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Biomedical subjects

M Ohnishi

Publications and source records attributed to M Ohnishi.

At least 109 records · Page 6Linked to original sources
PubMed1998

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells.

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (alpha, beta-1, beta-2, beta-3, beta-4 and beta-5), PP2C alpha was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2C alpha was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2C alpha is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.

Animals↗
PubMed1998

Mutational analysis of the domain structure of mouse protein phosphatase 2Cbeta.

The structures of five distinct isoforms of mammalian protein phosphatase 2Cbeta (PP2Cbeta-1, -2, -3, -4 and -5) have previously been found to differ only at their C-terminal regions. In the present study, we performed mutational analysis of recombinant mouse PP2Cbeta-1 to determine the functional domains of the molecule and elucidate the biochemical significance of the structural differences in the isoforms. Differences in affinity for [32P]phosphohistone but not for [32P]phosphocasein were observed among the five PP2Cbeta isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cbeta-1, caused a 35% loss of activity against [32P]phosphohistone but no loss of activity against [32P]phosphocasein. Deletion of up to 78 amino acids from this end did not cause any further alteration in activity, whereas deletion of 100 amino acids totally eliminated the activity against both [32P]phosphohistone and [32P]phosphocasein. On the other hand, deletion of 11 amino acids from the N-terminal end caused a 97% loss of enzyme activity, and further deletions caused a total loss of activity. Substitution of any of the six specific amino acids among 16 tested in this study, which were located among the 250 N-terminal residues, caused 98-100% loss of enzyme activity. Among these amino acids, three (Glu-38, -60 and -243) have recently been reported to be essential for the binding of metal ions in the catalytic site of the PP2C molecule [Das, Helps, Cohen and Barford (1996) EMBO J. 15, 6798-6809]. These observations indicate that PP2Cbeta is composed of at least two distinct functional domains, an N-terminal catalytic domain of about 310 amino acids and the remaining C-terminal domain, which is involved in determination of substrate specificity.

Animals↗
PubMed1998

Selective inhibition of Ras interaction with its particular effector by synthetic peptides corresponding to the Ras effector region.

Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.

Adenylyl Cyclases↗
PubMed1998

Purification and characterization of procytotoxin of Pseudomonas aeruginosa. Dimer to monomer conversion of protoxin by proteolytic activation.

Cytotoxin of Pseudomonas aeruginosa is a cytolytic toxin that forms a pore on the target membrane by oligomerizing into a pentamer. This toxin is produced as an inactive precursor (proCTX) and is converted to an active form by proteolytic cleavage at the C terminus. We purified proCTX to apparent homogeneity and characterized it in a comparison with the active toxin. ProCTX bound to the erythrocyte membrane but did not form an oligomer on the membrane, hence the lack of hemolytic activity in proCTX. Circular dichroic experiments showed that active and proCTX have similar beta-sheet dominant structures. Intrinsic fluorescence analysis indicated that a molecule-buried tryptophan residue(s) of proCTX was exposed to the surface of the molecule as a result of conversion to the active form. In analytical gel filtration, chemical cross-linking, and analytical ultracentrifugation experiments, dimer to monomer conversion occurred with proteolytic activation.

Animals↗
PubMed1998

Wound healing of scleral self-sealing incision: a comparison of ultrasound biomicroscopy and histology findings.

PURPOSE: The goal of this study was to compare the morphologic findings of wound healing in scleral self-sealing incisions using ultrasound biomicroscopy and histology. METHODS: Using a slit-knife, we made a scleral self-sealing incision in the rabbit eye. At various time points postoperatively, ultrasound biomicroscopy was performed to evaluate wound healing; the eyes then were enucleated and studied histologically. We also performed ultrasound biomicroscopy at various time points postoperatively in patients who received a scleral self-sealing incision during cataract surgery. RESULTS: In rabbit eyes, on days 1 and 2 postoperatively, we detected the scleral wound; thereafter, detection became increasingly difficult. On day 7 postoperatively, the wound was undetectable. By light-microscopic observation, the scleral wound was open at 1 day postoperatively. On day 2 postoperatively, fibrovascular tissue barely extended into the wound; on day 5 postoperatively, connective tissue extended through the full thickness of the wound. On day 7 postoperatively, the connective tissue became dense and aligned with the lamella. In human eyes, using ultrasound biomicroscopy, the scleral incision was detectable until 5 days postoperatively, but undetectable at 7 days postoperatively. CONCLUSIONS: Ultrasound biomicroscopy demonstrates the stages of wound healing of scleral self-sealing incisions. We believe that careful observation is necessary for approximately 7 days following self-sealing incision cataract surgery.

Animals↗
PubMed1998

Characterization of the subsite structure of the beta-glucosidase from Aspergillus niger, an aspect of the mechanism of carbohydrate recognition.

Steady-state kinetics on the reaction catalyzed by the beta-glucosidase of Aspergillus niger were carried out to evaluate the kinetic parameters, Km and ko, for phenyl beta-D-glucosides. The ko/Km values, which may relate to productive binding at subsites, were found to correlate with the substituent constant pi (hydrophobicity), suggesting that subsite 2 has a hydrophobic character. A "hydrophobic-driven" mechanism is considered to contribute to the productive E-S complex for recognition of the substrate.

Aspergillus niger↗
PubMed1998

Comparison of the acute effects of a selective endothelin ETA and a mixed ETA/ETB receptor antagonist in heart failure.

OBJECTIVE: Both the selective endothelin (ET) ETA receptor and mixed ETA/ETB receptor antagonists improve haemodynamics in patients and experimental models with congestive heart failure (CHF) and reduce the mortality in CHF rats. However, it remains unclear which of these antagonists is superior in the treatment of CHF. In addition, there is little information as to whether these ET receptor antagonists contribute to the neuroendocrine regulation and body fluid balance. We therefore investigated the cardiorenal and neurohumoral benefits of selective ETA receptor and mixed ETA/ETB receptor antagonists in CHF. METHODS: We administered acutely either the selective ETA receptor antagonist FR139317 (FR, n = 6, 1 and 10 mg/kg) or the mixed ETA/ETB receptor antagonist TAK-044 (TAK, n = 6, 1 and 3 mg/kg) to conscious dogs with CHF induced by rapid right ventricular pacing for ten days. RESULTS: Both FR and TAK decreased the cardiac pressures and the plasma atrial natriuretic peptide level and increased the cardiac output and urinary sodium excretion. FR increased the urine flow rate in association with an increased glomerular filtration rate and renal plasma flow, while TAK reduced the plasma aldosterone level. Neither antagonist increased the plasma renin activity or norepinephrine levels. CONCLUSIONS: These ETA/ETB antagonist. However, the long-term administration of a mixed ETA/ETB receptor antagonist would improve not only the haemodynamics but also prevent fluid retention by suppressing secretion of aldosterone during the treatment of chronic CHF.

Aldosterone↗
PubMed1998

Inhibition in vitro linoleic acid peroxidation and haemolysis by caffeoyltryptophan.

Antioxidant activities of caffeoyltryptophan were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging system, the superoxide anion generation system and the superoxide anion-mediated linoleic acid peroxidation system. At 10 microM, caffeoyltryptophan showed greater scavenging activity on DPPH than dl-alpha-tocopherol or ascorbic acid. DPPH radical scavenging activity of caffeoyltryptophan increased dose-dependently at concentrations ranging from 1 to 50 microM; 1 mol of caffeoyltryptophan reacted with ca 4 mol of radical. Caffeoyltryptophan caused 80% inhibition of superoxide anion generation at 50 microM. The inhibitory activity of caffeoyltryptophan was as strong as that of 5-caffeoylquinic acid. Caffeoyltryptophan inhibited the formation of conjugated diene from linoleic acid. The inhibitory activity increased in the order caffeic acid < 5-caffeoylquinic acid < caffeoyltryptophan < dl-alpha-tocopherol. Effects on the in vitro haemolysis and peroxidation of mouse erythrocytes induced by H2O2 were also examined. Caffeoyltryptophan exhibited strong inhibitory activities; Tryptophan was ineffective in these systems. These data suggest that caffeoyltryptophan may be a natural antioxidant in the human diet and, as such, may intervene in toxicological processes that are mediated by radical mechanisms.

Animals↗
PubMed1998

Interleukin-6 spillover in the peripheral circulation increases with the severity of heart failure, and the high plasma level of interleukin-6 is an important prognostic predictor in patients with congestive heart failure.

OBJECTIVES: We 1) evaluated whether interleukin-6 (IL-6) is produced in the peripheral circulation in patients with congestive heart failure (CHF), 2) estimated the factors for increased IL-6, and 3) clarified the prognostic role of high plasma levels of IL-6 in patients with CHF. BACKGROUND: Although plasma levels of IL-6 have been reported to increase in patients with CHF, and production of IL-6 in endothelial cells and vascular smooth muscle cells has been postulated from in vitro studies, the origin of the increase of IL-6 in CHF remains unknown. Moreover, the prognostic value of a high plasma level of IL-6, independent of classic neurohumoral factors, remains to be elucidated. METHODS: A comparison was made of the plasma levels of IL-6 between the femoral artery and the femoral vein in 13 normal subjects and in 80 patients with CHF. In another study, we measured plasma IL-6 in 100 patients with CHF and follow-up data. RESULTS: Plasma IL-6 levels increased significantly from the femoral artery to the femoral vein in normal subjects and in patients with CHF. Arteriovenous IL-6 spillover in the leg increased with the severity of CHF. Among the hemodynamic variables and the various neurohumoral factors, the plasma norepinephrine (NE) level showed an independent and significant positive relation with the plasma IL-6 level in patients with CHF. Moreover, treatment with beta-adrenergic blocking agents showed an independent and significant negative relation with plasma IL-6 levels. In 100 patients, plasma IL-6 (p < 0.0001), NE (p = 0.0004) and left ventricular ejection fraction (0.015) were significant independent prognostic predictors by Cox proportional hazards analysis. CONCLUSIONS: Our findings indicate that the IL-6 spillover in the peripheral circulation increases with the severity of CHF and that the increase in plasma IL-6 is mainly associated with the activation of the sympathetic nervous system. High plasma levels of IL-6 can provide prognostic information in patients with CHF, independent of left ventricular ejection fraction and plasma NE, suggesting an important role for IL-6 in the pathophysiology of CHF.

Adrenergic alpha-Agonists↗
PubMed1998

Community pharmacy practice in Japan--results of a survey.

OBJECTIVE: To survey the present condition of community pharmacies as future sites for pharmacy students' externship in Japan. METHOD: A questionnaire consisting of 55 questions was sent to 425 graduates from Kobe Gakuin University, School of Pharmacy, who owned or worked in community pharmacies. RESULTS: Of the 85 responders, about half were owners and half employees of pharmacies. Ninety per cent of pharmacy owners operated three and fewer pharmacies. Fifty per cent of pharmacies only dispensed drugs, 32% handled both OTC drugs and dispensing, and 18% handled only OTC drugs. Among the 44 dispensing pharmacies, 16 were one-to-one type pharmacies, 13 were located in front of the big medical institutions, nine dispensed prescriptions from various medical institutions and five were hospital-owned pharmacies. Forty-five per cent of pharmacies employed 1-4 part-time pharmacists and 52% employed 1-4 pharmacist assistants. Thirty-one per cent of prescriptions came from internal medicine departments and the daily number of prescriptions dispensed by each pharmacy was in the range 10-99 for 41% of the pharmacies and 100-199 for 36% of the pharmacies. The average daily number of prescriptions dispersed by each pharmacist was in the range 30-39 for 29% of pharmacies and in the range 20-29 for 22% of pharmacies. Pharmaceutical information was provided at 73% of pharmacies and patients were counselled orally on their medication at 80% of pharmacies. Patients' medication histories were recorded at 88% pharmacies. Only 15% of pharmacies conducted patients' medication counselling at their home, but 34% of pharmacies were planning to start this service. Community pharmacists attended very few professional meetings or continuing education programmes and only 20% of them obtained information through computers. Forty-seven pharmacists out of the 85 obtained their information from medical representatives of pharmaceutical companies and 32 pharmacists through marketing specialists of wholesalers. Ninety per cent of community pharmacists who responded thought that separation of prescribing and dispensing functions would progress further in the future and 50% of them thought positively about the future social status of pharmacists. Most of our graduates who responded were willing to accept pharmacy students from Kobe Gakuin University as externs at their pharmacies. CONCLUSION: Despite the low response rate, our survey suggests that community pharmacy in Japan requires substantial improvement. This is to ensure that pharmacists working in that sector can provide the quality information that is required for the optimum management of patients and that the environment is suitable for pharmacy externships.

Data Collection↗
PubMed1998

Chronic effects of a novel, orally active endothelin receptor antagonist, T-0201, in dogs with congestive heart failure.

Endothelin (ET)-1 increases in plasma during congestive heart failure (CHF). Some ET antagonists improve hemodynamics, suggesting its potential benefits in the treatment of CHF. We examined the acute and chronic effects of a new ET receptor antagonist, T-0201 (Tanabe Seiyaku Co. Ltd., Japan), in CHF. To confirm the in vivo effects of T-0201, we observed the inhibitory effects of T-0201 (1-100 micrograms/kg) on the response of blood pressure to exogenously administered ET-1 (0.75 nmol/kg) in conscious normal dogs. Pretreatment with T-0201 significantly inhibited the ET-1-induced initial hypotension that is mediated by ETB receptors, and attenuated the subsequent hypertension, which is primarily mediated by ETA receptors. Thus, T-0201 at a dose of 100 micrograms/kg not only works as a potent ETA antagonist but also shows antagonist activity for ETB receptors in dogs. To evaluate the chronic therapeutic effects of T-0201, we administered T-0201 (0.3 mg/kg/day; n = 5) orally to dogs with CHF induced by rapid right ventricular pacing (22 days, 270 beats/min) for 15 days, beginning 8 days after pacing. T-0201 significantly prevented the deterioration of cardiorenal function during the development of CHF, expressed as a decrease in cardiac pressure and an increase in cardiac and urine output. These results suggest that chronic antagonism of both ET receptors prevents the progressive exacerbation of CHF.

Animals↗
PubMed1998

Bradycardia and hypotension associated with severe hemorrhage are reversed by morphine given centrally or peripherally in anesthetized rats.

BACKGROUND: Severe simple hemorrhage (blood loss in the absence of tissue damage and nociception) leads to a reflex bradycardia and hypotension. Earlier studies showed that this reflex can be attenuated by prior administration of morphine. However, some patients may receive morphine, e.g., for analgesia after they have suffered severe hemorrhage. The aim of this study was to determine whether an established bradycardia and hypotension could be reversed by morphine. METHODS: Four groups of male Wistar rats (236-258 g) were anesthetized with alphadolone/alphaxalone (16-19 mg x hg x h(-1) intravenously). All groups received a hemorrhage of 40% total blood volume (BV) at 2% BV x min(-1). After the loss of 27% BV, bradycardia and hypotension were established equally in groups I and II and III and IV. Groups I (n=8) and III (n=10) received 0.9% saline (20 microL intracerebroventricularly or 1 mL x kg(-1) intravenously, respectively), whereas groups II (n=10) and IV (n=10) received morphine (10 microg intracerebroventricularly or 0.5 mg x kg(-1) intravenously, respectively). RESULTS: In groups I and III, heart rate and mean arterial blood pressure continued to fall, whereas the bradycardia was completely reversed and the hypotension partly reversed in groups II and IV after treatment with morphine. CONCLUSION: Morphine, administered centrally or peripherally, can reverse the bradycardia and markedly can attenuate the hypotension associated with severe hemorrhage. However, any benefit may be more apparent than real because other studies suggest that mortality may be increased.

Acid-Base Equilibrium↗
PubMed1997

Comparison of the effects of selective endothelin ETA and ETB receptor antagonists in congestive heart failure.

OBJECTIVES: This study was designed 1) to determine the extent to which endogenous endothelin (ET) affects hemodynamic, hormonal and body fluid balance through ETA and ETB receptors in congestive heart failure (CHF); and 2) to assess the therapeutic benefits and adverse effects of ET receptor antagonists for ETA and ETB on cardiorenal and neurohormonal variables. BACKGROUND: ET has two receptors, ETA and ETB, both of which are distributed in various tissues and cells. In vascular beds, ETA receptors mediate vasoconstriction, whereas ETB receptors mediate vasorelaxation. However, ETB receptors also exist in smooth muscle and mediate vasoconstriction. METHODS: We administered either the ETA receptor antagonist FR139317 (FR [n = 8], 1 and 10 mg/kg body weight) or the ETB receptor antagonist RES-701-1 (RES [n = 8], 0.2 and 1.5 mg/kg) to dogs with CHF induced by rapid ventricular pacing. The effects of both antagonists on cardiorenal and hormonal functions were studied. RESULTS: FR decreased cardiac pressures and the plasma atrial natriuretic peptide (ANP) level and increased cardiac output (CO). Urinary flow rate and urinary sodium excretion increased in association with an increase in the glomerular filtration rate and renal plasma flow (RPF). In contrast, RES increased cardiac pressures and decreased CO. It also decreased the plasma aldosterone level and RPF. Neither antagonist affected plasma norepinephrine levels. CONCLUSIONS: Endogenous ETs increase cardiac pressures and the retention of body fluid through ETA receptors in CHF. The vasodilative action through ETB receptors is overall functionally more important than the constrictive action through ETB receptors. ETs may regulate the secretion of ANP and aldosterone. Our findings suggest that selective ETA receptor antagonists have potential therapeutic benefits affecting both hemodynamic variables and diuresis, whereas ETB receptor antagonists have adverse hemodynamic effects, with the possibility of preventing fluid retention through suppression of aldosterone secretion in dogs with CHF.

Analysis of Variance↗
PubMed1997

Morphine blocks the bradycardia associated with severe hemorrhage in the anesthetized rat.

Progressive hemorrhage in the absence of tissue injury produces a biphasic response: an initial tachycardia, vasoconstriction and maintenance of arterial blood pressure by the baroreflex, followed by bradycardia, vasodilatation and hypotension due to the activation of a second 'depressor' reflex. The present study has investigated the effect of morphine (a mu-opioid receptor agonist) on the cardiac chronotropic response to a progressive hemorrhage at 2% total estimated blood volume (BV) min(-1) in the anesthetized rat. In control rats (20 microl saline intracerebroventricularly, i.c.v.) heart period initially decreased significantly (P < 0.05) by a maximum of 5.4 +/- 0.8 ms from a baseline of 147.3 +/- 2.2 ms after a blood loss of 8.3 +/- 1.5% BV, and then increased significantly by a maximum of 43.0 +/- 5.5 ms above the baseline after the loss of 34.5 +/- 1.6% BV. Blood pressure was initially maintained and then fell during the hemorrhage. The increase in heart period was prevented by treatment with morphine (10 microg i.c.v.), and the fall in blood pressure delayed significantly. These effects of morphine were prevented by pretreatment with naloxone (20 microg i.c.v.). Intravenous (i.v.) administration of morphine (10 microg) had no effect on the response to hemorrhage. However, a clinically relevant dose of 0.5 mg x kg(-1) morphine (i.v.) abolished the bradycardia and delayed the fall in blood pressure associated with hemorrhage. These results indicate that morphine, acting on central nervous opioid receptors, can abolish the bradycardia and delay the hypotension associated with progressive hemorrhage, a pattern of response reminiscent of the effects of musculo-skeletal injury on the response to blood loss.

Anesthesia↗
PubMed1997

Attenuation of compensation of endogenous cardiac natriuretic peptide system in chronic heart failure: prognostic role of plasma brain natriuretic peptide concentration in patients with chronic symptomatic left ventricular dysfunction.

BACKGROUND: Patients with congestive heart failure (CHF) have high plasma levels of atrial natriuretic peptide (ANP), mainly from the atrium, and brain natriuretic peptide (BNP), mainly from the ventricle. We examined the prognostic role of plasma BNP in chronic CHF patients in comparison with plasma ANP and other variables previously known to be associated with high mortality. We also evaluated the relationship between mortality and plasma cGMP, a biological marker of ANP and BNP. METHODS AND RESULTS: The study subjects were 85 patients with chronic CHF (left ventricular ejection fraction <0.45) who were followed for 2 years. The plasma levels of ANP, BNP, cGMP, and norepinephrine increased with the severity of CHF. Among plasma levels of ANP, BNP, cGMP, and norepinephrine and clinical and hemodynamic parameters, only high levels of plasma BNP (P<.0001) and pulmonary capillary wedge pressure (P=.003) were significant independent predictors of the mortality in patients with CHF by Cox proportional hazard analysis. Although plasma levels of ANP and BNP were threefold or fivefold higher in nonsurvivors than in survivors, there was no difference in plasma cGMP level between nonsurvivors and survivors. CONCLUSIONS: These findings indicate that plasma BNP is more useful than ANP for assessing the mortality in patients with chronic CHF and that the plasma levels of BNP provide prognostic information independent of other variables previously associated with a poor prognosis. Our findings also suggest that the compensatory activity of the cardiac natriuretic peptide system is attenuated as mortality increases in chronic CHF patients with high plasma levels of ANP and BNP.

Adult↗