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Biomedical subjects

J Okada

Publications and source records attributed to J Okada.

At least 55 records · Page 3Linked to original sources
PubMed1998

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum.

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol-10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol-10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1-500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.

Administration, Oral↗
PubMed1998

Pharmacological properties of the CO2/H+-sensitive area in the ventral medullary surface assessed by the effects of chemical stimulation on respiration.

We recently discovered that CO2/H+-sensitive neurons in the ventral medullary surface (VMS) are immunoreactive to glutamate, glutamic acid decarboxylase (GAD), calcineurin and cAMP. We then tested the hypothesis that glutamate, GABA, calcineurin and cAMP affect the activity of CO2/H+-sensitive neurons in the VMS. Using male Wistar rats anesthetized with urethane and pentobarbital, we checked for changes in relative tidal volume (VT) and respiratory frequency (f) in response to injecting the VMS with a variety of test agents dissolved in mock CSF. Respiratory changes occurred immediately and were dose-dependent. (1) 200-1600 pmol Glutamate increased VT but decreased f. The glutamate effect was never abolished by concomitant injection of AP5, a NMDA receptor antagonist, but was abolished by CNQX, an AMPA receptor antagonist, indicating predominance of AMPA receptors in the CO2/H+-sensitive neurons in the VMS. (2) 200-1600 pmol GABA decreased both VT and f. The GABA effect was never abolished by concomitant injection of saclofen, a GABA(B) receptor antagonist, but was abolished by bicuculline, a GABA(A) receptor antagonist, indicating predominance of GABA(A) receptors in the CO2/H+-sensitive neurons in the VMS. (3) 4-32 microg Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 2B, and 200-1600 pmol FK506, selective inhibitor of calcineurin, had no effect on respiration when they were applied extracellularly, but 400-3200 pmol BAPTA-AM, an intracellular Ca2+-chelating agent, decreased both VT and f, indicating involvement of intracellular Ca2+ in the excitatory mechanisms of respiration. (4) 100-800 pmol IBMX, an enhancer of intracellular cAMP, decreased both VT and f, indicating involvement of cAMP in the inhibitory mechanisms of respiration. These results indicate that the CO2/H+-sensitive neurons in the VMS contain glutamate and/or GABA in cytoplasma, possess AMPA and/or GABA(A) receptors on surface of plasma membrane, and compose the internal circuit, and that their activities are regulated by Ca2+ and cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Difference in topology and numbers of barosensitive catecholaminergic and cholinergic neurons in the medulla between SHR and WKY rats.

We hypothesized that there may be a significant difference in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto (WKY) and spontaneously hypertensive SHR rats. Using the double-immunoreactive (IR) method, the topology and numbers of barosensitive neurons that contain glutamate (Glu), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) were compared between the two strains. The control rats were sham-operated only for cannulation of the trachea and femoral artery/vein. The test rats were injected with the pressor agent phenylephrine to raise blood pressure and stimulate arterial baroreceptors. In both the control and test experiments, the c-Fos/Glu-, GAD-, TH- and PNMT-IR neurons were found in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM), while the FosB/ChAT-IR neurons were found in the NTS, dorsal motor nucleus of the vagus (DMX) and nucleus ambiguus (AMB). In the control experiment, no significant difference in numbers was recognized in any of the double-IR neurons between the two strains. In the test experiment, the numbers of FosB/ChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. The numbers of c-Fos/TH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY. These results suggest that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR, and that a larger number of barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR.

Animals↗
PubMed1998

Topology and immunohistochemistry of proton-sensitive neurons in the ventral medullary surface of rats.

We aimed to clarify the topology and immunohistochemistry of CO2/H+-sensitive neurons in the ventral medullary surface (VMS), the central chemoreceptor area in rats. Inhalation of 3 and 7% CO2 in air significantly decreased pH in arterial blood and increased paCO2, which caused hyperpneic and tachypneic responses. Following inhalation of 3 and 7% CO2 in air for 5 min, the density of c-Fos-immunoreactive (IR) neurons increased stepwise not only in the 3rd-5th divisions of the VMS (between the caudal end of the nucleus corporis trapezoidei and the caudal end of the area postrema), but also in the rostroventromedial medulla (RVMM). Following inhalation of 7% CO2 in air for 5 min, glutamate-, glutamic acid decarboxylase (GAD)-, calcineurin- and cAMP-IR neurons were found not only in the VMS, but also in the RVMM. The topology of these neurons was similar to that of the c-Fos-IR neurons. No immunoreactivity was found for serotonin, substance P, somatostatin, cholecystokinin-octapeptide, methionine-enkephalin, choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, NO-synthase, S-100, calbindin-D, calmodulin, or parvalbumin. The densities of c-Fos-, glutamate-, GAD-, calcineurin- and cAMP-IR neurons were almost zero in the 1st division of the VMS, but became higher along the 2nd-4th divisions of the VMS. Regression lines of the density against the 1st-4th divisions of the VMS were significantly linear. These results indicate that H+-sensitive neurons are common in the 4th-5th divisions of the VMS, and that they are glutamatergic, GABAergic, and containing calcineurin and cAMP.

Administration, Inhalation↗
PubMed1998

Preferential selection of heterologous G3-VP7 gene in the genetic background of simian rotavirus SA11 detected by using a homotypic single-VP7 gene-substitution reassortant.

Introduction of segmented genomes into virion is an important process in viral replication of rotavirus. We previously studied the assortment of the VP7 gene segment (encoding outer capsid protein VP7) in the genetic background of simian rotavirus SA11 (G serotype 3, G3) and found the preferential selection of homologous G3 VP7 gene over VP7 gene of heterologous G serotype (G1, G2 or G4). In the present study, in order to clarify whether or not VP7 gene derived from different G3 rotavirus (heterologous G3-VP7 gene) is also preferentially selected in the SA11 background, a single-VP7 gene-substitution reassortant was prepared from SA11 through multiple steps of coinfection with rotaviruses in vitro. The isolated reassortant, SNR1, possessed VP7 gene derived from canine G3 rotavirus K9 and all other gene segments of SA11 origin, and showed an identical growth characteristic to that of SA11. Amino acid sequence of K9 VP7 gene showed a high degree of identity (93.6%) to SA11 VP7 gene. In analysis by mixed infection and multiple passages of SNR1 and a single VP7 gene (with G1, G2 or G4 specificity) reassortant in the SA11 background, the G3-VP7 gene became predominant at early passage numbers. However, in mixed infection with SA11 and SNR1, homologous G3-VP7 gene (SA11-VP7 gene) was preferentially selected into progenies over heterologous one (K9-VP7 gene). These results together with our previous findings suggested that G3-VP7 gene, irrespective of origin of species, was functionally adapted to the genetic background of SA11, although the homologous gene had a better fit with other SA11 genes than did heterologous one, providing suggestions for efficaciousness of multivalent reassortant rotavirus vaccine.

Amino Acid Substitution↗
PubMed1998

Molecular discrimination of enterotoxin C subtype in clinical isolates of Staphylococcus aureus.

The subtype of staphylococcal enterotoxin C (SEC) of 33 S. aureus clinical isolates was determined by polymerase chain reaction and direct DNA sequencing of a portion of the SEC gene encoding the SEC subtype-specific region. With the exception of a single strain with the SEC2 gene, all other strains showing different biologic and genetic properties were proved to possess the SEC3 gene.

DNA Primers↗
PubMed1998

Glomerular thromboxane contributes to pressor response in deoxycorticosterone acetate-salt hypertension.

To assess the role of renal thromboxane in a salt sensitive pressor response in hypertension, urinary excretion of thromboxane and its release from isolated glomeruli and renal papillae were examined in deoxycorticosterone acetate treated rats with normal (0.6%, n = 12) and high (4%, n = 12) salt diets for 8 weeks. Mean blood pressure, measured directly by an implanted aortic catheter, was higher in the high salt diet group than in the normal salt diet group (146 +/- 2 vs 119 +/- 2 mmHg, P<0.01). Urinary excretion of thromboxane B2 and 6-keto-prostaglandin F1alpha in the high salt group were significantly higher than those in the normal salt diet group, but there was no difference in urinary excretion of prostaglandin E2 between the two groups. Release of thromboxane B2, 6-keto-prostaglandin F1alpha, and prostaglandin E2 from isolated glomeruli in the high salt diet group increased significantly by 104%, 55%, and 74%, respectively, compared with the normal salt diet group. Stepwise multiple linear regression analysis showed that significant contributory factors for mean blood pressure in deoxycorticosterone acetate treated rats were urinary excretion of sodium (F=14.187, P<0.01) and release of thromboxane B2 from isolated glomeruli (F=4.135, P<0.05). The unstandardized coefficient (R) calculated from the regression function using these two factors was 0.875 and R2 was 0.765. The manifest synthesis of thromboxane in renal glomeruli has an important role on salt sensitive pressor response in deoxycorticosterone acetate-salt hypertension of rats.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Experience and clinical usefulness of BACTEC MGIT 960.

Recently, an automatic instrument for culturing acid-fast bacteria in liquid media has been introduced in Japan to accelerate and increase the detection of the bacteria as well as the work e ciency of test lab technicians. We compared the MGIT (Mycobacterium Growth Indicator Tube) method and the two conventional culture methods used in our lab by testing 176 specimens (130 cases) submitted for acid-fast testing between July 27 and September 18, 1998. BACTEC MGIT 960 (Nippon Becton Dickinson) was used in the MGIT method while Ogawa K medium and Kudo PD medium were used for our conventional culture methods. Of the 176 specimens, 26 specimens were MGIT positive, 22 specimens were Ogawa K medium positive, and 21 specimens were Kudo PD medium positive. Among these three culture methods, M. tuberculosis was found in 8 specimens, M. avium was found in 9 specimens, M. intracellulare was found in 4 specimens, and other acid-fast bacteria were found in 5 specimens. It took an average of 13.5 days to detect M. tuberculosis by the MGIT method, 23.0 days by the Ogawa K medium, and 24.3 days by the Kudo PD medium, suggesting that the MGIT method is signi cantly faster than the conventional methods (p < 0.05). The MGIT method took an average of 5.7 days to detect M. avium and M. intracellulare while the two conventional methods required more than 20 days, suggesting that the MGIT method is signi cantly faster than the conventional methods (p < 0.05). The BACTEC MGIT 960 was extremely useful for clinical diagnosis in some of the cases and our results indicated that the MGIT method led to more rapid diagnosis and earlier treatment than the conventional methods.

Adult↗
PubMed1998

[Survey of the sensitivities of clinical isolates to antibacterial agents (annual report)].

Research groups were formed in 21 institutions nationwide to investigate carbapenem resistance. The activities of various antibacterial agents, principally carbapenems were tested against clinical isolates collected from these institutions. The broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) of 17 antibacterial agents for 1,282 strains of 11 bacterial species isolated at all institutions between October and December 1995. The results were as follows: 1. Carbapenems exhibited strong antibacterial activities against MSSA and Streptococcus pneumoniae. Their activities against Enterococcus faecalis were comparable to that of ABPC. Carbapenems showed low activities against MRSA. 2. OFLX exhibited the greatest antibacterial activity against Haemophilus influenzae, followed by MEPM. The antibacterial activities of the other carbapenems were comparable to those of FMOX and CTM. 3. The carbapenems showed high activities against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Bacteroides fragilis group. Their activities were greater than that exhibited by other beta-lactam antibacterial agents. The carbapenems also exhibited greater antibacterial activities against Serratia marcescens than the other beta-lactam antibacterial agents, but some resistant strains were detected. 4. The antibacterial activities of carbapenems against Pseudomonas aeruginosa were comparable to those of CAZ, AZT, AMK.

Carbapenems↗
PubMed1997

In vitro study of H+-sensitive neurons in the ventral medullary surface of neonate rats.

We hypothesized that the direct stimulus of the central chemoreceptor neurons is the CO2/H+-induced change in intracellular pH (pHi). If it is true, pHi responses during hypercapnic stimulation should be exhibited in the central chemoreceptor neurons in the ventral medullary surface (VMS) and some neurons in the CO2/H+ sensitive regions such as the nucleus tractus solitarii of the medial dorsal medulla (MDM). To test this hypothesis, the cultured VMS and MDM neurons (control) derived from one day-old neonate rats were labeled with H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and were exposed to perfusate of various pHs. The H+-sensitive neurons were determined by a rapid decrease in the intracellular BCECF fluorescence intensity. In almost all the MDM neurons (99.6%) and 94% of the VMS neurons, the intracellular BCECF fluorescence intensity remained unchanged when the extracellular pH (pHo) was decreased. In contrast, in 0.4% of the MDM neurons (8/1800) and in 6% of the VMS neurons (111/1800), the intracellular BCECF fluorescence intensity decreased when the pHo was decreased from 7.4 to 7.2. This subpopulation of MDM and VMS neurons were considered to be H+-sensitive neurons. The H+-sensitive neurons in the VMS showed positive immunoreactivity to glutamate (57%, 17/30) and glutamic acid decarboxylase (23%, 7/30), but no immunoreactivity to choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, somatostatin, serotonin and substance P. These results indicate that the H+-sensitive neurons are present specifically in the VMS, and are mainly glutamatergic and GABAergic.

Animals↗
PubMed1997

Barosensitive cardioinhibitory neurons in the medulla: comparison of FosB/ChAT-positive neurons with CT-HRP-labeled neurons.

The purpose of this study was to survey the distribution pattern of barosensitive cardioinhibitory preganglionic neurons in the medulla. This was done using Wistar rats anesthetized with fentanyl/midazolam. After stimulating arterial baroreceptors by blood pressure increase due to phenylephrine, c-Fos, FosB, c-Jun and JunD/choline acetyltransferase (ChAT)-positive neurons were surveyed in the medulla. After placing HRP conjugated by cholera toxin (CT-HRP) on the sino-atrial node, CT-HRP-labeled neurons were surveyed in the medulla. In the phenylephrine pressor test experiment, we ascertained that in the target neurons fosB was more sensitive to baroreceptor stimulation than any other immediate early genes such as c-fos, c-jun and junD. Using the FosB/ChAT method, we succeeded in determining sites of the barosensitive cardioinhibitory neurons that had never been found with the c-Fos/ChAT method. The distribution pattern of the FosB/ChAT-positive neurons was compared with that of the CT-HRP-labeled neurons. We found that the distribution pattern of the FosB/ChAT-positive neurons in the dorsal motor nucleus of the vagus nerve (DMX) and ambiguus nucleus (AMB) was similar to that of the CT-HRP-labeled neurons at the level between the caudal end of the area postrema (AP) and the caudal end of the nucleus of the trapezoid body. This suggests that the barosensitive FosB/ChAT-positive neurons in the DMX and AMB mediate the cardioinhibitory baroreceptor reflex.

Animals↗
PubMed1997

Six cases positive for anti-centromere antibodies with ulcer and gangrene in the extremities.

We report six cases that were positive for anti-centromere antibodies, with ulcer and gangrene in the extremities but mild or no skin thickening. The patients were five women and one man, and the mean age at onset of gangrene was 56 yr. Raynaud's phenomenon was found in five patients and calcinosis cutis in two patients. Three patients did not satisfy the criteria for systemic sclerosis and CREST syndrome in this study. Ulcer and gangrene occurred in the fingers in three patients, and in the fingers and toes in two patients. The gangrene was refractory to treatment, and amputation of fingers or toes was inevitable in five patients. Regardless of cutaneous lesion, the presence of anti-centromere antibodies may cause the same pathological presentation of vascular damage as seen in systemic sclerosis.

Adult↗
PubMed1997

[Effect of daily administration of oral etoposide for non-small cell lung cancer treated with concurrent radiation therapy].

The purpose of this study was to explore the effect of daily administration of oral etoposide (25 mg) for patients with non-small cell lung cancer treated with concurrent radiation therapy. Planned endpoints were response, survival and toxicity. Forty-one patients with non-small cell lung cancer were divided into 25 patients given daily oral etoposide (25 mg) with concurrent radiation therapy (ERT group) and 16 patients given radiation therapy alone (RT group). Etoposide was administrated in the morning throughout radiation therapy. The median total irradiated dose was 63.1 Gy in the ERT group and 64.0 Gy in the RT group. Twenty-four patients completed therapy in the ERT group and 16 in the RT group. Three (15%) ERT patients achieved complete response (CR) and 9 (45%) achieved partial response (PR). In the RT group, no patients achieved CR and 9 (69%) achieved PR. One-year survival was 22.6% with ERT and 23.0% with RT. The prognosis of stage III patients in the ERT group was worse than that in the RT group because radiation pneumonitis and radiation esophagitis were more severe with ERT. In conclusion, the ERT group had better local response but worse in survival than the RT group. Complications of ERT were severe enough to cause death in some patients. ERT had no clear advantage over RT.

Administration, Oral↗
PubMed1997

[Survey of sensitivities of clinical isolates to antibacterial agents (annual report)].

Research groups were formed in 20 institutions nationwide to investigate carbapenem resistance of clinical isolates. Activities of various antibacterial agents, principally carbapenems, were tested against clinical isolates collected from these institutions. The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of 17 antibacterial agents for 1,326 strains of 11 bacterial species isolated at the institutions between October and December 1994. The results are as follows: 1. Carbapenems exhibited strong antibacterial activities against MSSA and Streptococcus pneumoniae. Their activities against Enterococcus faecalis were comparable to that of ABPC. Carbapenems showed low activities against MRSA. 2. OFLX exhibited the greatest antibacterial activity against Haemophilus influenzae, followed by MEPM. Antibacterial activities of the other carbapenems were comparable to those of FMOX, CTM, and ABPC. 3. The carbapenems showed high activities against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Bacteroides fragilis group. Their activities were greater than those exhibited by other beta-lactam antibacterial agents. The carbapenems also exhibited stronger antibacterial activities against Serratia marcescens than the other beta-lactam antibacterial agents, but some resistant strains were detected. 4. The antibacterial activities of carbapenems against Pseudomonas aeruginosa were comparable to those of CAZ, AZT, AMK.

4-Quinolones↗
PubMed1996

Improved ex vivo/in vitro lymph node cell proliferation assay in guinea pigs for a screening test of contact hypersensitivity of chemical compounds.

Simple and efficient ex vivo/in vitro screening systems for contact allergens are developed for alternative to conventional animal tests. We have previously proposed an ex vivo/in vitro proliferation assay as a first stage screening method with advantages over existing alternatives, using lymph node cells (LNC) from sensitized guinea pigs of the Hartley strain. In this study, we have first confirmed, by histochemical analysis using in vivo bromodeoxyuridine (BrdU) and pyronin staining, that the ex vivo/in vitro LNC proliferation reflects in vivo response of lymph nodes to contact allergens. Furthermore, to improve the LNC assay, we then have investigated several experimental conditions for their influences on the LNC assay, demonstrating that, (1) the subscapular and the cervical LNC responded highly to contact allergens, (2) among three cervical lymph nodes the superficial dorsal cervical lymph nodes were the most reactive, (3) several vehicles alone used for animal sensitization exhibited little influence on the LNC proliferation, (4) employment of stimulation index offset the inter-batch fluctuation of the LNC proliferation in the control animals as baseline proliferation. Under optimized experimental conditions as above, experimentally determined stimulation indexes of several contact allergens correlated well with their sensitizing potential estimated by conventional animal tests. Therefore, the ex vivo/in vitro LNC proliferation assay should be a simple and efficient alternative to conventional guinea pig testings including the guinea pig maximization test (GPMT).

Animal Testing Alternatives↗
PubMed1996

Barosensitive and chemosensitive neurons in the rat medulla: a double labeling study with c-Fos/glutamate, GAD, PNMT and calbindin.

The purpose of this study was to survey distribution and density of the barosensitive and chemosensitive neurons in the medulla of rats anesthetized with fentanyl/midazolam, using immunohistochemical methods. After stimulation of the arterial baroreceptor or the chemoreceptor, we identified c-Fos-labeled neurons with immunoreactions to antisera of glutamate. PNMT, GAD and calbindin in the nucleus tractus solitarii (NTS) and the ventrolateral medulla (VLM). The double labeled neurons were located in the medical part of the NTS, and in the lateral part of the paragigantocellular reticular nucleus and the ventral division of the ambiguus nucleus. Main findings were as follows: (1) No significant difference was found in distribution and density of glutamatergic, adrenergic and calbindin-containing neurons between the barosensitive and chemosensitivie types; (2) a few GABAergic neurons were distributed almost evenly in the NTS and VLM, and in these neurons the barosensitive type outnumbered the chemosensitive one; (3) glutamatergic and calbindin-containing neurons were dominant in the NTS; adrenergic neurons in the VLM. (4) as for the adrenergic neurons in the NTS, the chemosensitive type significantly outnumbered the barosensitive one. This study showed that distribution and density of the barosensitive neurons, either glutamatergic, adrenergic, or calbindin-containing neurons, overlapped with those of the chemosensitive corresponding neurons, suggesting presence of the neural matrix of the cardiopulmonary interaction. Exceptionally, the number of the barosensitive GABAergic neurons was significantly larger than that of the chemosensitive GABAergic ones.

Animals↗