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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 235 records · Page 13Linked to original sources
PubMed1995

Mouse MTH1 protein with 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase activity that prevents transversion mutation. cDNA cloning and tissue distribution.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. We isolated cDNA for mouse 8-oxo-dGTPase, using as a probe human MTH1 (Escherichia coli mutT homolog) cDNA. The nucleotide sequence of the cDNA revealed that the mouse MTH1 protein (molecular weight of 17,896) comprises 156 amino acid residues. When the cDNA for mouse 8-oxo-dGTPase was expressed in E. coli mutT- mutant cells devoid of their own 8-oxo-dGTPase activity, an 18-kDa protein, which is cross-reactive with an anti-human MTH1 antibody, was formed. In such cells, the level of spontaneous mutation frequency that was elevated reverted to normal. High levels of 8-oxo-dGTPase activity were found in liver, thymus, and large intestine, whereas all other organs examined contained smaller amounts of the enzyme. In embryonic stem cells, an exceedingly high level of the enzyme was present.

Amino Acid Sequence↗
PubMed1995

Morphological abnormalities in the hippocampus of the weaver mutant mouse.

The lamination of the hippocampus in the homozygous B6CBA weaver mouse (wv/wv) was compared with that in normal B6CBA littermates (+/+) and C57BL/6J mice using Nissl and Timm's staining. In Nissl-stained preparations, the normal littermates exhibit a compact, regular arrangement of pyramidal cells in area CA3 of the hippocampus. In contrast, in homozygous weaver mutant mice, the pyramidal cell layer of area CA3 frequently appears to be thicker than normal with an apparent increase of neuropil, as evidenced by the presence of cell-free spaces within the layer. Also, small ectopic clusters of pyramidal cells and sometimes the subdivision of the pyramidal cell layer into 2 or 3 layers were found throughout the dorsoventral extent of the hippocampus. In Timm's stained preparations of the normal mouse hippocampus, two clearly separated bundles of axons were seen emerging from the hilus: one bundle running above the pyramidal cell layer of area CA3 (i.e., the suprapyramidal mossy fiber layer, SPMFL), and the second bundle running below the pyramidal cell layer (i.e., the infrapyramidal mossy fiber layer, IPMFL). In contrast, in some homozygous weaver mice, the origin of the mossy fiber bundles is clearly different from normal; specifically, mossy fibers emerge in a diffuse fashion from the area between suprapyramidal and infrapyramidal mossy fiber layers. In other weaver mice, short, discontinuous bundles diverge from the infrapyramidal mossy fiber layer and invade the thickened pyramidal cell layer. In addition, ectopic pyramidal cells are situated below the IPMFL in area CA3. The morphological changes observed in hippocampus of weaver mutants are likely to be secondary to a more basic genetic defect.

Animals↗
PubMed1995

Polymorphisms and probable lack of mutation in a human mutT homolog, hMTH1, in hereditary nonpoliposis colorectal cancer.

The human MTH1 gene, a homolog of the E. coli mutator gene mutT, encodes 8-oxo-dGTPase that degrades a mutagenic substrate for DNA synthesis. To determine whether this gene is associated with hereditary nonpolyposis colorectal cancer (HNPCC), we examined the hMTH1 sequence in 32 HNPCC cases by means of polymerase chain reaction-single strand conformation polymorphism and sequencing analyses. Three different DNA variants were identified in normal and the corresponding tumor DNA. The first variant, a G to A transition at codon 83, changes valine to methionine and was detected in 9 HNPCC cases. The same change was detected in 5 of 30 unrelated healthy individuals, suggesting that it is not associated with a marked HNPCC predisposition. The second variant is a silent C to T transition at codon 119. Another C to T transition at 31 bp upstream of the beginning of exon 4 was also found. However, specific mutations in hMTH1 were detected in neither normal nor tumor cells from HNPCC patients. These results indicate that hMTH1 is not directly involved in HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Cyclic AMP-regulated synthesis of the tissue inhibitors of metalloproteinases suppresses the invasive potential of the human fibrosarcoma cell line HT1080.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in regulating the activity of matrix metalloproteinases (MMPs). Tumor cell invasion and metastasis closely correlate with the activities of two members of MMPs, MMP2 and MMP9, both of which degrade type IV collagen in basement membranes. We herein report that the treatment of HT1080 cells with 8-bromo-cAMP and other chemicals that activate cyclic adenylase activity induces the expression of TIMP1 and TIMP2 both at the mRNA and the protein levels and that this induction of TIMPs correlates with suppression of invasive phenotypes of HT1080 cells. Treatment with various cAMP-elevating reagents induced the expression of TIMPs and MMP2 in HT1080 cells, whereas the expression of MMP9 was not significantly affected. The protein amounts of TIMP1, TIMP2, and MMP2 secreted into the medium from HT1080 cells treated with 1 mM 8-bromo-cAMP were 7.9-, 9.3-, and 8.5-fold higher than those secreted from untreated cells, respectively. Induction of these mRNAs by 8-bromo-cAMP was blocked by HA1004, a protein kinase A inhibitor, but not by calphostin C, a protein kinase C inhibitor. Cycloheximide abolished the induction of TIMPs and MMP2 mRNAs by 8-bromo-cAMP, indicating that the induction depends on a newly synthesized protein(s) whose expression may be regulated by cAMP. Type IV collagenolytic activity and the invasiveness of HT1080 cells, both of which were suppressed by 8-bromo-cAMP, were efficiently restored when the cells were exposed to anti-TIMP antibodies, demonstrating the importance of the increased levels of TIMP1 and TIMP2 proteins for the cAMP-mediated suppression of both type IV collagenolytic activity and the invasiveness of HT1080 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.

Blotting, Western↗
PubMed1995

Stabilization of cyclin E and cdk2 mRNAs at G1/S transition in Rat-1A cells emerging from the G0 state.

mRNAs for cyclin E and Cdk2 have a role in the commitment to DNA replication in the cell cycle, and are induced in Rat-1A cells by serum stimulation. Cyclin E and cdk2 genes are transcribed in quiescent cells, but their transcripts rapidly turn over and levels are kept low. The rate of transcription of the cdk2 gene is slightly increased after serum stimulation, while that of cyclin E is fairly constant. At the G1/S transition of serum-stimulated cells, transient stabilization of the two types of mRNAs occurs, an event which may lead to induction of each mRNA. Artificial expression of an immediate-early protein delta FosB results in proliferation of quiescent Rat-1A cells, and this is accompanied by an efficient induction of cyclin E and cdk2 mRNAs. In delta FosB-expressing cells, two types of mRNAs are stabilized to the same extent seen in serum-stimulated cells. The expression of cyclin E and cdk2 genes is upregulated by stabilization of their transcripts, at least in part. We propose that delta FosB may have a role in regulation of progression of the cell cycle in serum-stimulated Rat-1A cells by triggering stabilization of mRNAs for cyclin E and Cdk2.

Animals↗
PubMed1995

Genotype and phenotype of severe mitochondrial cardiomyopathy: a recipient of heart transplantation and the genetic control.

Comprehensive analyses of mitochondrial (mt)DNA of a recipient of heart transplantation at age 7 because of severe cardiomyopathy revealed three germ line point mutations, each one in the 12S rRNA gene, in the CO1 gene and in the cytochrome b gene, respectively. As the somatic mutation, extensive fragmentation of mtDNA associated with 212 kinds of deletions was detected in contrast to 5 kinds in an age-matched negative control. A recipient's positive control having almost the same base-substitutions and mutations with the recipient except one in the CO1 gene also developed severe cardiomyopathy died at age 20. The close relation between phenotype and mtDNA genotype provides the basis of our understanding of cell death and premature ageing.

Adult↗
PubMed1995

Coronary microvascular response to intracoronary administration of nicorandil.

Nicorandil is an antianginal drug that causes potent coronary vasodilation of both epicardial and resistance vessels. To measure the dose-response kinetics of bolus injections of intracoronary nicorandil and to compare the vasodilatory response to nicorandil with that of intracoronary papaverine in humans, coronary blood flow velocity was measured in 30 patients using a 3Fr intravascular Doppler catheter. Continuous intravenous nitroglycerin 6 to 8 micrograms/min was infused to achieve maximal vasodilation of the epicardial vessels. Bolus doses of nicorandil dissolved in warmed saline solution were injected into the left (0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mg) and right (0.1, 0.2, 0.4, 0.8, 1.0, and 1.5 mg) coronary arteries. Intracoronary nicorandil caused a dose-dependent increase in coronary flow velocity and a decrease in coronary vascular resistance. Maximal vasodilatory effects equivalent to those obtained with 12 +/- 2 mg of intracoronary papaverine were induced with nicorandil 1.5 mg in the left coronary artery, and effects similar to those of 10 +/- 2 mg of papaverine were produced with nicorandil 1.0 mg in the right coronary artery. The time from injection of nicorandil to the onset of maximal hyperemia and duration of hyperemia were significantly shorter after nicorandil than after papaverine in both coronary arteries. Each dose of nicorandil produced no clinical symptoms and fewer changes in systemic hemodynamics and electrocardiographic QT intervals than did papaverine. These results suggest that a bolus administration of intracoronary nicorandil can safely, quickly, and reliably induce maximal coronary hyperemia comparable to that achieved with intracoronary papaverine in humans.

Adult↗
PubMed1995

Generation and elimination of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate, a mutagenic substrate for DNA synthesis, in human cells.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a potent mutagenic substrate for DNA synthesis. The present study deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is rapidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP. (3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lower rates: Km for 8-oxo-dGMP is 10 times lower than that for dGMP, the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP, which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior.

Cell-Free System↗
PubMed1995

Multiple mitochondrial DNA deletions in a patient with mitochondrial myopathy and cardiomyopathy but no ophthalmoplegia.

Deletions of muscle mitochondrial DNA are known in mitochondrial myopathy patients who have chronic progressive external ophthalmoplegia (CPEO). A 41-year-old patient with no apparent family history of this condition suffers from hypertrophic cardiomyopathy, slight muscle atrophy, and weakness of the extremities, but not from CPEO. A muscle biopsy showed the presence of ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. This combination of clinical features in our patient is atypical in mitochondrial myopathy with demonstrable deleted muscle mitochondrial DNA. Pleomorphic clinical expression is suggested.

Adult↗
PubMed1995

Alpha-fetoprotein-producing gastric carcinoma: biological properties of a cultured cell line.

We describe a gastric carcinoma cell line that has been maintained in vitro for more than 10 years and retains the capacity to produce a large amount of alpha-fetoprotein. This cell line was isolated from a metastatic lymph node of a 63-year-old male patient with advanced gastric carcinoma (T2N3P0H0M0) who showed high serum levels of alpha-fetoprotein. The primary tumor was moderately differentiated tubular adenocarcinoma and the lymph node was poorly differentiated adenocarcinoma without any particular pattern. The cultured cells grew as densely packed islet-like colonies with small polygonal cells. Electron microscopy revealed cells abundant in cytoplasmic organelles, with some cellular attachments being tight with junctional complexes and some being loose across intercellular spaces. The free cell surface had microvilli. The population doubling-time was 152 h at passage 58. Chromosomal analysis revealed the modal number to be 77, with numerous karyotype abnormalities. The tumorigenicity of the cultured cells in athymic nude mice was positive only when they were subcutaneously transplanted beneath a plastic plate, but when the cells were transplanted subcutaneously or administered by intrasplenic injection in intact or weakly irradiated nude mice, no tumorigenicity was shown. The cell line produced tumor-associated antigens, such as alpha-fetoprotein, carcinoembryonic antigen, and tissue polypeptide antigen. This cell line may be useful for comparative studies of different types of gastric carcinoma and alpha-fetoproteins of different origins.

Adenocarcinoma↗
PubMed1995

Protein phosphatase mRNA expression in Purkinje cells of staggerer and reeler mutant mice.

We used in situ hybridization to search for the expression of three protein phosphatase (PP) mRNAs in Purkinje cells of normal mice, and staggerer (sg/sg) and reeler (rl/rl) mutant mice, two strains with known Purkinje cell disorders. The expression of the mRNAs was comparable in the normal and rl/rl Purkinje cells, but considerably reduced in the sg/sg. We interpret this finding as indicating: (a) the staggerer mutant gene may directly affect the phosphatase component of the protein phosphorylation-dephosphorylation cycle in the sg/sg Purkinje cells; or (b) the reduced mRNA expression may be a secondary phenomenon, resulting from abnormal Purkinje cell function due to the lack of synaptic input.

Animals↗
PubMed1995

Intercellular adhesion molecule-1 induction: a sensitive and quantitative marker for cardiac allograft rejection.

OBJECTIVES: Rats with abdominal heterotopic heart transplants were studied to determine whether cardiac allograft rejection could be assessed by immunoscintigraphy targeting intercellular adhesion molecule-1 (ICAM-1), which was induced on allografted organ cells in association with rejection. BACKGROUND: It is important to detect early rejection before development of myocyte necrosis. Although a variety of methods for the detection of cardiac rejection have been investigated, histologic inspection of biopsied samples is still used routinely for clinical diagnosis of rejection. METHODS: DA rat (RT-1a) hearts were transplanted into PVG rats (RT-1c). Immunohistologic examination of the allografts demonstrated that ICAM-1 induction on vascular endothelial cells was observed as early as 4 days after transplantation in this combination. Thirty-nine allografted rats and seven isografted rats were studied. One day after injection of 100 microCi of 111Inlabeled anti-ICAM-1 monoclonal antibody (1A29), planar images were obtained. RESULTS: Rejecting allografts showed increased radiotracer uptake and could be identified on the images as early as 5 days after transplantation. In contrast, nonrejecting cardiac allografts and isografts did not show specific uptake. Mildly rejecting allografts, with mononuclear cell infiltration but without significant myocyte necrosis, could be scintigraphically identified, and the level of radiotracer uptake reflected the histologic severity of rejection. Accumulation of 111In-labeled monoclonal antibody of isotype-matched irrelevant specificity was not detected in the rejecting allografts. CONCLUSIONS: These data indicate that ICAM-1 induction can be assessed quantitatively by radioimmunoscintigraphy. Radioimmunoscitigraphy is a sensitive method for early detection and assessment of cardiac allograft rejection.

Abdomen↗
PubMed1995

Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli.

8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is a potent mutagenic substrate for DNA synthesis. The accumulation of 8-oxo-dGTP in the nucleotide pool induces G:C-->T:A transversion as well as A:T-->C:G transversion, and Escherichia coli cells possess mechanisms for preventing such mutations. The mutT gene product specifically hydrolyzes 8-oxo-dGTP to the monophosphate form while the mutM and the mutY gene products function to correct mispairs caused by incorporation of 8-oxoguanine into DNA. From analyses of forward mutations induced in cells lacking 8-oxo-dGTPase (MutT protein) and/or repair enzymes that suppress mutations caused by 8-oxoguanine in DNA (MutM and MutY proteins), cooperative functions of these proteins in control of the spontaneous mutagenesis became evident. In mutator strains lacking MutT and/or MutM proteins, 8-oxoguanine of DNA increased to a concentration expected from the increased rate of mutation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Establishment of a natural suppressor cell line producing soluble suppressor factor other than transforming growth factor-beta.

Natural suppressor (NS) cell line (Clone 59) was established from the bone marrow of adult C3H/Hej mice in the presence of WEHI-3 conditioned media. Clone 59 cells suppressed the generation of cytotoxic T lymphocytes from normal mouse spleen. This suppression was seen at a responder-to-suppressor cell ratio of 1000:1 and lacked antigen specificity or MHC restriction. Clone 59 cells expressed the 'null' surface phenotype (Thy1.2-, CD3-, Lyt-2-, L3T4-, surface Ig-, MAC-1-) by immunofluorescent staining. Clone 59 cells exhibited no cytolytic activity against NK cell-sensitive YAC-1 and natural cytotoxic L929 target cell lines. Non-specific suppression, with a cell-free supernatant from the Clone 59-NS cells, also was observed. The supernatant did not inhibit [3H]-thymidine uptake by CTLL-2 cells which were proliferating in response to IL-2. Anti-transforming growth factor-beta (TGF-beta) monoclonal antibody had no effect on suppression, suggesting that the non-specific suppression is mediated by some soluble factors other than TGF-beta. Clone 59 cells may be useful in identifying non-specific suppressor cells in adult bone marrow and studying their functional role in the regulation of tolerance and self-reactivity.

Animals↗
PubMed1995

Functional comparison of D-serine and glycine in rodents: the effect on cloned NMDA receptors and the extracellular concentration.

We compared the activity of free D-Ser on the potentiation of cloned NMDA receptors with that of Gly by using a Xenopus oocyte expression system. The extracellular concentration of free D-Ser and Gly was further studied by means of microdialysis. The ED50 values of D-Ser were three to four times lower than those of Gly in any combination of epsilon 1, epsilon 2, epsilon 3, or epsilon 4 and zeta 1. Site-directed mutagenesis of zeta 1 subunits revealed that some aromatic residues necessary for the action of Gly affected the ED50 value of D-Ser. This result showed that the residues play crucial roles in the action of D-Ser. In vivo microdialysis of rodent brain revealed that the extracellular concentration of free D-Ser in the frontal cortex (6.5 microM) was high enough to saturate the Gly site on the NMDA receptor, but that in the cerebellum was not. These findings suggest that D-Ser is a candidate of the endogenous potentiator of the NMDA receptor in the rodent frontal cortex.

Animals↗
PubMed1995

cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotides.

The enzyme, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase), is present in various organisms and plays an important role in control of spontaneous mutagenesis. This enzyme degrades 8-oxoguanine-containing deoxyribonucleoside triphosphate, a potentially mutagenic substrate for DNA synthesis, to the corresponding monophosphate. To obtain appropriate probes for expression of the gene in various tissues and also to construct appropriate experimental models for carcinogenesis, we cloned cDNA for rat 8-oxo-dGTPase and elucidated its structure. The nucleotide sequence of the cDNA revealed that the rat 8-oxo-dGTPase protein is composed of 156 amino acid residues. The molecular weight of rat 8-oxo-dGTPase, calculated from the predicted amino acid sequence, was 18,006, and the 8-oxo-dGTPase protein of this size was detected when the cDNA was expressed in 8-oxo-dGTPase-deficient Escherichia coli mutT- cells. The predicted amino acid sequence of the rat 8-oxo-dGTPase has a close homology with those of human and bacterial counterparts. Using the cDNA as a probe, part of the rat gene for 8-oxo-dGTPase was isolated and was found to consist of at least three exons and spanned about 10 kb. A genomic region containing the pseudogene was also isolated.

Amino Acid Sequence↗