Biomedical subjects

M Inoue

Publications and source records attributed to M Inoue.

At least 91 records · Page 5Linked to original sources

PubMed1998

Expression of thyrotropin receptor on clonal osteoblast-like rat osteosarcoma cells.

The expression of thyrotropin receptor (TSH-R) on various cells derived from bone, including osteoblast-like rat osteosarcoma cells (UMR106 cells), was investigated. TSH receptor mRNA was detected in UMR106 cells by Northern blot analysis. 125I-labeled TSH binding analysis revealed specific high- and low-affinity binding sites (association constants of 5.6 x 10(9) M(-1) and 3.0 x 10(7) M(-1), respectively) on UMR106 cells. Recombinant TSH, but not recombinant human chorionic gonadotropin, increased cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner in these cells. Furthermore, immunoglobulin Gs from patients with Graves' disease induced cAMP response in UMR106 cells, and the cAMP response index in this cell line correlated with thyroid-stimulating antibody (TSAb) activity detected by Chinese hamster ovary (CHO)-K1 cells transfected with rat TSH-R. We have also demonstrated that recombinant TSH increased cAMP production in human osteoblast-like osteosarcoma (MG63) cells and mouse primary osteoblastic cells. These results suggest that osteoblasts possess functional TSH-R and that abnormal bone metabolism in Graves' disease may be partly explained by the interaction of TSAb with TSH-R in osteoblasts in some patients.

Animals↗

PubMed1998

Reduction of the suppressive effects of gurmarin on sweet taste responses by addition of beta-cyclodextrin.

Cyclodextrins (CDs) have the remarkable ability to form inclusion complexes with a wide variety of guest molecules. In the present study, possible influences of CDs on gurmarin inhibition of the chorda tympani responses to sucrose were examined in C57BL mice. Responses to sucrose were suppressed to approximately 50% of control by treatment of the tongue with 30 micrograms/ml (approximately 7.1 microM) gurmarin. Rinsing the tongue with 15 mM beta-CD after gurmarin gave rapid recovery of the suppressed sucrose responses to approximately 85% of control, whereas 15 mM alpha- or gamma-CD did not. When gurmarin was mixed with beta-CD, the suppressive effects of gurmarin on sucrose responses were largely reduced. No such reduction was observed for mixtures with alpha- and gamma-CD. Gurmarin includes tyrosine and tryptophan residues whose aromatic rings are directed outward and can probably form inclusion complexes with beta-CD. Therefore, the observed reduction of the effects of gurmarin may be due to steric hindrances in inclusion complexes of gurmarin with beta-CD that may interfere with gurmarin binding to sweet taste receptors.

Animals↗

PubMed1998

Relationship between human in-vitro fertilization and intracytoplasmic sperm injection and the zona-free hamster egg penetration test.

The zona-free hamster egg penetration test (HEPT) is widely used for evaluating the fertilizing ability of human spermatozoa. However, the relationship between the HEPT and microassisted fertilization has yet to be determined. To evaluate the efficiency of HEPT in selecting the most appropriate method of in-vitro fertilization (IVF), including intracytoplasmic sperm injection (ICSI) in couples with male factor infertility, clinical laboratory data was analysed retrospectively. The patients were divided into groups according to the sperm penetration index as determined by the HEPT: group A (sperm penetration index = 0), group B (sperm penetration index < 15) and group C (sperm penetration index > or = 15). A total of 405 oocytes were collected and inseminated by conventional methods in 69 couples with male factor infertility. In all, 31 out of 148 (20.9%) oocytes fertilized in group A; 35 out of 117 (29.9%) in group B; and 73 of 140 (52.1%) in group C. The clinical pregnancy rates per transfer in groups A, B and C were 0% (0/13), 0% (0/14) and 25.9% (7/27) respectively. Both the fertilization rate and pregnancy rate in group C was significantly higher than in groups A and B. ICSI was carried out in a total of 57 couples and 334 oocytes in metaphase II stage were manipulated. The normal fertilization (2 pronuclear) rate per oocyte was 65.6 +/- 26.0% (mean +/- SD). Out of 127 oocytes, 76 (59.8%) fertilized in group A, 57 out of 87 oocytes (65.5%) in group B and 86 out of 120 oocytes (71.7%) in group C. Of the 56 transfers, 17 clinical pregnancies were obtained, giving an average pregnancy rate of 30.4% per transfer. The clinical pregnancy rates per transfer in groups A, B and C were 17.4% (4/23), 40.0% (4/10) and 39.1% (9/23) respectively. No significant differences were observed in the fertilization rates or in the pregnancy rates between the three groups. In addition, there were no differences in the fertilization and pregnancy rates between the ICSI and IVF patients in group C. These findings suggest that the results of the HEPT are well correlated with the fertilizing ability of human spermatozoa in the patients treated by conventional IVF. Couples suffering from male factor infertility with a sperm penetration index of < 15 (as determined by HEPT) should consider treatment with ICSI, while those with a sperm penetration index of > or = 15 should attempt conventional IVF.

Animals↗

PubMed1998

Evaluation of the death certificate follow-up method for the analysis of survival rate: data from Aichi Prefecture, Japan.

To evaluate the use, for studies on survival, of death certificates from population-based cancer registries in Japan, we compared 5-year survival rates by two different methods: passive, derived from death certificates, and active, using family register systems. Registered cancer cases from Aichi Prefectural Cancer Registry were used as a model. The study subjects comprised 9244 cancer patients (3830 males and 5414 females) newly diagnosed at the Aichi Cancer Center Hospital and recorded in the Aichi Prefectural Cancer Registry between 1983 and 1991. The passive follow-up method, using death certificates, identified 87-89% of deaths among the study subjects. The proportion of deaths which were not identified by the passive follow-up method did not vary greatly with age or gender, but was higher among patients in the earlier rather than the later stages of cancer. Overall, the absolute and relative effects of unregistered deaths on apparent survival rate vary with the absolute survival rate. The absolute and relative differences between the survival rates calculated by the two methods are explained more clearly when data are analyzed by cancer site. The results of the present study provide useful information for interpreting the survival rate following diagnosis of cancer estimated by the passive follow-up method, i.e. using death certificates from a population-based cancer registry.

Aged↗

PubMed1998

Zinc deficiency enhances interleukin-1alpha-induced metallothionein-1 expression in rats.

This study investigated whether interleukin-1alpha-induced metallothionein gene expression is affected by zinc deficiency. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) or a zinc-supplemented diet [50.8 mg zinc/kg; controls for the diet included pair-fed (PF) and ad libitum consumption groups (AL)] for 4 wk. All rats except those that served as controls for interleukin-1alpha administration, (injected with vehicle and killed at 0 h) were then injected subcutaneously with interleukin-1alpha (2 x 10(7) units/kg body wt) and killed at 3, 6, 12, 24 and 72 h after the injection. Compared with AL and/or PF rats, zinc depletion significantly reduced zinc concentrations in plasma and liver but not in kidney or intestine, and significantly reduced hepatic, renal, and intestinal metallothionein-1 mRNA levels analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR). Interleukin-1alpha injection reduced plasma zinc concentration and enhanced liver zinc concentration, but did not affect zinc levels in kidney or intestine. Metallothionein-1 mRNA was significantly elevated by interleukin-1alpha in liver, kidney and intestine of all groups; the levels in liver and kidney of ZD rats 6 h after the injection were significantly higher than those of AL or PF rats. Liver metallothionein protein levels were enhanced after interleukin-1alpha injection in both AL and ZD rats. Semiquantitative RT-PCR revealed significantly higher hepatic levels of interleukin-1 receptor type-I mRNA in ZD rats than in AL and PF rats but no differences in renal or intestinal tissues among groups before interleukin-1alpha challenge. In conclusion, zinc deficiency induces upregulation of metallothionein-1 gene expression in response to interleukin-1alpha challenge in rats.

Animals↗

PubMed1998

Hydatidiform moles associated with multiple gestations after assisted reproduction: diagnosis by analysis of DNA fingerprint.

In spite of the widespread use of assisted reproductive technology, there have been, to our knowledge, only two reported cases of molar pregnancies after gamete intra-Fallopian transfer and five reported cases after in-vitro fertilization and embryo transfer. We report here a case of a complete hydatidiform mole in a twin pregnancy after gamete intra-Fallopian transfer, as well as a case of a complete hydatidiform mole in a triplet pregnancy after in-vitro fertilization and embryo transfer. The genetic constitution of each conceptus was determined by examination of the restriction fragment length polymorphism of the DNA with four different single-locus probes. This analysis revealed that both hydatidiform moles were of androgenetic origin and probably of monospermic origin. Moreover, the analysis confirmed that the pregnancies were dizygotic and trizygotic pregnancies respectively. The diagnostic utility of the analysis of DNA polymorphism is discussed in cases of a molar pregnancy with coexisting fetuses.

Adult↗

PubMed1998

Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes.

The role of mitogen-activated protein kinase (MAPK) in the meiotic resumption of porcine oocytes was examined. First, using indirect immunofluorescence staining with a specific antibody against rat MAPK, we monitored the dynamics of the subcellular distribution of MAPK during meiosis initiation. We found that the inactive MAPK was already present in immature oocytes arrested at the G2 stage and that this inactive kinase was localized exclusively in the cytoplasm. At the G2/M transition stage, part of the MAPK moved into the germinal vesicle (GV) before germinal vesicle breakdown (GVBD). In addition, immunoblot analysis showed that the nuclear MAPK existed in an active form. To determine whether this active MAPK could induce GVBD, we microinjected active MAPK into immature porcine oocytes. The active MAPK injected into the cytoplasm was quickly inactivated and could not accelerate GVBD. In contrast, MAPK injection into the GV markedly accelerated GVBD. These results show that in porcine oocytes, 1) inactive MAPK localizes in the cytosol of immature GV oocytes, 2) part of the activated MAPK translocates into the GV just before GVBD, and 3) exogenous MAPK maintains its activity level in the GV and induces GVBD, indicating that MAPK mediates the maturation-inducing signal from the cytoplasm into the nucleus and induces meiosis reinitiation.

Animals↗

PubMed1998

Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells.

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1 alpha and ECE-1 beta. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1 alpha mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1 alpha mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress.

Animals↗

PubMed1998

Oxidative stress suppresses the endothelial secretion of endothelin.

To address endothelial function on vascular walls exposed to oxidative stress, we investigated the effect of oxidative stress on the secretion of endothelin-1 (ET-1) from cultured bovine carotid artery endothelial cells (BAECs). Concentrations of ET-1 in the media were measured by a specific radioimmunoassay and ET-1 mRNA expression was estimated by Northern blot analysis. Treatment of BAECs with 0.5-2.0 mM H2O2 for 3 h suppressed both ET-1 secretion and ET-1 mRNA expression in a dose-dependent manner compared to control. Attenuation of ET-1 mRNA expression by H2O2 was revealed to take place at the transcriptional level. The addition of NG-nitro-L-arginine-methyl ester (L-NAME) 10 microns, a specific nitric oxide synthase inhibitor, had no effect on H2O2-induced suppression of ET-1 mRNA expression. Suppression of ET secretion under oxidative stress observed in the present study is proposed to be a compensatory mechanism of endothelial cells to inhibit vasoconstriction and proliferation during oxidative stress.

Animals↗

PubMed1998

Thermal injury induces thymocyte apoptosis in the rat.

BACKGROUND: The thymus plays important roles in host defense, which may be impaired after burn injury. The effects of thermal injury on thymocytes were investigated in male Wistar rats. METHODS: Changes in thymus weight and content of glutathione and corticosterone were determined after burn injury. Apoptosis of thymocytes was detected by electrophoresis of DNA, and lymphocyte subsets were characterized by flow cytometry. The effects of adrenalectomy and the glucocorticoid receptor antagonist RU486 in burned animals were also studied. RESULTS: The weight of the thymus decreased progressively after burn injury, and this effect was accompanied by increases in the corticosterone concentration in plasma and the thymus and apoptosis of CD4+CD8+ thymocytes. Administration of RU486 or adrenalectomy inhibited burn-induced thymocyte apoptosis. CONCLUSION: Thermal injury increases the corticosterone concentration in plasma and the thymus and triggers thymocyte apoptosis.

Adrenalectomy↗

PubMed1998

Characterization of mRNA expression of IkappaB alpha and NF-kappaB subfamilies in primary adult T-cell leukemia cells.

Tax protein of HTLV-1 activates the transcriptional capacity of the NF-kappaB family, resulting in up-regulation of various genes, which are linked to phenotypic alterations of HTLV-1-infected T cells. To understand NF-kappaB regulation in HTLV-1-infected leukemic cells in vivo, we analyzed expression of NF-kappaB and IkappaB alpha in primary cells isolated from ATL patients. Using competitive polymerase chain reaction, we observed an elevated expression of IkappaB alpha mRNA in all four ATL cases tested. In contrast to the elevated mRNA levels, the levels of IkappaB alpha protein were remarkably reduced in some of these cases, suggesting destabilization of IkappaB alpha protein. On the other hand, mRNA expression of p50/p105 and p65, subfamilies of NF-kappaB, was enhanced in primary cells isolated from some ATL patients. Furthermore, the expression patterns of NF-kappaB subfamily were variable among patients and also different from those in T cells isolated from uninfected individuals. Although the number of cases analyzed was limited, we can conclude from these observations that activation of NF-kappaB is restricted to a few subfamilies in vivo. These findings in vivo are strikingly different from those in HTLV-1-infected T cell lines in vitro, in which Tax is responsible for NF-kappaB activation. It is therefore suggested that the elevation of NF-kappaB expression in leukemic cells of ATL patients might not be supported mainly by the viral protein Tax.

Blotting, Western↗

PubMed1998

Lung cancer patients have increased 8-hydroxydeoxyguanosine levels in peripheral lung tissue DNA.

The 8-hydroxydeoxyguanosine (8-OH-dG) levels in the peripheral parts of human lung tissues were compared between lung cancer patients (n=70) and non-cancer patient controls (n=15). An increased level of 8-OH-dG was observed in the lung cancer group, in both the adenocarcinoma and non-adenocarcinoma (mainly squamous cell carcinoma) groups, as compared to the non-cancer control group. This result suggests that reactive oxygen species are partly involved in the induction of lung carcinomas (both adenocarcinoma and non-adenocarcinoma).

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Effects of inhaled nitric oxide on platelet-activating factor-induced pulmonary hypertension in dogs.

BACKGROUND: Platelet-activating factor (PAF), a lipid mediator released during endotoxin shock, induces pulmonary hypertension, systemic hypotension and cardiac dysfunction. In this study, we compared the effect of inhaled nitric oxide (NO) on PAF-induced pulmonary hypertension and NO metabolism with that on pulmonary hypertension induced by a stable thromboxane A2 mimetic, U46619. Since PAF-induced hypotension might be mediated by NO, the effect of inhaled NO combined with an intravenously administered NO synthase inhibitor, NG-nitro-L-arginine (L-NNA), on PAF-induced hemodynamic change was also investigated. METHODS: In a total of 28 beagles anesthetized with pentobarbital the following substances were intravenously administered: PAF 0.56 +/- 0.30 microgram.kg-1.min-1 (group PAF), L-NNA 10 mg.kg-1 + PAF 0.04 +/- 0.03 microgram.kg-1.min-1 (group L-NNA + PAF), U46619 0.60 +/- 0.11 microgram.kg-1.min-1 (group U46619) or L-NNA 10 mg.kg-1 + U46619 0.61 +/- 0.23 microgram.kg-1.min-1 (group L-NNA + U46619) to obtain a mean pulmonary arterial pressure (MPAP) of 25 mmHg. Nitric oxide was then inhaled at 5, 10, 20 and 40 ppm for 15 min at 15-min intervals in the order of increasing concentration. An additional 7 dogs (control group) inhaled NO at normal MPAP (17 mmHg). Hemodynamic and respiratory parameters, NOHb, NO2- + NO3-, and MetHb levels in blood were measured before and during NO administration. RESULTS: In the control group, hemodynamic and respiratory values did not change significantly during NO administration. In group PAF, NO significantly reversed the PAF-induced pulmonary hypertension. PAF induced a marked systemic hypotension and cardiac output reduction, but these changes were not affected by inhalation of NO. L-NNA pretreatment markedly decreased the dose of PAF required to maintain a MPAP of 25 mmHg, and abolished the PAF-induced hypotension. In group L-NNA + PAF, the diminishing effect of inhaled NO on pulmonary vascular resistance (PVR) was significantly greater than that in group PAF. U46619 induced pulmonary hypertension and increases in blood pressure, intrapulmonary shunt and peak airway pressure. L-NNA pretreatment did not change the dose of U46619 required to maintain a MPAP of 25 mmHg. The effects of inhaled NO on PVR decrease were similar in groups U46619 and L-NNA + U46619. No NOHb was detected in any group. NO2- + NO3- concentration increased during NO administrations. There were no significant differences in NO2- + NO3- concentration among groups. CONCLUSIONS: Inhaled NO at the dose of 5-40 ppm effectively reversed PAF-induced pulmonary hypertension, especially following pretreatment with L-NNA. Inhaled NO did not affect PAF-induced hypotension or cardiac dysfunction. These findings indicate that low concentrations of inhaled NO may be useful in reversing pulmonary hypertension in the endotoxemia where PAF may be one mediator.

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

PubMed1998

NER rat strain: a new type of genetic model in epilepsy research.

PURPOSE: We characterized and evaluated as an animal model of epilepsy NER, a new epileptic rat strain, which was developed by inbreeding rats with spontaneous tonic-clonic seizures in a stock of Crj:Wistar. METHODS: Animals were monitored through the inbreeding course, and video-EEGs were recorded selectively. External seizure-provoking stimuli were applied to NER and to a control parental strain. F1, F2, and backcross progenies were produced between NER and a nonepileptic unrelated strain. Pathologic study included hematoxylin-and-eosin (HE), Klüver-Barrera's, modified Bodian silver, and neo-Timm's staining. RESULTS: After the F9 generation, 94%-98% of NER exhibited spontaneous tonic-clonic convulsions, beginning with neck and forelimb clonus, wild jumping/running, opisthotonic posturing, and evolving to tonic, then clonic convulsion, followed by postictal flaccidity. Most seizure onsets occurred between 2-4 months of age, and the incidence was 0.45 +/- 0.21 seizures in 12 h. Ictal cortical and hippocampal EEGs were characterized by high-voltage spikes followed by diffuse spike-and-wave or polyspike-and-wave complexes. NER revealed seizure susceptibility to pentylenetetrazol, tossing, and transcorneal electroshock, but not to tactile, photic, or acoustic stimuli, or to transauricular electroshock. Mating experiments revealed that 0% (0/46) of the animals in F1, 25.5% (13/51) in F2, and 63.6% (56/88) in backcross progenies exhibited spontaneous tonic-clonic convulsions without sex difference. For all these epileptic traits, no pathologic changes were demonstrated in the CNS. CONCLUSIONS: NER frequently exhibited spontaneous convulsions, controlled by a major autosomal recessive gene for epilepsy, that are comparable to generalized tonic-clonic seizures in humans. This can serve as a new genetic model in epilepsy research.

Animals↗

PubMed1998

Evidence that folliculo-stellate cells mediate the inhibitory effect of Japanese kampo medicine, unkei-to, on growth hormone secretion in rat anterior pituitary cell cultures.

PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release. We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated. METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH3 cell were used to test the effect of Unkei-to on GH secretion. In reconstitutive coculture experiments, TtT/GF cells were mixed with GH3 cells at a ratio (TtT/GF cells: GH3 cells) of 1:99. From this mixture, cells were seeded onto plates at a density of 10(4) cells/well and were cultured for 5 days. The cells were then used in the experiments. RESULTS: Unkei-to at 20 micrograms/ml significantly inhibited GH secretion by normal anterior pituitary cells within 12 hr of incubation. In contrast Unkei-to stimulated GH secretion by GH3 cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved. To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH3 cells were cocultured with TtT/GF cells. The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion. CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells.

Animals↗

PubMed1998

The herbal medicine unkei-to stimulates cytokine-induced neutrophil chemoattractant production in the pituitary folliculo-stellate-like cell line (TtT/GF).

PROBLEM: We previously reported that a cytokine-induced neutrophil chemoattractant (CINC) was produced in the pituitary gland and that it influenced anterior pituitary hormone release. In this study we investigated the effect of Unkei-to, a Japanese herbal medicine, on CINC production in the rat anterior pituitary gland and the pituitary folliculo-stellate-like cell line (TtT/GF). METHOD OF STUDY: Dispersed normal anterior pituitary cells and the folliculo-stellate-like cell line TtT/GF were used to test the effect of Unkei-to on CINC secretion and CINC mRNA accumulation. Concentrations of CINC in the conditioned media were measured by an enzyme-linked immunosorbent assay, and levels of CINC mRNA were analyzed by Northern blot analysis. RESULTS: Unkei-to (20 micrograms/ml) significantly increased the secretion of CINC by normal anterior pituitary cells within 12 hr of incubation. Unkei-to also stimulated CINC secretion from TtT/GF cells in a time- and dose-dependent manner. Unkei-to (20 micrograms/ml) increased CINC mRNA accumulation in TtT/GF cells within 3 hr of incubation and also caused a 13-fold increase in the secretion of CINC from TtT/GF cells compared with the vehicle group within 24 hr of incubation. Finally, we found that some of the Unkei-to's ingredients, Evodiae fructus and Pinelliae tuber, markedly stimulated CINC secretion from TtT/GF cells. CONCLUSIONS: Our results will help to elucidate the mechanism behind the clinical effect of Unkei-to on the anterior pituitary gland. They also suggested the presence of special substances, which stimulate CINC secretion, within Unkei-to's ingredients such as E. fructus and P. tuber.

Animals↗

PubMed1998

[A mechanism of clarithromycin resistance in Helicobacter pylori].

The aim of this study was to elucidate the mechanism of clarithromycin (CAM) resistance in laboratory strains and clinical isolates of Helicobacter pylori. The CAM resistance in laboratory strains was induced in vitro by CAM exposure. The majority of CAM-resistant strains were highly resistant to CAM (MICs > 100 micrograms/ml). These CAM-resistant strains also showed cross resistance to azithromycin, rokitamycin and clindamycin. The sites of point mutations in these resistant strains were identified as follows; the conserved domain V of genes encoding 23S rRNA were amplified first by PCR and this PCR products (1.4 kb) were subsequently digested with BsaI and MboII and RFLP patterns were analyzed. 1.4 kb amplicons of CAM-susceptible strains yielded two DNA bands of 1000 bp and 400 bp when digested with BsaI but no digestion product was seen by MboII digestion. In contrast to this, two types of RFLP patterns were observed for the resistant strains induced in vitro by CAM; one was the formation of three bands (700 bp, 400 bp and 300 bp) after BsaI digestion, and the other was the formation of two bands (approximately 700 bp) by MboII digestion. RFLP patterns of CAM-susceptible and CAM-resistant clinical isolates obtained from patients before and after CAM medication were similar to those observed for the CAM-susceptible strains and CAM-resistant strains developed in the laboratory. These results strongly suggest that the CAM resistance of H. pylori was caused by point mutation of 23S rRNA.

Anti-Bacterial Agents↗

PubMed1998

Activation of Ca(2+)-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells.

The effects of cyanide (CN) on whole cell current measured with the perforated-patch method were studied in adrenal medullary cells. Application of CN produced initially inward and then outward currents at -52 mV or more negative. As the membrane potential was hyperpolarized, amplitude and latency of the outward current (Io) by CN became small and long, respectively. A decrease in the external Na+ concentration did not affect the latency for CN-induced Io but enhanced the amplitude markedly. The CN Io reversed polarity at -85 mV, close to the Nernst potential for K+, and was suppressed by the K+ channel blockers curare and apamin but not by glibenclamide, suggesting that Io is due to the activation of Ca(2+)-dependent K+ channels. Consistent with this notion, the Ca(2+)-mobilizing agents, muscarine and caffeine, also produced Io. Exposure to CN in a Ca(2+)-deficient medium for 4 min abolished caffeine- or muscarine-induced Io without development of Io, and addition of Ca2+ to the CN-containing solution induced Io. We conclude that exposure to CN produces Ca(2+)-dependent K+ currents in an external Ca(2+)-dependent manner, probably via facilitation of Ca2+ influx.

Adrenal Medulla↗