Biomedical subjects

Y Masuda

Publications and source records attributed to Y Masuda.

At least 289 records · Page 16Linked to original sources

PubMed1997

Chronotropic response to cardiac sympathetic nerve stimulation in spontaneously hypertensive rats.

In Sprague-Dawley rats, we attempted to develop an experimental model to stimulate the cardiac sympathetic nerve. After transverse thoracotomy under anesthesia, a miniature, bipolar electrode was placed on the right ansa subclavia and it was stimulated electrically. The nerve stimulation at 2, 4, and 8.3 Hz (2 ms in duration, 5 V) increased heart rate by 21.2 +/- 4.19, 34.2 +/- 5.33, and 47.8 +/- 9.25 beats min-1, respectively (p < 0.05). Tetrodotoxin abolished the chronotropic response evoked by nerve stimulation at 4 Hz (p < 0.05). The beta-adrenergic receptor antagonist propranolol abolished the chronotropic response (p < 0.005). These results indicate that the cardiac sympathetic nerve secretes norepinephrine as a neurotransmitter and that the electrical stimuli did not directly stimulate the cardiac tissues. Thus, our preparation is appropriate for stimulation of the cardiac sympathetic nerve. We then determined the extent of the positive chronotropic responses evoked by electrical stimulations of the cardiac sympathetic nerve in spontaneously hypertensive rats (SHR) and compared the results with those in Wistar-Kyoto rats (WKY). At 5-7 weeks of age, the extent of positive chronotropic responses at 2, 4, and 8.3 Hz in SHR was 16.1 +/- 3.5, 25.2 +/- 3.8, 32.2 +/- 4.3 beats.min-1, respectively, and the extent was not different from results in WKY at the same age. At 15-17 weeks of age, the positive chronotropic responses at 2, 4, and 8.3 Hz in SHR were 6.6 +/- 1.6, 15.7 +/- 2.8, and 22.3 +/- 4.0 beats.min-1, respectively, and were significantly less than those in WKY at the same age (p < 0.05). Also, responses in SHR at 15-17 weeks were significantly less than in SHR at 5-7 weeks (p < 0.05). The same degree of positive chronotropic response evoked by peripheral cardiac sympathetic nerve stimulation in both SHR and WKY at 5-7 weeks during the developmental phase of hypertension suggests that the overactivity of the sympathetic nerve known to occur during this phase in SHR may not be produced by the postganglionic nerve fiber per se, but rather by increased activity in the central nervous system. The reduced positive chronotropic response as hypertension is established may indicate that it is developed in the course of a high blood pressure state, but it is not the inherent nature of hypertension.

Animals↗

PubMed1997

Cerebrovascular selectivity and vasospasmolytic action of the novel calcium antagonist (+/-)-(E)-1-(3-fluoro-6, 11-dihydrodibenz[b,e]oxepin-11-yl)-4-(3-phenyl-2-propenyl)-piperazine dimaleate in isolated cerebral arteries of the rabbit and dog.

The cerebrovascular selectivity and vasospasmolytic action of AJ-3941 ((+/-)-(E)-1-(3-fluoro-6, 11-dihydrodibenz[b,e]oxepin-11-yl)-4-(3-phenyl-2-propenyl)-p iperazine dimaleate. CAS 143110-70-7), a new calcium antagonist, were studied in isolated rabbit and dog arterial preparations. In rabbit arterial ring preparations, AJ-3941 dose-dependently inhibited the contractions of various arteries caused by high K(+)-depolarization (high K+) and prostaglandin F2 alpha (PG). The inhibitory potency of AJ-3941 varied in different arteries, in descending order as follows: high K+: basilar > coronary > femoral > renal > mesenteric artery, PG: basilar > coronary > > femoral and renal artery. The median inhibitory concentration (IC50) in the basilar artery was over 40 times lower than that in the mesenteric or femoral artery for which the weakest inhibition in the examined arteries was observed. This selective action of AJ-3941 for cerebral artery was also observed in the frontal and middle cerebral arteries of dogs. The selectivity for the rabbit basilar artery was higher than those of flunarizine and nicardipine. Additionally, the contractile response of the rabbit basilar artery induced by phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), was greater than those of the arteries examined such as the coronary, femoral and mesenteric arteries. The response in the basilar artery was greatly reduced in Ca(2+)-free medium, while this was not the case in other arteries. AJ-3941 as well as H-7, an inhibitor of PKC, potently inhibited PDBu-induced contractile response in the basilar artery in the presence, but not in the absence of Ca2+ in the medium, whereas the existing calcium antagonists, diltiazem and nicardipine, did not inhibit the contractile response in both conditions. These results suggest that the PKC-dependent system which is mediated by influx of extracellular Ca2+ profoundly contributes to the contraction of the cerebral artery and that the cerebroselective-vasodilating effect of AJ-3941 may depend, at least partly, on the inhibition of the PKC-mediated contractile response. In rabbit basilar arteries, AJ-3941 caused a dose-dependent inhibition of the contraction induced by various vasospasmogens, such as endothelin-1 (ET), arachidonic acid, 15-hydroperoxy-eicosatetraenoic acid and the thromboxane A2-mimetic U-46619. Furthermore, when isolated basilar arteries of the dog were perfused intraluminally with AJ-3941 at the concentration that inhibits high K(+)- or PG-induced contraction in the rabbit basilar artery, AJ-3941 effectively antagonized the vasospasm induced by extraluminal application of PG or ET. However, when flunarizine, nicardipine, diltiazem or verapamil was used for intraluminal perfusion of the same preparations, none of these drugs exerted spasmolytic effect. These results indicate that AJ-3941 has cerebrovascular selective-vasospasmolytic action, and consequently is thought to be effective in cerebrovascular disorder such as vasospasm following subarachnoid hemorrhage.

Animals↗

PubMed1997

[The condition of PCBs and PCDFs in the blood of Yusho patients 20 years after the onset].

Blood samples of Yusho and control persons were analyzed for individual congeners of PCDDs, PCDFs, and PCBs by high resolution GC/MS. Concentrations of 2,3,4,7,8-penta-CDF, 1,2,3,4,7,8-hexa-CDF and 2,3,3',4,4',5-hexa-CB in Yusho blood were up to 56 times higher than the corresponding concentrations in the control blood. These high concentrations have persisted for 23 years after the incident. Concentrations of 3,3',4,4',5-penta-CB and 2,3',4,4',5-penta-CB in some Yusho blood were lower than the control blood. In Yusho blood, 2,3,4,7,8-penta-CDF contributed the highest toxicity (TEQ 77-248 ppt in lipid) among the congeners determined and toxic contribution of PCDFs was very large (41-77%) in the chlorinated pollutants. Thirty PCB congeners were identified in the blood of Yusho patients in 1996 by GC/MS. The average total PCB concentration in Yusho blood were 4.9 times higher than that of the controls. Characteristic PCB congeners in Yusho patients were 2,2',3,4,4',5-hexa-CB, 2,3,3',4,4',5-hexa-CB and 2,3,3',4,4',5'-hexa-CB and their concentration ratios to the controls were 8-19.

Adult↗

PubMed1997

[Tissue distribution of methylsulfonyl metabolites derived from Kanechlor 400 in mice].

Kanechlor 400, which caused the "Yusho disease", was i.p. administered to mice and methylsulfonyl (MeSO2) metabolites were investigated with respect to the concentration in liver and lung during 28 days after the administration. Major components were 3- and 4-MeSO2 derivatives from seven PCBs (IUPAC no. #31, #49, #64, #70, #101, #110, #132). In the liver, similar concentration ratio of 3- and 4-MeSO2 derivatives was observed, whereas seven 4-MeSO2 derivatives were selectively retained in the lung. Methylsulfone metabolites of triCB (#31) were rapidly formed and eliminated. The highest concentration of the metabolites in the lung was 4-MeSO2-2, 2', 4', 5-tetraCB. Concentration ratio of MeSO2-CBs to residual PCBs was 1:2.1 in the liver whereas 4.6:1 in the lung 28 days after the administration.

Animals↗

PubMed1997

Comparative study on formation of hydroxy and sulfur-containing metabolites from different chlorinated biphenyls with 2,5-substitution in rats.

2,4',5-Trichlorobiphenyl (TriCB), 2,3',4',5-tetrachlorobiphenyl (TetraCB), 2,2',4',5,5'-pentachlorobiphenyl (PentaCB), and 2,2',3',4',5,5'-hexachlorobiphenyl (HexaCB) were studied with regard to the fecal excretion and tissue distribution of their metabolites after intraperitoneal injection to rats. Major fecal metabolites were 3- and 4-hydroxy and 3- and 4-methylthio derivatives, the substitution ratios depending largely on the degree of chlorination. As the degree of chlorination increased, hydroxy products were more efficiently excreted, whereas the formation of methylthio metabolites greatly decreased. As a result, the excretion ratios of methylthio and hydroxy products varied with 2.8 for TriCB, 1.3 for TetraCB, 0.04 for PentaCB, and 0.02 for HexaCB. The 3-/4-hydroxy substitution ratios were 0.6 for TriCB, 1.4 for TetraCB, 21 for PentaCB, and 35 for HexaCB, whereas the 3-/4-methythio substitution ratios were 1.2 for TriCB, 0.8 for TetraCB, 0.18 for PentaCB, and 0.12 for HexaCB. The formation rate of 3- and 4-methylthio metabolites from each congener was correlated to the accumulation and distribution of 3- and 4-methylsulfonyl derivatives in tissues. The tissue/blood concentration ratios of methylsulfonyl metabolites showed that the 3-methylsulfonyl derivatives from higher chlorinated biphenyls had a relatively high affinity for liver and adipose tissue, whereas the 4-methylsulfonyl derivatives were selectively retained in the lung in all cases.

Animals↗

PubMed1997

[Evaluation of myocardial ischemia with echo planar magnetic resonance imaging (EPI): normal and ischemic heart].

We investigated the validity of Gradient recalled EPI(GRE-EPI) after bolus injection of contrast agent(Gadolinium 0.1 mmol/kg) to detect the ischemic zone of myocardium. Seven healthy volunteers and fifteen patients with coronary artery disease who had only one vessel disease more than 99% stenosis in AHA/ACC classification were studied. GRE-EPI was performed in a transaxial, long axis or short axis orientation of the left ventricle on a 1.5-T Signa Horizon Scanner. Images were obtained every heartbeat for 200 consecutive heartbeats. Contrast agent was injected into the antecubital vein in a bolus after 10 heartbeats. Signal intensity changes in the left ventricular blood and myocardium were measured during myocardial contrast perfusion study. In all healthy volunteers, signal intensity in myocardium was reduced by contrast agent following signal loss in the left ventricular blood and recovered with wash out of contrast agent. But in patients, reduction and recovery of signal intensity in the ischemic zone by contrast agent were significantly delayed and much less than that in the nonischemic zone. In conclusion, myocardial perfusion imaging with GRE-EPI was useful in evaluation of ischemic heart disease.

Coronary Circulation↗

PubMed1997

Post-treatment of transient focal cerebral ischemia in rats with the novel cerebrovascular-selective Ca2+ channel antagonist (+/-)-(E)-1-(3-fluoro-6,11-dihydrodibenz[b,e]-oxepine-11-yl)-4-(3-pheny l-2-propenyl)-piperazine dimaleate.

The efficacy of post-ischemic treatment with AJ-3941 ((+/-)-(E)-1-(3-fluoro-6,11-dihydrodibenz[b,e]-oxepine-11-yl )-4-(3-phenyl-2- propenyl)-piperazine dimaleate, CAS 143110-70-7), a cerebrovascular selective Ca2+ channel antagonist, on brain infarction induced by focal ischemia-reperfusion in rats was evaluated. Focal ischemia was induced by transient occlusion of middle cerebral artery (MCA) with a 3-0 nylon monofilament for 90 min. One day after MCA occlusion (MCAo), brain infarct size was determined by measuring 2,3,5-triphenyltetrazonium chloride-negative stained area of the serial brain sections. The development of cerebral infarction was observed in both regions of cortex and subcortex, such as striatum, caudatum, putamen, hippocampus and corpus callosum. Post-ischemic treatment with AJ-3941 (1 or 3 mg/kg p.o., 10 min and 3 h after the occlusion) significantly reduced the infarct size and volume in the ipsilateral hemisphere in a dose-dependent manner, compared to the solvent control. The reducing effect was observed mainly in the cortical region, and a significant reduction of the subcortical infarct volume was found at the higher dose (3 mg/kg). Post-ischemic treatment with the thromboxane A2 synthetase inhibitor, sodium ozagrel (150 micrograms/kg/min i.v. infusion, between 1 h and 24 h after the MCAo) did not reduce the infarct volume in the hemisphere after ischemia-reperfusion. AJ-3941 had only minimum effect on the elevation of body temperature during ischemia-reperfusion. These results indicate that post-ischemic treatment with AJ-3941 may ameliorate the brain injury after the transient focal cerebral ischemia, and they suggest that AJ-3941 has beneficial effects for treatment of ischemic cerebral damage, such as stroke.

Animals↗

PubMed1997

Laparoscopic Hill's vagotomy by the abdominal wall lifting method.

We performed laparoscopic Hill's vagotomy by the abdominal wall lifting method in nine patients with intractable duodenal ulcer. Our original I-type lifting bar is a curved stainless-steel rod 5 mm in diameter. One I-type lifting bar is inserted intraperitoneally into each of the right and left hypochondrial regions. An incision is made in the lesser omentum near the gastroesophageal junction, and the right esophageal wall and right crus of the diaphragm are dissected and exposed. The posterior trunk of the vagus nerve is identified and divided. Then the neurovascular bundle is dissected and divided repeatedly along the lesser curvature of the stomach from the first branch of the crow's foot to the gastroesophageal junction. The mean operating time was 163 min, with little blood loss. The reduction rate of basal acid output and maximal acid output was, respectively, 73.7 +/- 0.1 and 63.7 +/- 0.1%. Four weeks after surgery, gastroduodenoscopy revealed ulcer healing to a scar.

Adult↗

PubMed1997

Rare glomerular capillary regeneration and subsequent capillary regression with endothelial cell apoptosis in progressive glomerulonephritis.

Glomerulonephritis (GN) leading to glomerular sclerosis remains an important cause of renal failure. The glomerulus is a capillary network, but endothelial and vascular reactions during progressive GN are not well understood. We have, therefore, examined the morphological alterations of glomerular capillary network and endothelial cells during the progression of damaged glomeruli to glomerular sclerosis. A progressive model of anti-glomerular basement membrane (GBM) GN was induced in Wistar-Kyoto (WKY) rats with a single injection of anti-rat GBM antibody. Severe necrotizing glomerular injuries were observed between day 5 and week 3 with a reduction in the number of total glomerular endothelial cells and total glomerular capillary lumina per glomerular cross sections. In necrotizing lesions, the glomerular endothelial cells were lost with the destruction of the glomerular capillary network. Moreover, angiogenic capillary repair with proliferation of endothelial cells was rare in severely damaged regions of glomeruli. Subsequently, mesangial hypercellularity and marked mesangial matrix accumulation occurred with absence of the development of a capillary network, and the necrotizing lesions progressed to sclerotic scars until 8 weeks. Although active necrotizing lesions could not be seen in damaged glomeruli between week 4 and week 8, the number of apoptotic endothelial cells gradually increased in the glomerular capillaries (0.10 +/- 0.01 apoptotic endothelial cells/glomerular cross section at week 8 versus 0.00 +/- 0.00 control cells (mean +/- SEM; P < 0.05) with the progression of glomerular sclerosis. Whereas the number of apoptotic endothelial cells increased in the damaged glomeruli, the number of total glomerular endothelial cells decreased (9.3 +/- 3.0 cells/glomerular cross section at week 8 versus 24.8 +/- 3.0 cells in control (mean +/- SD); P < 0.001) with regression of glomerular capillaries (3.6 +/- 2.5 capillary lumina/glomerular cross section at week 8 versus 35.0 +/- 5.0 capillary lumina in control (mean +/- SD); P < 0.001). Finally, glomerular endothelial cells could not be detected in the sclerotic lesions in progressive anti-GBM GN in WKY rats. These data indicate that the destruction of the capillary network of glomeruli and subsequent incomplete angiogenic capillary repair leads to glomerular sclerosis in progressive GN. Endothelial cell apoptosis with glomerular capillary regression may also contribute to the development of glomerular sclerosis. Injury of the glomerular capillary network with endothelial cell damage, including apoptosis and subsequent incomplete capillary repair, plays an important role in the progression of glomerular sclerosis during anti-GBM GN in WKY rats.

Animals↗

PubMed1997

[Hemodynamic effects of amrinone in children during cardiopulmonary bypass and postoperative 12 hours].

Hemodynamic effects of amrinone in children during cardiopulmonary bypass (CPB) and postoperative 12 hours were studied. In 10 patients undergoing open heart surgery, 1 mg/kg of amrinone was infused as the initial CPB dose and 5 - 10 micrograms/kg/min of amrinone was continuously administered as the maintenance dose during CPB and postoperative 12 hours. Amrinone levels ranged from 0.9 to 1.4 micrograms/ml during CPB and postoperative 12 hours. After infusion of amrinone, mean arterial blood pressure decreased significantly, but other parameters did not show remarkable change. The administration of amrinone during CPB showed enough vasodilating effect and decreased the need of conventionally used other vasodilators (nitroglycerin or prostaglandin E1). The postoperative course of 10 patients was clinically uneventful. The administration of amrinone in 10 patients did not produce thrombocytopenia compared with the control group (5 patients) in the postoperative period. In conclusion, the administration of amrinone during CPB and postoperative 12 hours in children was useful in producing enough vasodilating effect without major side effect.

Amrinone↗

PubMed1997

[Typical normal cases and normal cases with abnormal image pattern in every myocardial SPECT radiopharmaceutical].

Working group of cardiac nuclear medicine was made as a Japanese part of society of international cardiac nuclear medicine under the cooperation between Japanese society of nuclear medicine and Japanese society of cardiology. We investigated typical normal cases and normal cases with abnormal image pattern in every myocardial SPECT radiopharmaceutical as one of the research activity of working group. From 11 faculties, 16 T1 cases, 14 BMIPP cases, 8 MIBG cases, 8 MIBI cases and 14 tetrofosmin cases were submitted as typical normal cases, and 12 T1 cases, 5 BMIPP cases, 12 MIBG cases, 10 MIBI cases and 5 tetrofosmin cases were submitted as normal cases with abnormal image pattern. We summarized the condition of SPECT data acquisition of each faculties. And we added the discussion from literature about how to discriminate normal cases with abnormal image pattern from abnormal cases. In MIBG, patterns of typical normal cases and normal cases with abnormal image pattern were slightly different from other 4 pharmaceuticals. In other 4 pharmaceuticals, diaphragmatic attenuation, breast attenuation, apical thinning and others were presented as normal cases with abnormal image pattern.

3-Iodobenzylguanidine↗

PubMed1997

[Evaluation of intramyocardial coronary flow velocity pattern before and after surgical repair of Bland-White-Garland syndrome by pulsed Doppler echocardiography].

Anomalous origin of the left main coronary artery from the pulmonary artery (Bland-White-Garland syndrome) is a rare congenital anomaly. Intramyocardial coronary flow dynamics by pulsed Doppler echocardiography were studied in three patients with this syndrome who underwent surgical repair by Hamilton's method. Before surgery, the intramyocardial flow at the ventricular septum showed a retrograde velocity pattern which had two peaks in systole and diastole in all patients. After surgery, two patients with successful repair showed a biphasic intramyocardial flow pattern which consisted of retrograde and antegrade flows in systole and diastole, respectively. In contrast, one patient who had a residual shunt between the left coronary artery and the pulmonary artery showed a biphasic pattern which had antegrade flow in systole and retrograde flow in diastole. These results may suggest that the evaluation of postoperative intramyocardial coronary flow velocity pattern by pulsed Doppler echocardiography is useful for detecting a residual shunt after surgical repair of Bland-White-Garland syndrome.

Adult↗

PubMed1997

Alacepril, an angiotensin-converting enzyme inhibitor, prevents cerebral vasospasm in subarachnoid hemorrhage model in rats.

The effects of angiotensin-converting enzyme (ACE) inhibitors was investigated on the development of cerebral vasospasm and on the endothelium-dependent relaxation in the rat subarachnoid hemorrhage (SAH) model. Alacepril or enalapril was used as an ACE inhibitor with or without a thiol moiety in the structure. SAH rats or sham-operated rats were produced by the injection of homologous blood or artificial cerebrospinal fluid into the cisternal magna, respectively. In the SAH rat, cerebral vasospasm was observed at 24 h after blood injection. Acetylcholine (Ach)-induced relaxation in basilar arteries from SAH rats significantly decreased compared to that from sham-operated rats, although the relaxation induced by 3-morpholinosydnonimine, sodium nitroprusside or papaverine did not decrease. These results suggest that the endothelium cell function of basilar arteries in SAH rats is damaged. Alacepril prevented both the development of cerebral vasospasm and the suppression in the Ach-induced relaxation of basilar artery in SAH rats. However, enalapril did not prevent the suppression of Ach-induced relaxation in SAH rats, despite the tendency to prevent cerebral vasospasm. Therefore, it is suggested that the preventive effect of alacepril on cerebral vasospasm could be based on its protective effect on endothelium-dependent relaxation system.

Acetylcholine↗

PubMed1996

Cloning and sequencing of a cDNA encoding a heat-stable sweet protein, mabinlin II.

A cDNA clone encoding a heat-stable sweet protein, mabinlin II (MAB), was isolated and sequenced. The encoded precursor to MAB was composed of 155 amino acid (aa) residues, including a signal sequence of 20 aa, an N-terminal extension peptide of 15 aa, a linker peptide of 14 aa and one residue of C-terminal extension. Comparison of the proteolytic cleavage sites during post-translational processing of MAB precursor with those of like 2S seed-storage proteins of Arabidopsis thaliana, Brassica napus and Bertholletia excelsa shows that the three individual cleavage sites between respective species are conserved.

Amino Acid Sequence↗

PubMed1996

Prevention by the new Ca2+ channel antagonist, AJ-3941, of loss of endothelium-dependent relaxation after subarachnoid hemorrhage in rats.

AJ-3941 ((+/-)-(E)-1-(3-fluoro-6,11-dihydrodibenz[b,e]-oxepine-11-yl ) -4-(3-phenyl-2-propenyl)-piperazine dimaleate; CAS No. 143110-70-7), a cerebrovascular-selective Ca2+ channel antagonist having anti-lipid peroxidative action, was reported to prevent cerebral vasospasm following subarachnoid hemorrhage in rats. The present study was undertaken to determine whether AJ-3941 protects the impairment of cerebroarterial endothelium-dependent relaxation which is concomitantly induced with cerebral vasospasm. Subarachnoid hemorrhage biphasically suppressed the response to acetylcholine in rat basilar artery, at 0.5 h (n = 4; P < 0.06) and 1 day (n = 5; P < 0.05) after subarachnoid hemorrhage. The reduction of the responses was correlated significantly to the degree of vasospasm determined angiographically. This reduction was accompanied by a 49% increase of arterial lipid peroxide contents. Endothelium-independent relaxation in subarachnoid hemorrhage rats was preserved in response to 3-morpholinosydnonimine, sodium nitroprusside and papaverine. AJ-3941 prevented (n = 6-8, P < 0.05) the suppression of the acetylcholine-induced response and the increase in lipid peroxide content in subarachnoid hemorrhage rats. These results suggest that AJ-3941 could exert its vasospasmolytic effect by preserving endothelial function through its anti-lipid peroxidative action, in addition to its inhibition of vasospasmogen-induced vasoconstriction related to intracellular Ca2+ mobilization.

Acetylcholine↗

PubMed1996

Geranylgeraniol causes a decrease in levels of calreticulin and tyrosine phosphorylation of a 36-kDa protein prior to the appearance of apoptotic features in HL-60 cells.

It was demonstrated recently that geranylgeraniol (GGO) has potent apoptosis-inducing activity in various lines of tumor cells, including HL-60 cells. In the present study, we found that GGO markedly inhibited the expression of a calcium-binding protein, calreticulin, prior to the induction of apoptosis in HL-60 cells. Furthermore, we also observed a significant decrease in the tyrosine phosphorylation of a 36-kDa protein that is a major tyrosine-phosphorylated protein in HL-60 cells. These findings suggest that decreases in levels of calreticulin and in the tyrosine phosphorylation of the 36-kDa protein might be associated with the induction of apoptosis by GGO in HL-60 cells.

Amino Acid Sequence↗

PubMed1996

Optimization of the rate of DNA hybridization and rapid detection of methicillin resistant Staphylococcus aureus DNA using fluorescence polarization.

The hybridization rate of two complementary single-stranded DNA 24-mers was determined at various NaCl concentrations using a fluorescence polarization method. The rate was taken as the rate constant of the second order reaction, obtained by mathematical fitting of the time course curves of hybridization. The rate increased with the NaCl concentration, plateauing in the concentration range of about 1-2 M. Over the temperature range of 46 degrees C to 56 degrees C, it was found that in 0.8 M NaCl, hybridization was complete in under 10 min and that the difference in the polarization values between specific hybridization and non-specific binding was greater at lower temperatures. Under the optimized conditions of 0.8 M NaCl at 46 degrees C, the time for DNA hybridization to reach 90% completion decreased to less than 5 min. The assay time for one sample was 10 min and the detection limit was of the order of 10(-10) M (40 fmol per assay). Under the optimized conditions, the DNA of methicillin resistant Staphylococcus aureus (MRSA), which had been multiplied by PCR, could be detected within 10 min.

Base Sequence↗

PubMed1996

The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.

1-Methyl-3-isobutylxanthine↗