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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.

Acids↗
PubMed2001

Expression and prognostic significance of O6-methylguanine-DNA methyltransferase in hepatocellular, gastric, and breast cancers.

BACKGROUND: O6-Methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine, a promutagenic DNA base damaged by endogenous and environmental alkylating agents. There are few reports that describe whether or not abnormal MGMT expression correlates with the prognosis in human solid cancers. METHODS: The expression of MGMT was immunohistochemically evaluated in 60, 62, 105, and 46 paraffin-embedded samples from patients with curatively resected hepatocellular, gastric, colorectal, and breast cancers, respectively. RESULTS: The expression of MGMT was a positive predictive factor for overall survival in hepatocellular (P = .005) and gastric cancers (P < .001) and for relapse-free survival in breast cancers (P < .001). MGMT-positive gastric tumors (n = 42) were correlated with the absence of serosal invasion (P = .045), lymph node metastasis (P = .006), intestinal type (P = .018), and low pathological tumor, node, metastasis stage (P < .001). All breast tumors that recurred locally after operation were MGMT negative (P = .004). The clinicopathologic characteristics of colorectal cancers with respect to MGMT expression did not significantly differ. CONCLUSIONS: The expression of MGMT is a predictive prognostic marker in patients with hepatocellular, gastric, and breast cancers. These findings may help to establish therapeutic strategies for patients with these types of solid cancer.

Aged↗
PubMed2001

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.

Animals↗
PubMed2001

Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Intracellular distribution of the antimutagenic enzyme MTH1 in the liver, kidney and testis of F344 rats and its modulation by cadmium.

Cellular distribution of the antimutagenic MTH1protein in the liver, kidney, and testis of Fischer rat was evaluated using the immunohistochemical staining with anti-MTH1 polyclonal antibody. The present investigation revealed a non-uniform distribution of MTH1 among cells and among the cytoplasmic, nuclear, and membranal structures of cells within a given tissue. A particularly strong expression of MTH1 was observed for the first time in the perinuclear acrosomic bodies of spermatocytes and in the acrosomic vesicles of sperm heads. Treatment of rats with a single sc dose of 20 micromol Cd(II)/kg body wt. produced histopathologic changes in these organs accompanied by redistribution of the cellular MTH1 protein between the cytoplasm and nuclei. The acute phase of Cd(II) toxicity, that in the liver and especially in the testes (but not in kidneys) led to cell necrosis, was accompanied by a characteristic decrease in the abundance of MTH1-expressing nuclei. Chronic toxicity without necrosis, persisting in the kidney over the entire 14-day study, as well as the survival and proliferation of cells, observed in the liver and testis after the necrotizing phase, were signified by increased number of nuclei expressing MTH1. Thus, unlike previous biochemical studies, immunohistochemistry managed to reveal alterations in the patterns of inter- and intracellular distribution of MTH1, associated apparently with the conditional changes in the dynamics of synthesis of nucleic acids, assisted by this protein.

Animals↗
PubMed2001

MGMT expression in murine bone marrow is a major determinant of animal survival after alkylating agent exposure.

Myelosuppression is commonly observed after alkylating agent chemotherapy due to low levels of O(6)-alkylguanine DNA alkyltransferase protein (AGT) in hematopoietic progenitors. Mice that lack AGT in all organs, O(6)-methylguanine-DNA methyltransferase gene knockout (MGMT(-/-)) mice are extremely hypersensitive to the methylating agent N-methyl-N-nitrosourea (MNU) and exhibit a 10-fold reduction in the LD(90). To determine whether bone marrow damage was the cause of the increased lethality, we transplanted 1 x 10(6) wild-type marrow into MGMT(-/-) mice and MGMT(-/-) marrow into wild-type mice and observed survival after MNU. Lethally irradiated MGMT(-/-) mice given > or = 25 mg/kg MNU 3 weeks after transplant of wild-type cells survived > 30 days (n = 11), whereas this dose was lethal to control MGMT(-/-) mice 9-12 days post treatment (n = 5). Conversely, lethally irradiated wild-type mice transplanted with MGMT(-/-) cells died after only 20-60 mg/kg MNU within 8-12 days (n = 6). No significant toxicities were found in other organs. Additionally, in an in vivo post transplant competition model, wild-type long-term repopulating cells had a > 200-fold competitive survival advantage over MGMT(-/-) cells, and after MNU treatment completely repopulated the mouse when transplanted at only one-tenth the cell number. We also observed a strong selection for transplanted marrow-derived wild-type stromal elements in the MGMT(-/-) background after drug treatment. These data indicate that alkylating agent hypersensitivity of MGMT(-/-) mice results from hematopoietic damage at the stem level. Thus, DNA repair involving AGT in hematopoietic cells is required for normal host survival following exposure to methylating and chloroethylating agents.

Alkylating Agents↗
PubMed2001

HOXD3 regulates expression of JAGGED1, a ligand for Notch receptors.

We generated transgenic mouse embryos expressing the human HOXD3 homeobox gene in the central nervous system (CNS) utilizing the Wnt1 expression vector. Whole mount in situ hybridization analysis revealed that the transgenic embryos at 10.5 days post coitum (dpc) expressed the HOXD3 gene in dorsal aspects of the CNS from the diencephalon to the spinal cord. Histological observation of sections showed that, in the spinal cord of the transgenic embryos at 10.5 dpc, there were few neuronal progenitor cells stretching from a luminal to basal side. This implies that Notch signaling which is involved in determining the courses of differentiation in the progenitors was disturbed within the CNS of the transgenic embryos. To elucidate what effects HOXD3 has on Notch signaling, we examined gene expression of Notch receptors and ligands using human erythroleukemia HEL and K562 cells transfected with the HOXD3 gene. Consequently, HOXD3 promoted expression of JAGGED1, a ligand for Notch receptors, in both the transfectants, suggesting that the JAGGED1 gene is a downstream target of HOXD3.

Animals↗
PubMed2001

Effects of substitution of conserved amino acid residues on the sugar-binding property of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans.

The 32-kDa galectin (LEC-1) of the nematode Caenorhabditis elegans (C elegans) is composed of two tandemly repeated homologous sequences, each containing a carbohydrate-recognition domain (CRD). Using the polymerase chain reaction (PCR) with LEC-1 cDNA as a template and "megaprimers", we performed site-directed mutagenesis to substitute conserved amino acid residues in these domains. The resultant mutated LEC-1s were produced in E. coli, and their binding abilities were estimated by affinity chromatography. When one of the conserved amino acid residues in the first lectin domain was substituted, the binding ability of the mutant protein to asialofetuin-agarose was reduced but still remained. The binding ability of such mutants was similar to that of the recombinant half molecule containing the second lectin domain (Ch). However, when mutations were introduced into the second lectin domain, the binding ability of these mutant lectins to asialofetuin-agarose was significantly reduced just like the half recombinant molecule containing the first lectin domain (Nh). The different effects of the substitution of amino acid residues on the two lectin domains suggest that the binding properties of the two sites are different and that LEC-1 acts as a "heterobifunctional crosslinker."

Animals↗
PubMed2001

Involvement of angiotensin II in progression of renal injury in rats with genetic non-insulin-dependent diabetes mellitus (Wistar fatty rats).

Wistar fatty (WF) rats have a genetic predisposition to hyperglycemia, polyuria, hyperinsulinemia, hyperlipidemia, obesity and nephropathy. These phenotypic characteristics are similar to those observed in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) nephropathy. In this study, the effects of two types of renin-angiotensin system inhibitors, an angiotensin II type 1-receptor antagonist (AT1A) and an angiotensin I-converting enzyme inhibitor (ACEI), on renal injury in WF rats were studied during the progressive phase of diabetic nephropathy. An AT1A, candesartan cilexetil (1 mg/kg), and an ACEI, enalapril (10 mg/kg), were administered orally once a day for 12 weeks, beginning when the rats were 27-week-old and already showed diabetic nephropathy and obesity. Both drugs prevented an increase in proteinuria during the experimental period. Furthermore, after 4-week intervention, the levels of proteinuria were markedly lower in drug-treated rats. At the end of the experiment, both drugs prevented the development of glomerular lesions without affecting glucose metabolism and obesity. In conclusion, the inhibition of angiotensin II activity ameliorated both existing proteinuria and the progression of proteinuria, resulting in preservation of glomerular structure. Thus angiotensin II plays important roles in the development and the progression of nephropathy in genetically obese diabetic WF rats.

Angiotensin II↗
PubMed2001

Iron content of rat serum ferritin.

The serum ferritin concentration was significantly higher in female than in male rats, reflecting higher iron stores in females than in males. The mean iron/protein ratio of serum ferritin was 0.018+/-0.008 (SD) (microg of Fe/microg of protein) in female rats and 0.011+/-0.011 in male rats, being much lower than that of liver ferritin (0.233+/-0.014 in females and 0.227+/-0.020 in males). Iron loading of rats significantly increased serum ferritin concentration, but did not influence the iron content of serum ferritin. These results indicate that rat serum ferritin contains only a small amount of iron independent of body iron stores.

Animals↗
PubMed2001

A non-arthropathic dose and its disposition following repeated oral administration of ofloxacin, a new quinolone antimicrobial agent, to juvenile dogs.

A non-arthropathic dose and disposition of ofloxacin, a potent new quinolone antimicrobial agent, were assessed in male juvenile (3-month-old) dogs, when administered orally at 5, 10 and 20 mg/kg/day once daily for 8 consecutive days. Ofloxacin concentrations in sera and articular cartilages were analyzed by high-performance liquid chromatography (HPLC). Macroscopically, arthropathy characterized by fluid-filled vesicles in articular surface of the humerus and femur was observed in animals receiving 10 and 20 mg/kg/day of ofloxacin, but not in those given 5 mg/kg/day. At 20 mg/kg/day, arthropathy of comparable severity also occurred on day 2. Microscopically, the cavity formation in the middle zone of the articular cartilage was first identified and then necrotic chondrocytes were found numerous around the cavity, followed by appearance of chondrocyte clusters. In pharmacokinetics, peak serum concentration (Cmax) and area under the concentrations (AUC0-24) were increased in a dose-dependent manner. However, no remarkable differences in these two parameters were noted between a single and repeated treatments, suggesting no accumulation of the drug. The articular ofloxacin concentration 2 hr after treatment was approximately 1.8 (day 2) to 2.0 times (day 8) higher than the serum concentration. Based on these results, a non-arthropathic dose of ofloxacin in male juvenile dogs following an 8-day treatment is considered to be 5 mg/kg/ day, and its Cmax, AUC0-24 and articular cartilage concentrations 2 hr after treatment were 3.4 microg/ml, 35.1 microg-hr/m/ and 7.0 microg/g, respectively, under these experimental conditions. Thus, arthropathy due to ofloxacin may be predicted by monitoring serum drug concentration.

Administration, Oral↗
PubMed2001

[Outcome of patients with significant coronary stenosis but without ischemic evidence on exercise myocardial perfusion scintigraphy].

OBJECTIVES: The rates of cardiac events and coronary revascularization were evaluated in patients with significant coronary stenosis of more than 75% by the American Heart Association (AHA) classification but no ischemic evidence by exercise myocardial perfusion scintigraphy. METHODS: Subjects were 171 patients (113 males, 58 females, mean age 66 +/- 9 years) undergoing coronary angiography and without scintigraphic evidence of myocardial ischemia. They were divided into two groups according to the severity of coronary artery stenosis based on AHA classification. Group A was composed of 139 patients with more than 75% stenosis (101 patients with 75% stenosis and 38 patients with more than 90% stenosis), and Group B was composed of 32 patients with 50% stenosis. Cardiac events including angina pectoris (n = 63), myocardial infarction (n = 1), heart failure (n = 2) and cardiac death (n = 0), coronary revascularization and predictive factors were evaluated during follow-up of 34 +/- 21 months. Furthermore, the interval between coronary revascularization and exercise myocardial perfusion scintigraphy was estimated. RESULTS: The rates of cardiac events (45%) and coronary revascularization (29%) in Group A were significantly higher than the rate of cardiac events (9%, p < 0.05) and coronary revascularization (6%, p < 0.05) in Group B. Only percentage stenosis and the number of diseased vessels affected the rates of cardiac event and coronary revascularization. CONCLUSIONS: Patients with significant coronary stenosis, but without ischemic evidence by exercise myocardial perfusion scintigraphy, have a relatively high rate of cardiac event and coronary revascularization, especially in patients with severe stenosis or multivessel disease. However, coronary revascularization should not be performed in all patients with significant coronary stenosis.

Aged↗
PubMed2001

Presence of potential nickel-responsive element(s) in the mouse MTH1 promoter.

The murine MTH1 gene codes for MTH1, an 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) which hydrolyzes 8-oxo-dGTP, a promutagenic product of reactive oxygen species' attack on the nucleotide pool. This gene is regulated by oxidative stress. Therefore, we hypothesized that MTH1 expression can be affected by carcinogenic nickel(II), known to induce such stress. Three plasmid constructs, carrying different upstream regions of the mouse MTH1 and the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently transfected into NIH 3T3 cells and the CAT protein was measured in nickel(II) acetate-treated and untreated cells. Nickel concentration-dependent increase of CAT protein level was observed for low Ni(II) concentrations, up to 400 microM Ni(II), in cells transfected with pHI103 plasmid (-5969 to +530 of the MTH1 sequence) only. Cells transfected with the pHI104 (-1331 to +530) or pHI108 (-151 to +530) plasmids did not respond to nickel(II) whatsoever. This finding demonstrated that the MTH1 sequence between -5969 and -1331 contained element(s) responsive to nickel(II) treatment. DNA sequencing revealed the presence of AP-1, NF-kappaB, and ATF-1 binding sites in both the -5969 to -1331 and -1331 to +530 regions. In contrast, two (CA)n repeats (-5642 to -5582 and -2078 to -2031), a family of B2 (-5428 to -5247) and B1 (-4559 to -4420) short interspersed repeated elements, and an (AT)n repeat (-5243 to -5230) were identified only in the -5969 to -1331 sequence. The results suggest that up-regulation of murine MTH1 expression by nickel(II) is controlled by the repeat sequences, potential candidates for nickel-responsive elements.

3T3 Cells↗
PubMed2001

[Strategy for post coronary artery bypass grafting in patients with bypass graft stenosis: comparison of percutaneous transluminal coronary angioplasty for the native coronary artery, internal mammary artery and saphenous vein graft].

OBJECTIVES: The strategy for post coronary artery bypass grafting (CABG) was investigated in patients with graft stenosis. METHODS: The study included 123 post-CABG patients with graft stenosis. The patients were divided into three groups according to target vessels; saphenous vein graft (SVG; n = 72), internal mammary artery (IMA; n = 21) and native coronary artery (n = 30). Furthermore, SVG lesions were divided into proximal anastomosis (n = 23), body (n = 40) and distal anastomosis (n = 9). The procedural success rate and late patency rate were compared between the three groups. Furthermore, the relationships between pre percutaneous transluminal coronary angioplasty (PTCA) percentage diameter stenosis, procedural success rate and late patency rate were evaluated. RESULTS: Procedural success rate was similar in the three groups, but late patency rate was higher in the IMA group. Procedural success rate and late patency rate were significantly lower in proximal anastomoses compared to other sites of SVG stenoses, IMA group and native coronary artery group (p < 0.05). Totally occluded native coronary artery lesions had a high procedural success rate compared with occluded IMA and SVG lesions, but the late patency rate was not higher. Procedural success rate showed no significant difference for 75-99% stenotic lesions, but the late patency rate was significantly higher in the IMA group (p < 0.05). Patients in the stenting group had a greater late patency rate compared with the balloon angioplasty group. There was no significant difference in late patency rate between the IMA group and SVG group. CONCLUSIONS: Late patency rate of the IMA is higher than that of the native coronary artery. SVG with proximal anastomosis and severe stenosis shows a significantly lower late patency rate than the native coronary artery. Therefore, PTCA should be considered for the native coronary artery in the absence of chronic total occlusion.

Aged↗
PubMed2000

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗