Search PubMed⌕ Search

Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 361 records · Page 20Linked to original sources
PubMed1991

Oscillatory muscarinic acetylcholine responses of Xenopus oocytes are desensitized by protein kinase C and sensitized by protein phosphatase 2B.

The oscillatory current response to acetylcholine (ACh) in Xenopus laevis oocytes, mediated by endogenous muscarinic ACh receptors, is known to be mildly desensitized by repetitive ACh applications. Pretreatment of oocytes with staurosporine (an inhibitor of protein kinases) was found not only to abolish this desensitization but also to positively and progressively potentiate oscillatory ACh responses. This sensitization by staurosporine was suppressed by 12-O-tetradecanoylphorbol 13-acetate (an activator of protein kinase C). In staurosporine-untreated (control) oocytes, intracellularly injected calcineurin (an isozyme of Ca2+/calmodulin-dependent protein phosphatase 2B) or Ca2+ enhanced oscillatory ACh responses, while trifluoperazine (a calmodulin inhibitor) suppressed the ACh responses but did not affect oscillatory responses to intracellularly injected inositol 1,4,5-trisphosphate. These results suggest that, as far as short-term changes in receptor responsiveness are concerned, endogenous muscarinic ACh receptors in Xenopus oocytes are desensitized by phosphorylation by protein kinase C and sensitized by dephosphorylation by Ca2+/calmodulin-dependent protein phosphatase 2B.

Acetylcholine↗
PubMed1991

Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit.

Biotin carboxylase [biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14] is the enzyme mediating the first step of the acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] reaction. We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned. The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene. Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme. The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation. The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]; therefore, all these enzymes probably function through the same mechanism of reaction.

Acetyl-CoA Carboxylase↗
PubMed1991

Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase.

cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction.

Amino Acid Sequence↗
PubMed1991

Inhibitory effect of cadmium and mercury ions on transcription of the ada gene.

The effect of Cd2+ and Hg2+ on transcription of the ada gene was examined using an in vitro system reconstituted with Ada protein and methylated DNA, together with their effect on methyltransferase activity of the Ada protein. Both Cd2+ and Hg2+ inhibited transcription of the ada gene over the doses which did not inhibit methyltransferase activity.

Bacterial Proteins↗
PubMed1991

Endogenous angiotensin II receptors in Xenopus oocytes and eggs.

Angiotensin II (AII) induced strongly desensitizing oscillatory Cl- inward currents in both follicle-enclosed and collagenase-treated Xenopus oocytes. The AII response was abolished by EGTA and attenuated by pertussis toxin. Treatment of oocytes with collagenase transiently reduced both the ratio of oocytes responsive to AII and the amplitude of AII responses, followed by restoration to original levels in 3-4 days. The response to adrenaline, which is mediated by endogenous beta-adrenoceptors in follicle cells, however, was irreversibly abolished by collagenase treatment. These results suggest that endogenous current-mediating AII receptors in oocytes are coupled with phosphatidylinositol hydrolysis and localized in the oocyte or in a cellular structure distinct from that for endogenous beta-adrenoceptors. Progesterone-matured Xenopus eggs also responded to AII, and this AII-induced depolarization resembled the fertilization potential in the eggs, suggesting a possible role of AII receptors in processes of fertilization or growth of the eggs.

Acetylcholine↗
PubMed1991

Desensitization of endogenous angiotensin II receptors in Xenopus oocytes: a role of protein kinase C.

The inward chloride current induced by angiotensin II (AII) in Xenopus oocytes shows strong and homologous desensitization, and was suggested to be mediated by phosphatidylinositol (PI) hydrolysis (Sakuta et al., 1991, Eur. J. Pharmacol. Mol. Pharmacol. 208, 31). As a model of agonist-induced desensitization of receptors coupled with PI hydrolysis, the mechanism of the desensitization of endogenous AII receptors in oocytes was investigated. Incubation of collagenase-treated oocytes with staurosporine significantly augmented the peak amplitude of AII responses, prolonged their duration, and increased the ratio of oocytes responsive to AII. Moreover, staurosporine-pretreatment made oocytes be consistently responsive to every application of AII. These effects of staurosporine were inhibited by incubation of staurosporine-treated oocytes with 12-O-tetradecanoylphorbol 13-acetate (TPA) but not with dibutyryl cAMP. TPA also attenuated AII responses in staurosporine-untreated control oocytes. These results suggest that staurosporine suppresses the desensitization of endogenous AII receptors in oocytes by blocking protein kinase C (PKC), and the desensitization is likely to be due to phosphorylation by PKC of the receptors or the molecules comprising an AII receptor complex.

Acetylcholine↗
PubMed1991

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli.

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.

Alleles↗
PubMed1991

Synaptic receptors and intracellular signal transduction in the cerebellum.

Glutamate receptor subtypes mediating excitatory synaptic neurotransmission in the cerebellar cortex are briefly reviewed from molecular biological, electrophysiological and pharmacological points of view. In particular, molecular biological findings of a novel family of AMPA-selective glutamate receptors are introduced, and the pharmacological and electrophysiological properties and the identity of cerebellar N-methyl-D-aspartate-sensitive receptors probably existing on Purkinje cells are discussed in comparison with well-established cerebral NMDA receptors. As possible intracellular mechanisms of the long-term depression of parallel fiber-Purkinje cell neurotransmission, the perspective of the roles of novel messengers, nitric oxide and arachidonic acid, is particularly commented based on recent information about cerebral long-term events. The specificity and possible independence of cerebellar excitatory amino acid receptors and linked intracellular second messengers are also suggested, taking the highly active guanylate cyclase system in Purkinje cells and other cerebellum-specific proteins into consideration.

Animals↗
PubMed1991

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235.

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.

Aldehyde Reductase↗
PubMed1991

Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair.

Escherichia coli possesses 2 types of O6-methylguanine-DNA methyltransferases, one inducible and the other constitutive. These enzymes are coded by the ada and the ogt genes, respectively. Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5'-flanking region of the nirR gene, on the E. coli chromosome. To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes. In either the ada+ or the ada- background, the ogt mutation had no effect on cell survival after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. On the other hand, ada- ogt- cells were more prone to mutation as compared to the ada- ogt+ cells exposed to MNNG. The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt+ plasmid into the cells produced a 2-3-fold decrease in the frequency of spontaneous mutation. O6-Methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents.

Base Sequence↗
PubMed1991

Characterization of O6-methylguanine-DNA methyltransferase in transgenic mice introduced with the E. coli ada gene.

The characteristics of O6-methylguanine-DNA methyltransferase (O6-MTase) produced in transgenic mice, in which the introduced E. coli ada gene was expressed under the control of the metallothionein promoter, were investigated. Liver extracts from transgenic homozygotes showed approximately 3 times the control activity, a marked increase of up to about 8 times the non-transgenic control levels being observed 10 h after zinc treatment. Examination of the substrate specificity of the enzyme revealed that the activity in the transgenic mice is due to the introduced foreign gene. The enzyme possessed methylphosphotriester-DNA methyltransferase as well as O6-MTase, characteristic of the E. coli Ada protein. Comparison of differences in biological response between transgenic and non-transgenic mice after treatment with the alkylating carcinogen methylnitrosourea (MNU) at various doses revealed transgenic mice to be more capable of repairing O6-MTase activity, only showing signs of exhaustion at very high levels of exposure. In non-transgenic mice, on the other hand, the basal level of O6-MTase was low, and the activity was hardly detectable when the animals were treated with MNU.

Animals↗
PubMed1991

Application of boronated anti-CEA immunoliposome to tumour cell growth inhibition in in vitro boron neutron capture therapy model.

An immunoliposome containing a 10B-compound has been examined as a selective drug delivery system in boron neutron-capture therapy. Liposomes, conjugated with monoclonal antibodies specific for carcinoembryonic antigen (CEA) were shown to bind selectively to cells bearing CEA on their surface. The immunoliposomes attached to tumour cells suppressed growth in vitro upon thermal neutron irradiation and suppression was dependent upon the concentration of the 10B-compound in the liposomes and on the density of antibody conjugated to the liposomes. The results suggest that immunoliposomes containing the 10B-compound could act as a selective and efficient carrier of 10B atoms to target tumour cells in boron neutron-capture therapy.

Antibodies, Monoclonal↗
PubMed1991

Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

Bacterial Proteins↗
PubMed1991

Endomyocardial biopsy findings in cases with pericarditis or perimyocarditis.

In order to determine the presence or absence of myocarditis in cases with viral or idiopathic pericarditis, a study was conducted as one of our series on endomyocardial biopsy. There were two groups of patients, pericarditis cases (n = 8), and patients with perimyocarditis (n = 6). In the former group, it was confirmed that cardiac sarcoplasmic enzymes were not released during the acute stage of the disease. In the latter, there was positive evidence of the enzyme release. Also, employing our method of categorizing the possibility of myocarditis at the histopathological level, we found that the category 'highly suggestive' of myocarditis was absent in all eight cases with pericarditis. However, in cases with perimyocarditis, this category was assigned in four out of six cases (67%), indicating a high incidence. The category, 'slightly suggestive', was seen in three cases of the former (38%) and two cases of the latter group (33%). It is concluded that in patients with pericarditis, the release of cardiac sarcoplasmic enzyme is an important diagnostic element in the diagnosis of perimyocarditis even if the clinical features reveal a predominance of pericarditis. In patients with perimyocarditis, progression to residual cardiac disease, such as conduction disturbance or congestive heart failure, is likely.

Adult↗
PubMed1991

Histochemical demonstration of heavy metals in the hippocampal formation embedded in Quetol 523M.

To facilitate improvement of investigations on the distribution of mossy fibers in the hippocampal formation, a method is described using Timm's stained preparations after methacrylate embedding with the hydrophilic resin, Quetol 523M. Fixation with a mixture of formaldehyde and glutaraldehyde yielded satisfactory staining results and good structural preservation. During the course of histochemical experiments employing Timm's staining, examinations revealed that sulfide silver reaction products were consistently present in both the mossy fibers themselves and their terminals associated with the dendrites of pyramidal cells in tissue sections of 1-2 microns in thickness. The results obtained also revealed that variations of the mossy fiber system occurred in the neurological mutant mouse dreher (dr). The bundles of mossy fibers forming the intrapyramidal synaptic field may be considered to reflect genotype-dependent differences in the mutation. The present method is adequate for allowing the histochemical demonstration of mossy fibers and their giant boutons by light microscopy.

Animals↗
PubMed1991

[A case of polyarteritis nodosa with bilateral hilar lymphadenopathy].

A 55-year-old male was admitted with non productive cough and fever which had continued for 6 weeks. The patient had symptoms of peripheral neuralgia. Chest X-ray revealed bilateral hilar lymphadenopathy (BHL) and reticular shadows in both lung fields. Other laboratory abnormalities included hematuria, RBC cast, high BUN, leukocytosis and thrombocytosis. Destruction of the internal membrane of arterioles was observed in a livedo reticularis on the right lower extremity. Renal angiography showed irregularity in the diameter, discontinuation and narrowing of peripheral arteries of both kidneys. These findings suggested the existence of "angiitis". These data were compatible with the diagnosis of polyarteritis nodosa (PN). Prednisolone (60 mg/day) administration resulted in the improvement of his symptoms and laboratory findings. A case of PN with lymph node swelling has been reported, however PN with BHL has not yet been reported. This is the first report of PN with BHL.

Humans↗
PubMed1991

[Effect of natural panting frequency and cheek support on the plethysmographic determination of thoracic gas volume in patients with chronic obstructive pulmonary disease].

The authors studied the effects of natural panting frequency (NF) and the cheek support on the plethysmographic measurement of thoracic gas volume (TGV) in 8 normal subjects (non-smokers) and 46 patients with chronic obstructive pulmonary disease (COPD). The patients were divided into 2 groups according to the degree of airway obstruction (group I; specific airway conductance (SGaw) greater than 0.1 (n = 18), group II; SGaw less than 0.1 (n = 28)). TGV was measured with a pressure-type body plethysmograph (BP). NF was 2.00 +/- 0.43 Hz (mean +/- SD) in control subjects, 1.92 +/- 0.78 Hz in group I, and 1.39 +/- 0.59 Hz in group II, respectively, indicating lower NF in the patients with severe airway obstruction. In control subjects and group I, the differences between TGV at NF and at 0.5-1.0 Hz (TGVNF-TGV1.0) were -0.01 +/- 0.07L, and -0.06 +/- 0.16L, respectively, and cheek support did not alter the difference. On the other hand, in group II, the difference was slightly larger than other groups in spite of the lower NF, and this overestimation was abolished by cheek support (0.13 +/- 0.25L-----0.06 +/- 0.27L, p less than 0.05). These results suggest that, in patients with severe airway obstruction, TGVNF may be overestimated even if NF is relatively low. This overestimation may be mainly due to the extrathoracic airway compliance including the cheek.

Adult↗