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M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 199 records · Page 11Linked to original sources
PubMed1996

Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.

Alleles↗
PubMed1996

Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release.

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

Animals↗
PubMed1996

Effect of mutY and mutM/fpg-1 mutations on starvation-associated mutation in Escherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine.

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starved Escherichia coli cells is markedly raised in mutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carrying mutM or fpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together with mutY, however, mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.

Catalase↗
PubMed1996

Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

Animals↗
PubMed1996

The abnormal distribution of mossy fiber bundles and morphological abnormalities in hippocampal formation of dreher(J) (dr(J)/dr(J))mouse.

The organization of pyramidal cells and mossy fibers in the hippocampal formation of homozygous dreher(J) mutant mice was investigated using Timm's and Golgi methods. Five clear abnormalities were found: (1) some pyramidal cells were located below the infrapyramidal mossy fiber layer, (2) mossy fibers emerged in diffuse fashion from between the suprapyramidal and infrapyramidal mossy fiber layers, and their fibers invaded within the pyramidal cell layer, where they traveled as 3-6 small, usually quite short, bundles, (3) some normally situated pyramidal cells had unusual contacts with mossy fibers at two or three places on their apical and/or basal dendrites, (4) some normally situated pyramidal cells had abnormal dendritic trees typified by the occurrence of fine-caliber dendritic branches extending out of the apical dendrite or the apical portion of the soma, and (5) a few Timm positive fibers extending from the dentate hilus to the dentate molecular layer in both dreher(J) and control mice were observed. These abnormalities indicate that in the hippocampal formation a variety of cell populations and neuronal circuits can be indirectly modified by the dreher mutation.

Animals↗
PubMed1996

Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter.

Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids. Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E. coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity. Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent. This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter. Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents. An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine. This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter. Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter. We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.

Amino Acid Sequence↗
PubMed1996

A novel function for nucleoside diphosphate kinase in Drosophila.

Nucleoside diphosphate (NDP) kinase is an enzyme that transfers the gamma-phosphate of nucleoside triphosphates to nucleoside diphosphates. Besides this well-defined role, recent evidence suggests that NDP kinase may be implicated in a wide variety of essential cellular processes. In this paper, we showed that the NDP kinase of Drosophila exhibited protein kinase activity as well as autophosphorylation. Ovalbumin was phosphorylated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) or ATP. Protein kinase activity was not detected in NDP kinase mutant, abnormal wing discs (awd). These results suggest that this activity could be one of the functions of NDP kinase essential for normal fly development, since awd gene is lethal.

Animals↗
PubMed1996

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Amino Acid Sequence↗
PubMed1996

Eosinophil chemotactic activity in bronchoalveolar lavage fluid obtained from Toxocara canis-infected rats.

We examined eosinophil chemotactic activity (ECA) in bronchoalveolar lavage fluid (BALF) obtained from rats infected with Toxocara canis. For 4 weeks after infection, the number of eosinophils was determined in peripheral blood and BALF. ECA was assayed using a microchemotaxis chamber. Eosinophils in peripheral blood and BALF increased markedly after infection, peaking at 12 days and 2 weeks, respectively. ECA in BALF also increased significantly and peaked 2 weeks after infection. Partial characterization revealed that ECA was heat labile, lipid soluble, and resistant to trypsin digestion. Two ECA peaks were identified by molecular sieve column chromatography: one near the egg albumin marker (MW 45,000) and the other observed after elution with quinacrine (MW 472.9). Treatment with a specific leukotriene (LT) B4 receptor antagonist (ONO-4057), a platelet activating factor (PAF) receptor antagonist (TCV-309), and an anti-interleukin (IL)-5 monoclonal antibody (TB13) significantly reduced the ECA, suggesting that LTB4, PAF, and IL-5 contribute to the accumulation of eosinophils in the lungs of rats infected with T. canis.

Animals↗
PubMed1996

Cardiac sarcoidosis: diagnostic, prognostic, and therapeutic considerations.

Cardiac involvement in patients with sarcoidosis is an important consideration for those who are concerned with this strange disease. Sarcoidosis is not an acute malignant disease but may be noticed at the time of sudden, expected death as fatal myocardial sarcoidosis at autopsy. Even with modern advances in our ability to diagnose heart disease, cardiac sarcoidosis is still often overlooked because of its subclinical disease progression. In view of this, an extensive review of previously published literature and of our own case analyses has been carried out because of the authors' long-term experience with performing Konno's endomyocardial biopsy, which was originally developed in 1962 at the author's institution. However, the sensitivity of endomyocardial biopsy in detecting sarcoid granuloma is low (20-30%), and, instead, various kinds of nongranulomatous pathologies are often seen. During the course of our research it was found that there might exist a racial difference in cardiac sarcoidosis. Cardiac death was much more frequent in Japanese patients. The possibility that heart disease in sarcoidosis is caused by cor pulmonale due to advanced pulmonary fibrosis should be reevaluated because only a limited amount of background data is available. The author's review clarified the fact that cardiac sarcoidosis is caused by myocardial or pericardial involvement, resulting in various kinds of bradyarrhythmias or tachyarrhythmias and/or congestive heart failure. Electrocardiographic (ECG) and Holter monitor readings provide a simple and effective method for early detection of this disease. The incidence of ECG abnormalities in a total of 963 sarcoidosis patients was 22.1%, which was more frequent than that of the sex- and age-matched healthy control subjects (17.9%; p < 0.025). Echocardiography and radionuclide studies also provide useful clinical information. Careful follow-up and early corticosteroid administration followed by small maintenance doses may prevent the progression of the disease and improve prognosis. Owing to the progress in antiarrhythmic drugs and pacemaker implantation, the primary cause of death in cardiac sarcoidosis has changed from sudden death (1976 report) to congestive heart failure (1985 report).

Adrenal Cortex Hormones↗
PubMed1996

Radiofrequency catheter ablation of supraventricular tachycardias in patients with hypertrophic cardiomyopathy.

Supraventricular tachycardias, as well as ventricular tachycardia, are critical patients with hypertrophic cardiomyopathy. Anti-arrhythmic agents often induce arrhythmias, known as pro arrhythmia. Radiofrequency catheter ablation has become a well-established therapy for the treatment of supraventricular tachycardia. The reported success rate is over 90%, although catheter mapping in the left ventricle is difficult in patients with hypertrophic cardiomyopathy because of the marked left ventricular wall thickening associated with bizarre myocardial hypertrophy with disorganization, the abundant capillary muscle networks, and annoying ventricular arrhythmias. This report documents catheter mapping technique of radio frequency ablation in patients with hypertrophic cardiomyopathy and supraventricular tachycardias.

Adult↗
PubMed1996

DNA-repair methyltransferase as a molecular device for preventing mutation and cancer.

Alkylation of DNA at the 0(6) position of guanine is regarded as one o f the most critical events leading to induction of mutations and cancers in organisms. Once 0(6)-methylguanine is formed, it can pair with thymine during DNA replication, the result being a conversion of the guanine.cytosine to an adenine.thymine pair in DNA, and such mutations are often found in tumors induced by alkylating agents. To counteract such effects, organisms possess a mechanism to repair 0(6)-methylguanine in DNA. An enzyme, 0(6)-methylguanine-DNA methyltransferase, is present in various organism, from bacteria to human cells, and appears to be responsible for preventing the occurrence of such mutations. The enzyme transfers methyl groups from 0(6)-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing DNA lesions in a single-step reaction. To elucidate the role of methyltransferase in preventing cancers, animal models with altered levels of enzyme activity were generated. Transgenic mice carrying the foreign methyltransferase gene with functional promoters had higher levels of methyltransferase activity and showed a decreased susceptibility to N-nitroso compounds in regard to liver carcinogenesis. Mouse lines deficient in the methyltransferase gene, which were established by gene targeting, exhibited an extraordinarily high sensitivity to an alkylating carcinogen.

Amino Acid Sequence↗
PubMed1996

Closed intramedullary osteotomy for rotational deformity after long bone fractures.

Ten patients with rotational deformities after fractures of the femur or tibia were treated by closed intramedullary osteotomy. Eight had an external rotation deformity of the femur and 2 had deformities of the tibia. The rotational deformity varied from 30 degrees to 60 degrees and 6 patients had shortening of 15 mm to 30 mm. The osteotomies were carried out with an intramedullary saw and were fixed by an interlocking nailing. Shortening was corrected at the same time. At follow up the average rotational deformity was less than 5 degrees and shortening less than 15 mm. All the patients felt that they had a normal range of movement and equal length. There were no complications, except for one case of delayed union in which consolidation occurred at 24 months without any further procedures.

Adolescent↗
PubMed1996

MutT-related error avoidance mechanism for DNA synthesis.

Mutator mutants that show an increased frequency of spontaneous mutation have led to the elucidation of the multiple pathways of spontaneous mutagenesis. 8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. MutT protein of Escherichia coli and related mammalian enzymes specifically degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of transversion mutation. The gene encoding the human enzyme, designated MTH1 (for mutT homologue 1), maps to chromosome 7p22. These proteins may be responsible for genomic stability.

Bacterial Proteins↗
PubMed1996

Organization and expression of the mouse gene for DNA repair methyltransferase.

06-Methylguanine-DNA methyltransferase (MGMT) is present in various organisms, from bacteria to human cells, and plays an important role in preventing mutations caused by alkylating substances. To understand better the regulatory mechanism involved in the expression of the gene and to construct a mouse model to investigate roles of the enzyme in carcinogenesis, the genomic sequence for mouse methyltransferase was isolated and characterized. The gene consists of 5 exons and spans over 180 kb, whereas mRNA for the enzyme was less than 1 kb. The promoter region for the gene is GC-rich, contains many Sp1 recognition sequences and lacks typical TATA and CCAAT boxes. Primer extension and S1 mapping revealed the existence of multiple transcription initiation sites, among which a major site was defined as +1. The putative promoter region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and the construct was introduced into mouse NIH-3T3 cells. Deletion analyses revealed that a sequence from -262 to + 56 carries the basic promoter activity. In addition, an adjacent region, spanning from +56 to +95, carries an E2F-like element that greatly stimulates the frequency of transcription. Alteration of TTTTGGGGC to TTAACGGGC considerably reduced the activity.

3T3 Cells↗
PubMed1996

Analysis of recombinant human tumour necrosis factor-alpha-induced CD4 expression on human eosinophils.

We examined the hypothesis that one of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), could induce expression of the adhesion molecule CD4 on human eosinophils. We further examined the effector function of CD4 and the mechanisms regulating CD4 expression. Human eosinophils were cultured with various concentrations of recombinant human TNF-alpha (rhTNF-alpha) with or without various drugs for 24 hr. After culture, eosinophils were stained for CD4 using a monoclonal antibody and then analysed by flow cytometry. Eosinophil-derived neurotoxin (EDN) release as eosinophil degranulation was examined by cross-linking of CD4 on eosinophils. The rhTNF-alpha induced CD4 expression on human eosinophils in a dose- and time-dependent fashion; rhTNF-alpha-induced CD4 expression was significantly inhibited by 10(-6) M cycloheximide, 10(-8) M dexamethasone, or 10(-6) M herbimycin A. Recombinant human interferon-gamma inhibited rhTNF-alpha-induced CD4 expression in a dose-dependent manner. However, cross-linking of CD4 on eosinophils did not evoke EDN release, suggesting that newly expressed CD4 molecules on human eosinophils do not play any role in triggering degranulation. Our data indicate that TNF-alpha-induced CD4 expression on human eosinophils is dependent on protein synthesis and may be dependent on tyrosine kinase activity.

Benzoquinones↗