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Biomedical subjects

N Ogata

Publications and source records attributed to N Ogata.

At least 199 records · Page 11Linked to original sources
PubMed1991

[Primary culture of mammalian brain neurons and its application to patch-clamp recording].

A method is described for the primary culture of neurons from adult mammalian brain. The primary culture consists of five processes: 1) preparation of brain slices, 2) microdissection of the discrete area, 3) enzymatic treatment of the tissue, 4) dissociation of cells by mechanical agitation of the tissue fragments, and 5) plating and feeding of dissociated cells. The cells could be maintained in culture for more than several weeks. Whereas neurons freshly dissociated from the adult brain do not respond to exogenously applied neurotransmitter substances, probably due to destruction of receptors by the enzymatic treatment, the neurons regained the ability to respond to a variety of neurotransmitters when they were cultured. Cultured neurons from adult mammalian brain are proving to be an excellent model for physiological as well as pharmacological investigations on the central nervous system.

Animals↗
PubMed1991

Point mutation, allelic loss and increased methylation of c-Ha-ras gene in human hepatocellular carcinoma.

Somatic alterations of the c-Ha-ras gene were examined in 21 Japanese patients with hepatocellular carcinoma. Restriction endonuclease analysis by double digestion with MspI and HpaII revealed that DNAs from two of 21 hepatocellular carcinoma tissues were affected by nucleotide substitution at the twelfth amino acid coding sequence of the c-Ha-ras gene. DNAs from cirrhotic noncancerous liver tissue, but not leukocytes, of one of these patients possessed the mutation, whereas DNAs from noncirrhotic liver tissue and leukocytes of the other patient did not. In one of the nine patients harboring heterozygosity for c-Ha-ras-related BamHI-fragments, the loss of one allele was demonstrated as a somatic change not only in DNA from the tumor tissue but also in DNA from the cirrhotic nontumorous tissue. In two of the 19 patients comparatively examined for digestion patterns of c-Ha-ras locus with HpaII and MspI, extensive methylation was observed as a somatic modification in both DNAs from the tumor and the cirrhotic nontumorous tissues. These results thus indicate that the genetic lesions affecting the c-Ha-ras gene do occur in human hepatocellular carcinoma and probably serve as one of the multiple steps in the process of hepatic carcinogenesis.

Adult↗
PubMed1990

[Transcatheter embolization therapy of pseudoaneurysms in patients with chronic pancreatitis].

Chronic pancreatitis is known to cause vascular disorders including pseudoaneurysm of peripancreatic arteries. Although the incidence of pseudoaneurysms due to pancreatitis detected by angiography has been reported as high as 10% in western literature, they are still considered rare in Japan. We reported two cases of pseudoaneurysm caused by chronic pancreatitis, one in splenic artery and the other in gastroduodenal artery, successfully treated by embolization. The embolizations were performed by occluding proximal splenic artery for splenic arterial aneurysm and gastroduodenal artery distal and proximal to the orifice for the aneurysm of gastroduodenal artery with stainless steel coils. Since the surgical therapy has high mortality rate, we consider transcatheter embolization is the treatment of choice for pseudoaneurysms caused by chronic pancreatitis.

Aneurysm↗
PubMed1990

[Right anterior caudo-cranial oblique (RACCO) view of portal branches].

The right caudocranial oblique (RACCO) view which is obtained by tilting the image intensifier 25 degrees caudally and 30 degrees to the right while the patients lie in supine position is proposed for improved depiction of the portal venous system. Transarterial portography with digital subtraction angiography was performed in the RACCO and posteroanterior (PA) views in 32 patients. Comparison of the two views revealed that the RACCO view was superior to the PA view of the left main branch in 25 patients (78%) and of the right posterior lobe branch in 24 (75%) patients. Visibility of more distal portal branches, however, did not always improve by RACCO view. RACCO view will be useful for the evaluation of portal systems of primary and secondary hepatic neoplasms as a special view following portogram in PA projection when more detailed information is necessary on proximal portal branches.

Angiography, Digital Subtraction↗
PubMed1990

Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian-virus-40-pBR322 recombinant plasmid.

To study the relationship between the structural organization and function of the eukaryotic genome, DNA associated with nuclear matrix was analysed by using a transformed rat fibroblast cell line. The nuclear matrices were prepared from the isolated nuclei of pSV1-FR, a rat fibroblast cell line transformed by a pBR322-based recombinant plasmid containing an early gene region, which codes for large T-antigen, of simian virus 40. This transformed cell contained a single copy of the plasmid sequence integrated into the chromosomal DNA of the host cell. The early gene of this plasmid was constitutively expressed, as demonstrated by positive immunofluorescence staining of the cell for large T-antigen and by RNA-blot analysis for its specific mRNA. DNAs were extracted from whole isolated nuclei and nuclear-matrix preparations of the cells, and the relative amounts of the sequence similar to that of the plasmid were compared between these DNA preparations. By employing dot hybridization and Southern-blot analyses we found that the plasmid sequence was more enriched in the DNA extracted from the nuclear matrices than in the DNA extracted from the whole nuclei. When an albumin gene sequence that was not transcribed in this cell line was compared similarly as a control, we found no significant enrichment of this sequence in the DNA associated with the nuclear matrix. Our results strongly support the concept that a transcriptionally active gene is preferentially associated with the nuclear matrix.

Animals↗
PubMed1990

A comparison of the molecular structure of integrated hepatitis B virus genomes in hepatocellular carcinoma cells and hepatocytes derived from the same patient.

To elucidate critical genetic elements in the development of hepatocellular carcinoma associated with hepatitis B virus DNA integration, a single integrant in hepatocellular carcinoma cells and one species of multiple integrants in hepatocytes, both obtained from the same patient, were compared structurally using molecular cloning techniques. Both hepatitis B virus integrants showed similar inverted repeat sequences consisting of two defective virus genomes. The recombination of viral DNAs seemed to be mediated by short regions of base homology near the direct repeat 1 and at other regions of the virus genomes in both integrants. The virus component in the junction with host DNAs was the cohesive end region in each identical end of the viral integrant in hepatocellular carcinoma cells and in one end of the viral integrant in hepatocytes. The structure of the integrant in hepatocellular carcinoma cells was characterized by an inverted, duplicated conformation composed not only of integrated virus genomes but also of flanking cellular sequences. It was shown to be the so-called "alpha dimer" of satellite DNA. In contrast, the flanking, nonreiterated cellular DNA in the hepatocyte-derived clone did not show discernible rearrangement. These findings suggest that a common mechanism underlies the integration of hepatitis B virus DNA so that a similar organization of inverted repeat genomes is found in hepatocellular carcinoma cells and in hepatocytes. The unstable nature of cellular DNA where DNA integration occurs may be important in generating chromosome alterations found in hepatocellular carcinoma.

Adult↗
PubMed1990

Sodium current kinetics in freshly isolated neostriatal neurones of the adult guinea pig.

Neurones of the neostriatum were freshly dispersed from the adult guinea pig brain. A fast, transient inward Na+ current (INa) was analysed using the whole-cell patch-clamp technique. Upon depolarizations, INa developed with a sigmoidal time course, which was described by m3 kinetics. INa showed an activation threshold of about -60 mV, a peak current at -30 to -20 mV, and a reversal of polarity at +60 mV. The steady-state activation (m infinity) curve for INa had a slope factor of about 9 mV for a change in the rate constant by a factor of e) in the range between -50 mV and -20 mV. Conversely, the backward rate constant (beta m) decreased as the membrane was depolarized (about 31 mV for an e-fold change). The steady-state inactivation (h infinity) curve was well expressed by the Boltzmann's equation with a half-inactivation potential of -62 mV and a slope parameter of 6 mV. The time course of INa decay followed a second-order process, whereas the recovery from inactivation was described as a first-order process. The tau h curve showed a bell-shaped configuration with a maximum value at -60 mV. The forward rate constants (alpha h) decreased as the membrane was depolarized (about 17 mV for an e-fold change) in the range between -50 mV and -20 mV. The backward rate constants (beta h) increased as the membrane was depolarized (about 10 mV for an e-fold change). There was a significant overlap between m infinity and h infinity curves, suggesting a steady influx of Na+ (window current).

Animals↗
PubMed1990

Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells.

1. The effects of chlorpromazine on the voltage-activated sodium and type I (transient) calcium channels were studied in cultured mouse neuroblastoma cells (N1E-115) using the whole-cell patch-clamp technique. 2. Chlorpromazine (2-10 x 10(-6) M) blocked both the sodium channel current and the calcium channel current as carried by Ba2+ in a reversible and dose-dependent manner. 3. The block was not associated with any change in the time course of the activation and inactivation of the sodium and calcium channel currents. 4. The dose-response relationships for the block of these channels measured with a holding potential of -120 mV indicated a 1:1 binding stoichiometry with apparent dissociation constants of 2.5 +/- 10(-6) M and 1.5 +/- 10(-5) M for the sodium and calcium channels, respectively. 5. The block was dependent on the holding potential for both channel currents. The apparent dissociation constant for the sodium channel was decreased to 0.65 +/- 10(-6) M when the membrane was held at -80 mV. The apparent dissociation constant for the calcium channel was decreased to 3.2 +/- 10(-6) M when the membrane was held at -60 mV. 6. The steady-state inactivation curve for the sodium channel was shifted by 12.4 +/- 1.8 mV to more negative potentials by exposure to 1 x 10(-6) M-chlorpromazine. The inactivation curve for the calcium channel was also shifted by 15.4 +/- 3.2 mV to more negative potentials by exposure to 1 x 10(-5) M-chlorpromazine. These results indicate a greater affinity of chlorpromazine for the inactivated state of the channels than for the resting state. 7. Chlorpromazine caused a marked use-dependent block of the sodium channel current, as demonstrated by a cumulative increase of the block during a train of depolarizing pulses. The use-dependent block was observed even with an interpulse interval as long as 2 s. 8. On the other hand, the block of the calcium channel current did not notably accumulate during a train of depolarizing pulses even when extremely prolonged (1 s) pulses were applied at a very short interpulse interval (200 ms). 9. The marked use dependence of the sodium channel block was due to a very slow repriming of the drug-bound sodium channels from inactivation, whereas the lack of use dependence of the calcium channel block was due to a rapid repriming of the drug-bound calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

[Evaluation of cardiac function at rest and under acute loading in patients with essential hypertension using pulsed Doppler echocardiography].

We studied 45 untreated patients with mild to moderate essential hypertension (EH) and 27 normotensive controls to assess the relationship between pulsed Doppler echocardiographic parameters and cardiac function, and the effects of acute changes in preload or afterload on these parameters. The following pulsed Doppler parameters were measured: as left ventricular (LV) inflow parameters, rapid filling peak velocity (R), presystolic peak velocity (A), and A/R; as LV outflow parameters, preejection period (PEP), ejection period (ET), ET/PEP, and peak velocity (PV). These parameters were compared with those relating to cardiac function, such as age, interventricular septal thickness (IVST), LV mass index (LVMI), % fractional shortening (%FS), LV end-systolic wall stress (WS), blood pressure, and heart rate. The changes in preload and afterload were induced by sodium nitroprusside (SNP) or phenylephrine infusion, which caused decreases or increases in the mean blood pressure by approximately 15%. 1. LV inflow parameters: Compared to normals, A and the A/R were higher in the group EH (p less than 0.01). IVST in the group EH correlated with R and the A/R, but LVMI did not correlate with these parameters. In normals, A correlated with %FS and WS, but did not in the group EH. R and the A/R correlated with age in normals, but not in the group EH. 2. LV outflow parameters: There were no significant differences between the two groups. PV in the group EH correlated positively with %FS, and negatively with WS. The ET/PEP in normals correlated with %FS and WS, but not in the group EH. 3. Effects of SNP: Reduction in preload caused a decrease in R and an increase in the A/R in both the groups (p less than 0.01). At the same time, reduction in afterload induced increase in PV. 4. Effects of phenylephrine: An increase in afterload did not change A, R or the A/R, though PV decreased significantly in normals and tended to decrease in the group EH. In summary, though the significance of the pulsed Doppler parameters of the LV was not always the same between the two groups, these parameters may be used to evaluate cardiac function in EH, if we pay attention to the influence of preload and afterload.

Adult↗
PubMed1990

Potent blocking action of chlorpromazine on two types of calcium channels in cultured neuroblastoma cells.

The effects of chlorpromazine on calcium channel currents were studied in cultured mouse neuroblastoma cells (N1E-115) using the whole-cell variation of the patch-clamp technique. Two types of calcium channel currents (type I and type II) as carried by barium ions were recorded separately on the basis of their different voltage- and time-dependent kinetics. Chlorpromazine reversibly reduced the amplitude of both types of calcium channel currents. The concentration dependence of the calcium channel block indicated a one-to-one binding stoichiometry for both type I and type II currents, with an apparent dissociation constant of 15 microM in both cases. The block of calcium channels was dependent on the holding potential for both type I and type II currents, being enhanced by depolarization. This voltage dependence of the block was due to a higher affinity of chlorpromazine for the inactivated state of the calcium channels than the resting state, as demonstrated by a hyperpolarizing shift of the steady-state inactivation curve. The activation kinetics were not affected by chlorpromazine in either type I or type II current. The time course of inactivation of the type I current was not changed by chlorpromazine, whereas that of the type II current was accelerated, suggesting an involvement of an open channel block. Chlorpromazine block of type I current was independent of the level of test depolarizing pulse, whereas that of type II current was augmented with an increase in depolarization. It was concluded that chlorpromazine binds preferentially to the inactivated form of both type I and type II calcium channels, without affecting the gating kinetics of channel activation.

Animals↗
PubMed1989

Psychotropic drugs block voltage-gated ion channels in neuroblastoma cells.

Effects of neuroleptics and tricyclic antidepressants on voltage-gated ion channels were investigated in the neuroblastoma cell line N1E-115 using the whole cell variation of patch electrode voltage-clamp techniques. Imipramine, chlorpromazine and haloperidol in micromolar concentrations blocked sodium, calcium and potassium channel currents in a reversible and concentration-dependent manner. The order of potency was chlorpromazine greater than imipramine greater than haloperidol. These direct blocking actions on neuronal ion channels might play a role in clinical effects of these drugs.

Animals↗
PubMed1989

[Degradation of cytoskeletal proteins in cerebral ischemia].

Degradation of neurofilament (NF) triplet proteins (Mw 200,000 (NF200), Mw 150,000 (NF150), Mw 68,000 (NF68], high molecular weight microtubule-associated proteins (MAP1 and MAP2) and other cytoskeletal proteins in rat brain during ischemia was investigated by sodium dodecyl sulfate (SDS)-gel electrophoresis and immunoblot methods using anti-NF200 monoclonal antibody. Selective degradation of NF200 and NF150 was observed during the initial 10 to 15 minutes of ischemia: and the degradation of MAP1 and MAP2 during the initial 15 to 30 minutes of ischemia. These degradations suggest that the activation of a protease occurs at the very early stage of cerebral ischemia, which are the earliest irreversible neuronal changes ever to be reported. Effect of ischemia on polymerization of microtubule proteins was also investigated by turbidity assay. A rapid decrease of the ability of polymerizing microtubules was observed during the initial 5 minutes of ischemia. This loss of polymerization ability was apparently independent of the degradation of MAP1 and MAP2.

Animals↗
PubMed1989

Change in cytoplasmic free Ca2+ concentration in rabbit platelets contacting with albumin coated and uncoated polystyrene surfaces.

Direct measurement of cytoplasmic free Ca2+ concentration in rabbit platelets in contact with polystyrene latexes was examined by monitoring an intracellular trapped fluorescent indicator dye, Fura 2, as a quantitative evaluation of platelet activation on material surfaces. It was found that an increase in cytoplasmic free calcium levels in platelets in contacting latexes was strongly reduced by the adsorption of albumin on the latex surfaces. Further, such an elevation of cytoplasmic free calcium levels was found to require a calcium influx from an extracellular medium across the platelet-plasma membrane.

Animals↗
PubMed1989

Block of sodium channels by psychotropic drugs in single guinea-pig cardiac myocytes.

1. Effects of imipramine and haloperidol on voltage-gated sodium channels were investigated in guinea-pig isolated ventricular myocytes by the whole-cell patch clamp technique. Some additional experiments were also performed with chlorpromazine for the purpose of comparison. 2. All test drugs in micromolar concentrations suppressed the amplitude of peak sodium current associated with step depolarization from a holding potential of -140 mV in a reversible manner. The order of potency was chlorpromazine greater than imipramine greater than haloperidol. 3. Dose-response curves obtained with a holding potential of -140 mV were best fitted by 2:1 stoichiometry in all three drugs and were shifted in the direction of lower concentrations when a holding potential of -90 mV was used. 4. The drug-induced block was not associated with any change in the time courses of sodium current activation and inactivation. 5. Steady-state sodium channel inactivation curve was shifted in the direction of more negative potentials by the drugs. 6. All three drugs also produced marked use-dependent block as demonstrated by a cumulative increase in the block during a train of depolarizing pulses. 7. The use dependence was due to a higher affinity of the drugs for the inactivated state of sodium channels than the resting state and to a very slow repriming of the drug-bound sodium channels from inactivation. 8. The steady-state and use-dependent block of voltage-gated sodium channels by psychotropic drugs may contribute to their cardiotoxic and perhaps antiarrhythmic effect.

Animals↗
PubMed1989

Modulation of sodium current kinetics by chlorpromazine in freshly-isolated striatal neurones of the adult guinea-pig.

1. The neurones of the striatum were freshly dissociated from the adult guinea-pig brain by enzymatic and mechanical treatments. Sodium channel current kinetics in these neurones were measured using a whole cell variation of the patch-clamp technique. 2. Chlorpromazine, a neuroleptic, in micromolar concentrations reversibly reduced the amplitude of the sodium currents. Activation and inactivation time constants were not affected. The inhibition followed one-to-one binding stoichiometry. 3. The concentration-response curve shifted to the left when the holding potential was less negative. The EC50 shifted from 4.8 microM to 0.9 microM when the holding potential was changed from -120 mV to -70 mV. 4. The steady-state activation curve of the sodium current was not affected by chlorpromazine, whereas the steady-state inactivation curve was shifted in the negative direction. Consequently, the window current which is normally present at a potential range around -50 mV was decreased in the presence of chlorpromazine. 5. Successive sodium currents evoked by a train of depolarizing pulses (30 ms duration) to -10 mV showed a cumulative decrease in size during the application of chlorpromazine. However, such 'use-dependent' block was not observed when the pulse duration was reduced to 1 ms. 6. The recovery from inactivation in the presence of chlorpromazine, was expressed as a second order process. The faster component was similar to the recovery time course of the normal sodium channels. The slower component accounted for the use-dependent effect of chlorpromazine. 7. The results indicate that chlorpromazine binds to the resting sodium channels producing steady-state block at a very negative holding potential. When the membrane is depolarized, chlorpromazine binds to the inactivated form of the sodium channels with much higher affinity and stabilizes them in the inactivated state, slowing their kinetics.

Animals↗
PubMed1989

Degradation of neurofilament protein in cerebral ischemia.

Degradation of neurofilament (NF) triplet proteins: NF200 (molecular weight (MW) 200,000), NF150 (MW 150,000), and NF68 (MW 68,000) as well as of other cytoskeletal proteins in the rat brain during ischemia was investigated. Sodium dodecyl sulfate-gel electrophoresis and immunoblot methods with anti-NF200 antibody were used for the study. Selective degradation of NF200 and NF150 was observed during the initial 10 to 15 minutes of ischemia. The degradation was demonstrated both in permanent ischemia caused by decapitation and in transient ischemia induced by four-vessel occlusion followed by reperfusion after 30 minutes of occlusion (modified Pulsinelli method). The degradation suggests that the activation of a protease occurs in the first 15 minutes of cerebral ischemia, which is the earliest irreversible neuronal change ever to be reported.

Animals↗