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Biomedical subjects

K Obara

Publications and source records attributed to K Obara.

At least 19 recordsLinked to original sources
PubMed1999

Evidence for beta3-adrenoceptor subtypes in relaxation of the human urinary bladder detrusor: analysis by molecular biological and pharmacological methods.

The purpose of the present study was to confirm the presence of beta3-adrenoceptor subtype in the relaxation of human urinary bladder detrusor tissue by reverse transcription-polymerase chain reaction (PCR); direct sequencing of the PCR product, in situ hybridization; and isometric contraction. Using reverse transcription-PCR, the mRNAs of three receptor subtypes (beta1, beta2, and beta3) were expressed in the human urinary bladder detrusor tissue. Direct sequencing of the PCR product of the above beta3-adrenoceptor revealed no mutation in the amplified regions. In situ hybridization with digoxygenin-labeled oligonucleotide probe revealed the presence of the mRNA of beta3-adrenoceptor subtype in the smooth muscle of the urinary bladder. The relaxant effects of isoproterenol (a nonselective beta-adrenoceptor agonist); ZD7114, BRL37344, and CGP12177A (putative selective beta3-adrenoceptor agonists); and SR59230A (a putative selective beta3-adrenoceptor antagonist) were tested using an isometric contraction technique. Isoproterenol in either the presence or absence of both atenolol (a beta1-adrenoceptor-selective antagonist) and butoxamine (a beta2-adrenoceptor-selective antagonist) revealed a relaxant effect on the carbachol-induced contraction of the human urinary bladder detrusor. Both BRL37344 and CGP12177A also revealed relaxant effects on the human urinary bladder detrusor, but ZD7114 did not elicit any relaxation. These results suggest that beta3-adrenoceptor may have some role in urine storage in the human urinary bladder.

Adrenergic beta-Agonists↗
PubMed1999

Pharmacological and molecular biological evidence for ETA endothelin receptor subtype mediating mechanical responses in the detrusor smooth muscle of the human urinary bladder.

The aim of this study was to characterize endothelin receptor subtypes of the detrusor muscle of the human urinary bladder. The receptor subtypes mediating endothelin (ET)-1-induced activity in the human detrusor smooth muscles have been characterized using isometric contraction and reverse transcription-polymerase chain reaction (RT-PCR). ET-1 (a non-selective ET receptor agonist; 10(-10) M to 10(-6) M) exhibited concentration-dependent contractions in human urinary bladder with a plateau at concentrations above 3x10(-7) M. Neither IRL1620 nor sarafotoxin S6c (both ETB-selective agonists; 10(-10) M to 10(-6) M) elicited contractile activity in the human urinary bladder detrusor smooth muscle. FR139317 (an ETA-selective antagonist; 10(-7) M to 10(-5) M) produced a marked shift to the right of the ET-1 concentration-response curve in human urinary bladder detrusor smooth muscle (from the Schild plot TpA2=7.96; slope=0.95). In contrast, RES701-1 (an ETB-selective antagonist; 10(-7) M to 10(-5) M) had no effect on the ET-1 concentration-response curve. RT-PCR revealed positive amplification of ETA receptor mRNA fragment, but not ETB. These results indicate that the ET-1-induced contractile effects of urinary bladder detrusor smooth muscle seem to be mediated mainly by the ETA receptor, not by the ETB receptor.

Aged↗
PubMed1998

Analysis of peripheral blood mononuclear cell stimulated with pyruvate dehydrogenase complex, T-cell receptors from patients with primary biliary cirrhosis.

Progressive destruction of the intrahepatic bile ducts in patients with primary biliary cirrhosis (PBC) is thought to be mediated by cytotoxic T cells which recognize certain epitopes, such as the pyruvate dehydrogenase complex (PDC). To clarify the T-cell repertoire in PBC, we analyzed T-cell receptor (TCR) Vbeta-chain messages expressed in peripheral blood mononuclear cells (PBMCs) stimulated with PDC and in liver biopsy specimens. PBMCs from 12 PBC patients and 6 healthy controls were examined. The TCR Vbeta repertoires of unstimulated PBMCs and PBMCs stimulated with PDC purified from bovine heart were analyzed, using the reverse transcriptase-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism (SSCP). Liver biopsy specimens from 5 PBC patients were also analyzed. In the PBC patients, several different T-cell clones, some of which showed the same mobility, were evident in both the PDC-stimulated and unstimulated PBMCs, as demonstrated by SSCP analysis. In addition, TCR clonality of infiltrating lymphocytes in the liver was also observed in PBC patients, showing common clonal T-cell accumulation with that seen in PBMCs stimulated with PDC. These data indicate that common clonal T-cell accumulation specific for PDC may be present in both peripheral PBMCs and the liver of patients with PBC.

Case-Control Studies↗
PubMed1998

A case with subcapsular hematoma of the liver following laparoscopic cholecystectomy.

Laparoscopic cholecystectomy (LC) is a safe and less traumatic procedure for benign cholecystic disease. However there have been some complications peculiar to LC. Here we report a case with postoperative subcapsular hematoma of the liver after LC. A 78-year-old woman was admitted with acute cholecystitis due to cholecystolithiasis. She underwent LC and liver dysfunction was noted after the operation. Ultrasonography (US) and computed tomography (CT) showed subcapsular hematoma of the liver. It was supposed that it had happened to occur during the operation, but we could not make the cause clear. The patient was improved with only conservative therapy and discharged. It is important to handle the forceps carefully close to the liver in the laparoscopic surgery in order to prevent this complication. We report a case with subcapsular hematoma of the liver following LC with a review of the literature.

Aged↗
PubMed1998

Localization of P2Y1 purinoceptor transcripts in the rat penis and urinary bladder.

PURPOSE: The aim of this study was to determine the expression and localization of the P2Y1 purinoceptor mRNA in rat penis and urinary bladder using reverse transcription polymerase chain reaction (RT-PCR), northern blotting and in situ hybridization (ISH). MATERIALS AND METHODS: RT-PCR: First strand cDNA was prepared from rat penis and urinary bladder dome total RNA and used for PCR with primers designed to amplify fragments of the P2Y1 purinoceptor cDNA sequence. Northern blotting: PCR products were subcloned into the pGEM-5Zf(+) plasmid vector, sequenced and random primer labeled using 32p. Labeled probe was hybridized. ISH: Digoxigenin labeled cRNA probes were synthesized by in vitro transcription. RESULTS: P2Y1 purinoceptor mRNA was detected by RT-PCR analysis in both rat penis and urinary bladder. RNA blotting using a P2Y1 purinoceptor cDNA probe revealed a single transcript of 4.2kb in both tissues. This band was the same size as that expressed by the heart, which contains high levels of P2Y1 purinoceptor (Burnstock, G.: Physiological and pathological roles of purines: an update. Drug. Dev. Res., 28: 195, 1993). By ISH, P2Y1 purinoceptor mRNA was localized in detrusor smooth muscle cells and blood vessels in urinary bladder. In penis, positive signals were detected in endothelial cells which line the lacunar space and blood vessels. No hybridization was seen in corpus cavernosum smooth muscle cells and urethra. CONCLUSION: These results indicate that mRNAs for P2Y1 purinoceptor are expressed in detrusor smooth muscle cells and blood vessels of rat urinary bladder. However, in penis, this receptor is expressed in endothelial cells which lines the lacunar space and blood vessels, but not expressed in corpus cavernosum smooth muscle cells and urethra.

Animals↗
PubMed1997

Effects of hypoxia on [Ca2+]i, pHi and myosin light chain phosphorylation in guinea-pig taenia caeci.

1. Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mM)-induced contracture to approximately 10% of the control value in guinea-pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC-P1), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle. 2. [Ca2+]i, measured using the ratiometric fluorescent dye fura-2, increased when 40 mM KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8%. There were no detectable decreases in [Ca2+]i during hypoxia. 3. MLC-Pi, measured using isoelectric focusing-polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9% of the total LC20 in Ca(2+)-free PSS to a peak value of 51% in 40 mM KCl-PSS. The steady-state value in hypoxia of 43% was not significantly different from that in control oxygenated conditions at the same point in time. 4. pHi, measured using the ratiometric fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF), under quiescent conditions (Ca(2+)-free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production. 5. As [Ca2+]i and MLC-Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea-pig taenia caeci appears to be directly related to energy limitation rather than any oxygen-sensing mechanism.

Animals↗
PubMed1997

Plasma endothelin-1 level in patients with renovascular hypertension - does the kidney with stenosis of the renal artery upregulate production of endthelin-1?

PROBLEM: To examine the value of plasma endothelin-1 (ET-1), we measured the level of plasma ET-1 activity of peripheral venous blood and selective renal venous blood in renovascular hypertension patients, and compared that activity with corresponding renin activity and split renal function. METHODS: ET-1 level, renin activity in selective renal venous blood and peripheral venous blood, and 99mTc-dimercaptosuccinic acid renal uptake as a split renal function test were measured in 11 patients (mean 42.1 years old) with renovascular hypertension (RVH) and 6 patients with both renal cell cancer (RCC) and essential hypertension. RESULTS: 1. In patients with RVH, resting peripheral venous plasma ET-1 ranged from 0.6 to 8.1 (mean 4.07) pg/ml and was higher than the normal level (p <0.01). However, the renal vein ET-1 ratio was nor correlated with the renal vein renin ratio. 2. In patients with RCC, resting peripheral venous plasma ET-1 was not different from the normal level. There was no step-up of plasma ET-1, or renin among renal veins, or the proximal and distal parts of the interior vena cava. 3. Both renal vein renin ratio and renal vein ET-1 ratios were inversely correlated with t99mTC-dimercaptosuccinic acid renal uptake as a split renal function examination in patients with RVH. 4. The peripheral plasma ET-1 level was correlated with the degree of stenosis of the renal artery in patients with RVH of a unilateral lesion, but not bilateral lesions. CONCLUSION: These results suggest that ET-1 may take part in the hypertensive mechanisms of RVH in addition to the renin-angiotensin system, but its significance in RVH still remains to be examined.

Adolescent↗
PubMed1997

Specific attenuation of the pressure-induced contraction of rat cerebral artery by herbimycin A.

In order to determine whether protein tyrosine kinase mechanisms are involved in pressure-induced contraction, we compared effects of three structurally unrelated tyrosine kinase inhibitors and orthovanadate, a tyrosine phosphatase inhibitor, on the pressure-induced contraction of the posterior cerebral artery isolated from rats. The change in vessel diameter was continuously measured with a width analyzer. Herbimycin A inhibited the pressure-induced contraction, while it only slightly inhibited contractions produced by potassium chloride or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (U46619). Genistein inhibited not only the pressure-induced contraction but also the U46619-induced one. Tyrphostin 23 significantly attenuated contractions in response to three different stimuli, i.e., pressure, potassium chloride and U46619. Orthovanadate potentiated the pressure-induced contraction. These results suggest that herbimycin A is a specific and potent inhibitor of the pressure-induced contraction and that a protein tyrosine kinase mechanism may play an important role in the genesis of the pressure-induced contraction of the rat cerebral artery.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Cerebral hemodynamics in patients with moyamoya disease and in patients with atherosclerotic occlusion of the major cerebral arterial trunks.

To determine the difference in cerebral hemodynamics between Moyamoya disease and atherosclerotic occlusion of the major cerebral arterial trunks, we measured local cerebral blood flow (CBF) and local CO2 reactivity (CO2R) by xenon-enhanced computed tomography (CT). A total of 11 adult patients with Moyamoya disease (mean age, 39.6 +/- 7.8 years) and eight patients with atherosclerotic occlusion of the major arterial trunks (mean age, 62.4 +/- 15.4 years) were studied. Regions of interest were frontal, temporal and occipital cortex, caudate, putamen and thalamus in each hemisphere. In patients with Moyamoya disease, local CBF values in the internal carotid artery territory (frontal and temporal cortex, caudate, putamen) were significantly higher than those in the occluded side of patients with atherosclerotic occlusion. Local CO2R values in the caudate and putamen were significantly higher than those in the occluded side of patients with atherosclerotic occlusion. These results suggest that the cerebral hemodynamics of Moyamoya disease differ from those of atherosclerotic occlusion of the major cerebral arterial trunks, and may be a result of the abundant collateral circulation through basal 'Moyamoya' vessels.

Adult↗
PubMed1997

Vanadate oxidation activates contraction in skinned smooth muscle without myosin light chain phosphorylation.

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.

Animals↗
PubMed1997

Enhanced interleukin-8 production by cultured Chang liver cells subjected to ethanol exposure and subsequent stimulation with tumor necrosis factor-alpha.

We investigated the production of interleukin-8 (IL-8) and chemotactic activity released from Chang liver cells subjected to long-term treatment with ethanol (Et) and subsequent stimulation with tumor necrosis factor-alpha (TNF-alpha). Chang liver cells were cultured in the presence of 5, 50 or 100 mmol/l Et for 4 weeks and then treated with recombinant TNF-alpha (1, 10, 100 U/ml). The culture supernatants were assayed for IL-8 using a sandwich ELISA and chemotactic activity was measured using a chemotactic chamber. Total RNA was also extracted from these cells and IL-8 mRNA was assayed by RT-PCR. In addition, TNF-receptor expression on the Et-treated cells was analyzed by flow cytometry. IL-8 levels in supernatants of cells stimulated with 100 U/ml of TNF-alpha for 48 h rose significantly with increasing concentrations of Et and values obtained were as follows: 4,918 +/- 244.4 pg/ml at 0 mmol/l Et, 5,335 +/- 266.2 pg/ml at 5 mmol/l Et, 8,726 +/- 873.4 pg/ml at 50 mmol/l Et and 9,134 +/- 866.0 pg/ml at 100 mmol/l Et. The chemotactic activity also increased with increasing concentrations of Et and was almost completely suppressed by anti-IL-8 antibody. Using semiquantitative analysis of radioactivity of IL-8 mRNA using a 32P gamma ATP-labeled primer for IL-8 mRNA, Et-treated cells were shown to have markedly higher levels of radioactivity than untreated cells. In addition, TNF-receptor expression was significantly higher in cells treated with 100 mmol/l Et. These data suggest that long-term Et treatment of Chang liver cells stimulated with TNF-alpha may enhance transcription of the IL-8 gene with up-regulation of the TNF receptor.

Antibodies↗
PubMed1997

Cold snare excision is a safe method for diminutive colorectal polyps.

Cold snare excision (CSE) has proved to be an effective method for the destruction of diminutive polyps of the colon and rectum. We investigated the correlation between polyp size and bleeding time at the resected end after CSE, and also an appropriate measuring method using CSE. Eighty patients with single polyps were examined. Each polyp was identified as being 5 mm in diameter or smaller using the open-biopsy forceps technique (OBFT). The size of the polyp was calculated using our measuring system (SMS). Of the polyps identified as being 5 mm in diameter or less using OBFT, 15% were 6 mm or more using the SMS. CSE was performed for each polyp, and the time taken for the hemostasis (bleeding time of the CSE; BTCSE) was determined. In seventy-seven polyps that were 6 mm or smaller in SMS, a correlation was noted between SMS and BTCSE. In three polyps that were 7 mm or more by SMS, hemostasis took 10 min or more after CSE, and required electrocoagulation. These results suggest that CSE is a safe method for the removal of polyps determined to be 6 mm in diameter or smaller using the SMS.

Aging↗
PubMed1997

Retroperitoneal laparoscopic adrenalectomy for functioning adrenal tumors: comparison with conventional transperitoneal laparoscopic adrenalectomy.

PURPOSE: We attempted to confirm the possibility and feasibility of laparoscopic adrenalectomy via the retroperitoneal approach, and to compare results of the transperitoneal and retroperitoneal approaches. MATERIALS AND METHODS: Three men and 8 women (mean age 39.6 years) with functioning adrenocortical tumors (primary aldosteronism in 5 and Cushing's syndrome in 6) underwent laparoscopic adrenalectomy via the retroperitoneal approach using a balloon dissection technique and a newly developed ultrasonic aspirator. Results were compared to those of 27 cases of transperitoneal laparoscopic adrenalectomy. RESULTS: Although the retroperitoneal approach was successful in all 5 patients with primary aldosteronism, it succeeded in only 2 of the 6 cases of Cushing's syndrome. In 3 Cushing's syndrome cases the retroperitoneal approach was changed to the transperitoneal laparoscopic approach due to difficulty in exploration. Open laparotomy was required in 1 case of left Cushing's syndrome because of an inadvertent pancreatic injury. Subcutaneous emphysema developed in 6 patients without hypercapnia or prolonged postoperative symptoms. Mean operative time and blood loss, and time to oral intake and ambulation were 248.3 minutes, 151.4 ml., and 1.55 and 2 days, respectively. There was no difference between retroperitoneal and conventional transperitoneal laparoscopic adrenalectomy in regard to these factors or to convalescence. CONCLUSIONS: Retroperitoneal laparoscopic adrenalectomy is feasible for primary aldosteronism. However, Cushing's syndrome is presently a much more difficult indication than primary aldosteronism for this new operative technique.

Adrenal Cortex Neoplasms↗
PubMed1997

[Locations of crossover breakpoints within the CMT 1 A-REP repeat in patients with hereditary neuropathy with liability to pressure palsy--detection by recombinant chromosome-specific polymerase chain reaction].

The crossover breakpoints for hereditary neuropathy with liability to pressure palsies (HNPP) are located in the CMT 1 A-REP repeat flanking a 1.5 Mb region of chromosome 17p11.2-12. Three-unrelated HNPP patients have breakpoints in a 3.2 kb novel fragment of recombinant chromosome in the CMT 1 A-REP repeat. The fragment is detected by recombinant chromosome-specific polymerase chain reaction (PCR). Further analysis of PCR demonstrated a 1.2 kb novel junction fragment in the 3.2 kb region in all HNPP patients. The precise mapping of the breakpoints indicated that 2 patients were localized in a 700 bp interval of the 1.2 kb fragment 1.3kb telomeric to a marier transposon-like element (Kiyosawa and Chance, HMG 5: 745-753, 1996), suggesting that this region is a recombinational "hotspot" within the CMT 1 A-REP repeat for HNPP as well as CMT 1 A.

Adult↗
PubMed1996

Local cerebral blood flow in motor neuron disease: correlation with clinical findings.

Local cerebral blood flow (CBF) measured by xenon-enhanced CT was correlated with the clinical findings in 21 patients with motor neuron disease (mean +/- SD age, 60 +/- 10 years). In 11 patients, CBF was also measured after intravenous injection of 2 mg thyrotropin releasing hormone (TRH). There were no significant differences in CBF of the cerebral cortex, basal ganglia, thalamus, and subcortical white matter with respect to age, duration of illness, or presence (n = 9) or absence (n = 12) of bulbar palsy. In patients with upper motor neuron disturbance (n = 10), however, local CBF in the frontal cortex was significantly lower (p < 0.05) than in those without it (n = 11). Within the frontal cortex, CBF reduction was marked in the upper posterolateral part (p < 0.05) that included the motor and premotor areas. Intravenous administration of TRH did not significantly alter the local CBF in any of the brain regions examined. An additional patient with motor neuron disease and severe dementia showed marked CBF reduction in the frontal lobe, which was in common with but much greater than the reduction in those exhibiting subcortical dementia (e.g., progressive supranuclear palsy). We conclude that in motor neuron disease patients with upper motor neuron involvement, the selective reduction of CBF in brain regions that include primary motor and premotor areas may reflect functional disturbance or neuronal degeneration in these regions. The ameliorating effect of TRH in patients with motor neuron disease, if any, appears not to be related to increases in local CBF or activation of brain function.

Aged↗
PubMed1996

Interleukin-6 induces proliferation of rat hepatocytes in vivo.

BACKGROUND/AIMS: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo. METHODS: IL-6 was injected at a dose of 200 micrograms/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using 3H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR. RESULTS: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased 3H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing 3H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase 3H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration. CONCLUSIONS: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.

Animals↗