidine in the rat.

Dexmedetomidine (Dex) is an alpha 2-adrenergic agonist that decreases cerebral blood flow (CBF) when administered systemically. It is unclear whether cerebral vasoconstriction is mediated by a local effect on cerebral vessels or by a remote neural mechanism. In the present study, we compared the pial arteriole responses to locally and systemically administered Dex with and without local application of the specific alpha 2-adrenergic antagonist atipamezole. Six groups of male rats (n = 7 each) were anesthetized with isoflurane and prepared for measurements of small (20-39 microns), medium (40-59 microns), and large (60-79 microns) pial arteriole diameter by intravital microscopy or for regional CBF measurement by the radiolabeled-microsphere method. Local application of Dex caused dose-dependent constriction that was significant starting at 10(-8) M for small and medium-sized arterioles and at 10(-7) M for large arterioles. Constriction to 10(-5) M in small [21 +/- 2% (SE)], medium (21 +/- 2%), and large (15 +/- 1%) arterioles was almost completely blocked by local application of 10(-4) M atipamezole. Intravenous administration of Dex at 1 microgram/kg decreased CBF and caused modest arteriolar constriction that began to resolve 8 min after administration. A dose of 10 micrograms/kg constricted arterioles of all sizes with constriction beginning to resolve after approximately 10 min. Local application of atipamezole (10(-4) M) slightly blunted the response to 1 micrograms/kg of intravenous Dex but did not substantially limit constriction after 10 micrograms/kg. These data demonstrate that pial arterioles are capable of substantial constriction to Dex by a local alpha 2-adrenergic mechanism. However, the inability of locally applied atipamezole to substantially inhibit the vasoconstrictor response to systemically administered Dex suggests that Dex might also cause vasoconstriction indirectly through actions at other sites in the central nervous system.

Adrenergic alpha-Agonists↗

PubMed1997

Localization of low-KM cAMP phosphodiesterase in rat nephron segments.

To evaluate the roles of cAMP degradation on hormonal actions in the kidney, we examined the segmental distribution and activities of cAMP-phosphodiesterase (PDE), a key enzyme for cAMP hydrolysis, in dissected segments of the rat nephron and characterized the isozyme compositions with subtype-specific inhibitors. Summary of the nephron distribution of PDE activities showed that cAMP-PDE activities were detected in all nephron segments and the distal convoluted tubules (DCTs) had the highest activity. Both in proximal convoluted tubules (PCTs) and medullary collecting ducts (MCDs), more than 80% of the total cAMP-PDE activities were inhibited by a nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). In PCTs, rolipram (type-IV PDE inhibitor) was an equally potent inhibitor of IBMX, while 8-methoxymethyl-IBMX (MM-IBMX, type-I PDE inhibitor) and cilostamide (type-III PDE inhibitor) inhibited cAMP-PDE by 35 and 57%, respectively. In MCDs, inhibition by rolipram (40%) was less than that by MM-IBMX (70%) or cilostamide (66%). Rolipram potentiated the parathyroid hormone (PTH)-induced cAMP content in PCTs most effectively. Rolipram and MM-IBMX increased the vasopressin (AVP)-stimulated cAMP content equally in MCDs. Cilostamide had no effect on the cAMP content stimulated by PTH or AVP in PCTs and MCDs. These results indicate that PDE activities were unevenly distributed along the rat nephron and cAMP-PDE was composed of at least three isoforms, namely type I, III and IV in PCTs and MCDs. Among these, type-IV PDE was responsible for hydrolysis of cAMP in PCTs, and type-I and IV PDE were responsible for that in MCDs.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Comparison of bactericidal effects of commonly used antiseptics against pathogens causing nosocomial infections. Part 2.

Opportunistic infections caused by gram-negative rods (GNR), conventionally regarded as organisms with low or no pathogenicity, and intractable infections caused by various resistant organisms pose a great problem now. In view of this, we determined the bactericidal effects of 5 commonly used disinfectants using as the test strains Xanthomonas maltophilia and Serratia marcescens, chosen among other GNR since they often cause nosocomial infections. Regarding the bactericidal activities against X. maltophilia and S. marcescens, both sensitive strains and resistant strains were killed within 20 s of exposure to povidone-iodine and sodium hypochlorite. With chlorhexidine, 1 strain each of both species was not killed within 10 min of exposure at a concentration of 0.2%. Both sensitive strains and resistant strains of X. maltophilia were killed within 20 s of exposure to benzalkonium at 0.02%, while a concentration of 0.1% was required for benzalkonium to kill S. marcescens within 20 s. With Tego-51, both sensitive strains and resistant strains of X. maltophilia were killed within 20 s at 0.02%, while 1 strain of S. marcescens was not killed within 20 s at a concentration of 0.1%. In the use of disinfectants, comparative bactericidal effects of various disinfectants against clinical isolates should be taken into consideration.

Anti-Infective Agents, Local↗

PubMed1997

Clinical features and virological findings in children with primary human herpesvirus 7 infection.

OBJECTIVE: To elucidate clinical features of patients with primary human herpesvirus 7 (HHV-7) infection and serologic and virologic findings between HHV-7 and human herpesvirus 6 (HHV-6). MATERIALS AND METHODS: During a 19-month observation period, 71 infants and children (35 boys and 36 girls with a mean age of 14.5 months [range, 1 month to 48 months]) who had acute febrile respiratory illness with or without skin rash were examined clinically and virologically. Heparinized blood samples were used for isolation of HHV-6 and HHV-7 and detection of both virus DNA sequences by a nested polymerase chain reaction amplification. Both virus antibody activities were measured by an indirect immunofluorescent assay. RESULTS: HHV-7 infection was observed in 15 (6 boys and 9 girls with a mean age of 12.9 months [range, 7 months to 27 months]), 1 of 10 with upper respiratory infection and 14 (28%) of 50 with febrile exanthem, whereas HHV-6 infection was in 22 (44%) of the 50. Fever (37.5 degrees C) was observed in all 15, with an average maximum body temperature of 38.7 degrees C (range, 37.6 degrees C to 39.8 degrees C), which persisted for 2.9 days (range, 1 to 5 days). Papular, macular, or maculopapular rash was observed in 14 (93%) of the 15, which appeared on day 2.9 of fever (range, days 2 to 5) on the face, trunk, and extremities and persisted for 2.7 days (range, 1 to 5 days). A convulsive seizure that persisted for a few minutes developed in 1 patient on the first day of elevation of fever. HHV-6 antibody was demonstrated in 13 (87%), and a simultaneous significant increase to HHV-6 antibody titers was observed in 8 (53%) of the 15 during primary HHV-7 infection. HHV-7 and HHV-6 DNAs were almost always detected in mononuclear cells (MNCs) during acute and convalescent phases, whereas HHV-7 DNA was positive in some plasma samples obtained during the acute phase of the disease. CONCLUSIONS: Primary HHV-7 infection occurred somewhat later than HHV-6, which was confirmed by the isolation of HHV-7 from blood and/or seroconversion to the virus. Clinical features of a virologically confirmed patient with primary HHV-7 infection were comparable with those of primary HHV-6 infection. Preexisting HHV-6 antibody increased significantly in the half of patients with primary HHV-7 infection. HHV-7 DNA was detected in peripheral blood MNCs and plasma in the acute phase and persisted in MNCs thereafter.

Antibodies, Viral↗

PubMed1997

[Reproductive and developmental toxicity studies of taltirelin hydrate (1) fertility study in rats by oral administration].

Fertility study of taltirelin hydrate, a thyrotropin releasing hormone analogue, was carried out in Wistar rats. Taltirelin hydrate was orally administrated at a dose of 0 (control), 0.15, 1.5, or 15 mg/kg. Male rats were given the drug from 63 days before mating to the day before autopsy (total of 121 days), and female rats were treated from 14 days before mating to day 7 of gestation. The females were sacrificed on day 21 of gestation and pregnancy outcome was determined. In the 15 mg/kg group, wet dog shaking behavior and hyperlocomotion were observed in males and females, and the food consumption was slightly decreased in male rats. These changes induced by taltirelin hydrate were not found in the 0.15 and 1.5 mg/kg groups. No adverse effects of taltirelin hydrate on reproductive function were detected in any groups. In the fetal examination, taltirelin hydrate had no teratogenic, lethal, or growth retardation effects in any groups. These results show that the no-toxic dose levels of taltirelin hydrate are 1.5 mg/kg for general toxicity in parent animals, and 15 mg/kg for reproductive function of parent animals and for development of their fetuses.

Administration, Oral↗

PubMed1997

[Reproductive and developmental toxicity studies of taltirelin hydrate (2) teratogenicity study in rats by oral administration].

Teratogenicity study of taltirelin hydrate, a thyrotropin releasing hormone analogue, was carried out in Wistar rats. Female rats were orally given taltirelin hydrate at a dose of 0 (control), 0.15, 1.5, or 15 mg/kg from day 7 to day 17 of gestation. Twenty-seven female rats in each group were sacrificed on day 21 of gestation and their fetuses were examined. The remaining 13 female rats in each group were allowed to deliver spontaneously and their newborns were examined. In the 15 mg/kg group, the dams (P) showed wet dog shaking behavior and hyperlocomotion. No adverse effect of taltirelin hydrate on the body weight gain, food consumption, water intake, and reproductive performance was observed in this group. In the 0.15 and 1.5 mg/kg groups, taltirelin hydrate did not show any adverse effects. In F1 generation groups, taltirelin hydrate had no teratogenic, lethal, or growth retardation effects in any groups. There were also no adverse effects of taltirelin hydrate on postnatal development, emotionality, coordinated activity, sensitivity, learning ability, and reproductive performance of F1 offspring, and development of F2 fetuses. These results show that the no-toxic dose levels of taltirelin hydrate are 1.5 mg/kg for general toxicity in dams, and 15 mg/kg for reproductive function of dams (P) and for development of F1 generation.

Administration, Oral↗

PubMed1997

[Reproductive and developmental toxicity studies of taltirelin hydrate (3) teratogenicity study in rabbits by oral administration].

Teratogenicity study of taltirelin hydrate, a thyrotropin releasing hormone analogue, was carried out in Japanese white rabbits. Female rabbits were orally given taltirelin hydrate at a dose of 0 (control), 0.15, 1.5, or 15 mg/kg from day 6 to day 18 of gestation. Females were sacrificed on day 29 and their fetuses were examined. Neither death nor adverse effects on food consumption in dams were found in any dose groups. In the 15 mg/kg group, rapid breathing and decreased body weight gain in dams were temporally observed. No adverse effect of taltirelin hydrate on reproductive function was detected in any groups. In the fetal examination, taltirelin hydrate had no teratogenic, lethal, or growth retardation effects in any groups. These results show that the no-toxic dose levels of taltirelin hydrate are 1.5 mg/kg for general toxicity in dams, and 15 mg/kg for reproductive function of dams and for development of their offspring.

Administration, Oral↗

PubMed1997

[Reproductive and developmental toxicity studies of taltirelin hydrate (4) perinatal and postnatal study in rats by oral administration].

Perinatal and postnatal study of taltirelin hydrate, a thyrotropin releasing hormone analogue, was carried out in Sprague-Dawley rats. Female rats were given taltirelin hydrate at a dose of 0 (control), 0.15, 1.5, or 15 mg/kg from day 17 of gestation to day 20 after delivery. All pregnant rats were allowed to deliver spontaneously and their offspring were examined. In the 15 mg/kg group, the dams showed the central nervous effects such as wet dog shaking during gestation periods. No adverse effect of taltirelin hydrate on the body weight gain, food consumption and reproductive performance was observed in this group. In the 0.15 and 1.5 mg/kg groups, the drug did not have any adverse effects. Taltirelin hydrate did not have any adverse effects on viability, growth, physical differentiation, functional and behavioral development (coordinated activity, auditory function, emotionality, learning ability, and spontaneous motor activity), and reproductive performance of F1 offspring, and development of F2 fetuses. These results show that the no-toxic dose levels of taltirelin hydrate are 1.5 mg/kg for general toxicity in dams, and 15 mg/kg for reproductive function of dams and for their offspring.

Administration, Oral↗

PubMed1997