Biomedical subjects

M Mizutani

Publications and source records attributed to M Mizutani.

At least 73 records · Page 4Linked to original sources

PubMed1999

Microsomal electron transfer in higher plants: cloning and heterologous expression of NADH-cytochrome b5 reductase from Arabidopsis.

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b5 reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b5, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b5 reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b5 reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b5 reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b5 (AtB5-A), whereas no Cyt b5 reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b5 with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b5 reductase and NADPH-Cyt P450 reductase can reduce Cyt b5 and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.

Amino Acid Sequence↗

PubMed1999

Disaccharide esters screened for inhibition of tumor necrosis factor-alpha release are new anti-cancer agents.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine playing a part in various pathological states. Non-toxic inhibitors of TNF-alpha release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF-alpha release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF-alpha release with synthetic disaccharide esters, such as 6,6'-di-O-alkanoyl-alpha, alpha-trehaloses (6,6'-diester-trehaloses), 4,4'-di-O-alkanoyl-alpha, alpha-trehaloses (4,4'-diester-trehaloses) and 6,6'-diamino-6,6'-dideoxy-N,N'-dialkanoyl-alpha, alpha-trehaloses (6,6'-diamide-trehaloses) bearing fatty acids of various chain lengths, and n-dodecyl-beta-D-maltoside as a disaccharide monoester. 6,6'-Diester-trehaloses and 4,4'-diester-trehaloses of C8 to C12 fatty acids, 6,6'-diamide-trehaloses of C8 to C14 fatty acids, and n-dodecyl-beta-D-maltoside all inhibited TNF-alpha release in a dose-dependent manner. The IC50 values are 7.4-14.8 microM for 6,6'-diester-trehaloses (C8 to C12), 14.6-21.6 microM 4,4'-diester-trehaloses (C8 to C12), 2.9-15.0 microM for 6,6'-diamide-trehaloses (C8 to C14) and 23 microM for dodecyl-beta-D-maltoside. Both 6,6'-di-O-octanoyl-alpha, alpha-trehalose (C8, designated as SS555) and n-dodecyl-beta-D-maltoside (C12) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor-bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n-dodecyl-beta-D-maltoside. Moreover, SS555 inhibited TNF-alpha gene expression mediated through inhibition of AP-1 activation, but not NF-kappa B activation. This paper reports that diester-trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n-dodecyl-beta-D-maltoside appear to be potential cancer-preventive agents of a new type.

3T3 Cells↗

PubMed1999

The relationship between gastrointestinal transit and motility in dogs with postoperative ileus.

We investigated the relationship between gastrointestinal (GI) transit and motility during postoperative ileus in dogs undergoing a single laparotomy. We combined X-ray radiography for a GI transit study with chronically implanted force transducers (FTs) for a GI motility study. Radio-opaque markers made of polyethylene and steel wires or barium sulfate were used to examine solid substance transit or liquid substance transit. For a while after the end of the operation, postoperative ileus was observed, with weak irregular contractions of the GI tract. Transmission of the contractions to the lower GI tract was then observed. The start point of interdigestive migrating contraction (IMC)-like motility was observed in the order of small intestine (I-IMC), duodenum (D-IMC), and stomach (G-IMC), and IMC proceeded gradually after the operation. The gastric emptying time of a solid marker was 73.6 +/- 2.3 h (n = 5), and depended on the time of first occurrence of G-IMC (r = 0.674, p = 0.006). The gastric emptying of the liquid marker was finished before the time of the first occurrence of G-IMC, and its small intestinal transit time correlated with the time of the first occurrence of G-IMC (r = 0.888, p = 0.018). Using combined X-ray radiography and FTs we found that recovery from postoperative ileus was aided by GI motility in which contractions were transmitted from the stomach to the lower GI tract, like IMC.

Animals↗

PubMed1999

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.

Alleles↗

PubMed1999

[Tests for Hardy-Weinberg equilibrium in Japanese population].

In population genetics, the absence of the departure from Hardy-Weinberg equilibrium (HWE) is usually tested when a population study of a certain DNA marker is performed to show the observed allele frequencies represent those of the whole population. The goodness-of-fit test (chi 2 test) assuming chi 2 approximation has frequently been used with classical blood type markers having a few alleles. However, new tests suitable for DNA markers having many alleles, such as homozygosity test, likelihood ratio test and Guo-Thompson's (G-T') exact test, have recently been devised. In the present study, appropriate tests for HWE was studied using population data of 206 Japanese individuals with 9 different short tandem repeat loci. Firstly, we found that the recommendation of NRC II for the treatment of rare allele frequencies (If a bin in the database contains fewer than five entries, it is pooled with adjacent bins so that no bin has fewer than five) is quite reasonable for personal identification in forensic sciences. Secondly, we proposed that homozygosity test, likelihood ratio test and G-T's exact test should be applied altogether and HWE of the sample population should be valid only when all of the three tests were cleared.

Asian People↗

PubMed1999

Allele distribution at nine STR loci--D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820--in the Japanese population by multiplex PCR and capillary electrophoresis.

Nine tetranucleotide short tandem repeat (STR) loci, D3S1358, vWA, FGA TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820, were analyzed in the Japanese population with a newly released kit for personal identification using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis. The observed heterozygosities were 0.67, 0.77, 0.82, 0.61, 0.62, 0.73, 0.78, 0.81 and 0.74, respectively, and the combined discrimination power of the nineplex was 0.9999999991. None of the nine loci deviated from Hardy-Weinberg equilibrium expectations using the chi-square test, homozygosity test, likelihood ratio test and exact test after the grouping of the alleles. The nine STR loci allele frequencies were significantly different from those of other ethnic populations.

Alleles↗

PubMed1998

Facilitation of gastric motility induced by portal infusion of hyper- and hypotonic solution in rats.

The effects of the portal infusion of hyper- and hypotonic solution on gastric motility in rats were investigated. The infusion of hypertonic saline into the portal vein (portal infusion) elicited a significant enhancement of gastric contractile activity. The portal infusion of water also produced this enhancement. However, the portal infusion of isotonic saline showed no significant enhancement; nor did the infusion of water and hypertonic saline into the jugular vein. Sectioning of the hepatic branch of the vagus nerve (hepatic vagus) eliminated the enhanced responses of the gastric motility. It is therefore concluded that hepatoportal osmoreceptive afferent signals affect the gastric motility by way of the hepatic vagus. These effects on osmolarity revealed that hypotonic stimulation is more effective than hypertonic stimulation for the enhancement of motility. Sectioning of the dorsal subdiaphragmatic vagus, which includes the dorsal gastric and celiac branch, did not eliminate these responses. Sectioning of the ventral gastric vagus, in contrast, did eliminate the responses. These results suggest that vagal preganglionic neurons in the left dorsal motor nucleus of the vagus play a role in enhancement of gastric motility observed in the present research.

Animals↗

PubMed1998

Factors responsible for inhibiting the motility of zoospores of the phytopathogenic fungus Aphanomyces cochlioides isolated from the non-host plant Portulaca oleracea.

In a survey of plant secondary metabolites regulating the behaviour of Aphanomyces cochlioides zoospores, we found that root extracts of Portulaca oleracea inhibited zoospore motility. Bioassay-directed fractionation of Portulaca constituents revealed that the inhibitory activity was dependent on the interaction of two chemically different factors. These were identified as a phenolic compound, N-trans-feruloyltyramine, which by itself was active as a zoospore stimulant, and an acidic compound, 1-linoleoyl-2-lysophosphatidic acid monomethyl ester, which had zoospore-repellent activity. When Chromosorb W AW particles coated with a mixture of these pure compounds were bioassayed in Petri dishes, the inhibitory effect on zoospore motility was identical with that caused by root tip or root extracts of P. oleracea. Inhibited zoospores rapidly settled to the bottom of the Petri dishes where they initially encysted, and then germinated within 1-2 h. This is the first report of factors which inhibit zoospore motility without killing or bursting the zoospores.

Chemotaxis↗

PubMed1998

Computer-Aided Diagnosis (CAD) for Mammography: Preliminary Results.

PURPOSE: To evaluate the CAD system for screening mammograms. MATERIALS AND METHODS: Mammograms of patients with asymptomatic breast cancerwere examined retrospectively. A total of 260 screening mammograms from 65 patients (bilateral cranio-caudal and medio-lateral-oblique views from each) were tested by using the CAD system (ImageChecker M1000 system, R2 Technology, Los Altos, CA, USA). Results: With 0.58 microcalcifications marks/film and 0.20 mass marks/film set as the false positive rate, 100% of microcalcifications cases and 79% of mass cases were detected correctly. Conclusion: The CAD system has the potential to become a second reader. Further developments are expected to improve the accuracy of detection of mass lesions

Journal Article↗

PubMed1998

Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1.

Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1alpha-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.

Amino Acid Sequence↗

PubMed1998

Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency.

Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.

Adenoviridae↗

PubMed1998

Laser-induced fluorescence excitation spectroscopy of N(2)(+) produced by VUV photoionization of N(2) and N(2)O.

Synchrotron radiation emitted from the UVSOR storage ring is monochromated by a grazing-incidence monochromator and introduced coaxially with the second harmonic of a mode-locked Ti:sapphire laser. Sample gases, N(2) and N(2)O, are photoionized into vibronically ground N(2)(+) with the fundamental light of the undulator radiation at 18.0 and 18.6 eV, respectively. Laser-induced fluorescence (LIF) excitation spectra of N(2)(+) from N(2) and N(2)O are measured in the laser wavelength region of the (B (2)Sigma(u)(+), v' = 0) <-- (X (2)Sigma(g)(+), v" = 0) transition at 389-392 nm. The LIF excitation spectra of N(2)(+) exhibit two maxima due to the P and R branches in which rotational bands are heavily overlapped. The rotational temperature is determined by simulating an LIF excitation spectrum by using the theoretical intensity distribution of rotation bands convoluted with the spectral width of the laser.

Journal Article↗

PubMed1998

Comparative study of behavioral development between high and low shuttlebox avoidance rats.

Previously, we reported that high- and low-avoidance animals (HAA and LAA, respectively), selectively bred for different avoidance response rates in a shuttlebox avoidance test, showed additional behavioral differences in wheel cage activity and in water maze performance after weaning. In the present study, physical and behavioral development were examined in HAA and LAA during the preweaning period. As compared to HAA, LAA offspring showed lower body weight, delayed eye opening, poorer performance in pivoting and negative geotaxis, and increased open-field activity. A fostering study indicated that these differences observed in eye opening, pivoting, negative geotaxis, open-field activity, swimming speed, shuttlebox avoidance and wheel cage activity were independent of maternal care. Only the pup weight was strongly dependent on the maternal line. These results indicate that behavioral differences between HAA and LAA observed in the pre- and postweaning periods may be linked to the avoidance genotype, but the difference in pup weight may be caused by maternal care.

Animals↗

PubMed1998

Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.

Animals↗

PubMed1998

Impairment of absorption of digoxin by acarbose.

This study was conducted to determine the effect of acarbose on serum concentrations of digoxin in healthy male volunteers. A randomized crossover design with three phases was used. In phase 1 participants received 0.5 mg of digoxin alone. In phase 2 they received 0.5 mg of digoxin 0.5 hours after a 200-mg dose of acarbose. In phase 3 they received 100 mg of acarbose 0.5 hours before each meal three times daily for 3 days. On the fourth day, they received 0.5 mg of digoxin 0.5 hours after a 100-mg dose of acarbose. Area under the concentration-time curve (AUC0-48) and mean maximum concentration (Cmax) were significantly lower and tmax significantly increased in phases 2 and 3 compared with phase 1. These results indicate that the absorption of digoxin is reduced by administration of acarbose, and that one of the major mechanisms of this interaction may be due to the pharmacodynamics of acarbose.

Absorption↗

PubMed1998

Influence of cellular incorporation of n-3 eicosapentaenoic acid on intracellular Ca2+ concentration and membrane potential in vascular smooth muscle cells.

Long-term treatment with n-3 eicosapentaenoic acid (EPA) has been shown to exert hypotensive effects and have beneficial effects on atherosclerosis. To elucidate one of the underlying mechanisms of these effects, intracellular calcium concentration [Ca2+]i, and resting membrane potential were measured in rat vascular smooth muscle cells (A7r5 cell) treated with EPA, using Ca2+-sensitive dye fura-2 AM and the patch clamp technique. The alterations in fatty acid compositions of phospholipids and cell migration after treatment with EPA (30 microM) for 6 h-7 days were also examined. After treating cells with EPA, the EPA and DPA (docosapentaenoic acid) content of the phospholipid fraction (mol.%) increased in a time-dependent manner. Alternatively, arachidonic acid (AA) decreased, and then the ratio of EPA and AA (EPA/AA) increased significantly. The resting [Ca2+]i decreased from 170 +/- 46 nM (n = 16) in control cells to 123 +/- 29 nM (n = 16) in cells treated with EPA (30 microM) for 7 days. Vasopressin (100 nM), endothelin-1 (100 nM) and platelet-derived growth factor (PDGF 5 ng/ml) evoked an initial peak of [Ca2+]i, followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular Ca2+. In EPA-treated cells, both the peak and the sustained rise of [Ca2+]i induced by these agonists decreased in comparison to the control cells. EPA treatment also decreased the transient [Ca2+]i rise evoked by these agonists in the absence of extracellular Ca2+. Under the current clamp condition, resting membrane potential was significantly higher in EPA-treated cells (-49.8 +/- 10.4 mV, n = 41) than in control cells (-44.6 +/- 7.4 mV, n = 41, P < 0.05), and the input resistance of the cell was lower in EPA-treated cells, while cell size and capacitance were not statistically different. In addition, long-term treatment with EPA for 7 days significantly inhibited PDGF-induced cell migration. These results suggest that cellular incorporation of n-3 eicosapentaenoic acid attenuates intracellular mechanisms related to changes of [Ca2+]i and affects membrane potential, thereby inhibiting migration of vascular smooth muscle cells. These actions of EPA may contribute to its vasorelaxant and antiatherosclerotic effects.

Animals↗

PubMed1998

Hypoxia and endothelin-1 induce VEGF production in human vascular smooth muscle cells.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a secreted mitogen for vascular endothelial cells, and it promotes vascular permeability and neovascularization in vivo. We investigated the mechanisms by which low oxygen tension modulates the expression of VEGF in human aortic vascular smooth muscle cells (h-SMC) in vitro. Moreover, we measured VEGF levels in the cultured medium with or without endothelin-1 (ET-1) using a newly developed, highly sensitive, enzyme-linked immunosorbent assay. Hypoxia resulted in a substantial induction of VEGF transcripts at 3 and 24 hr. VEGF levels were significantly higher when h-SMC were cultured in medium containing ET-1 than when cultured in medium without ET-1. In conclusion, hypoxia and ET-1 constitute potent stimuli for VEGF production in h-SMC.

Cell Hypoxia↗

PubMed1998

Frequent expression of human Thy-1 antigen on pre-B cell acute lymphoblastic leukemia with t(9;22).

Thy-1 (CDw90) is a phosphatidylinositol-anchored protein, and is expressed on human pluripotential hematopoietic stem cells. The expression pattern of this antigen on leukemia cells is still controversial. In this study, 72 adult patients with pre-B cell acute lymphoblastic leukemia (pre-B ALL) were examined for the expression pattern of Thy-1 by using indirect immunofluorescence and reversed transcription polymerase chain reaction (RT-PCR) methods. Twelve cases were judged positive on the basis of conventional immunophenotype criteria. Thirteen cases showed a weak clonal shift on the fluorogram, even though their positive percentages were from 6.7% to 14.9%. Thy-1 gene transcripts were detected in all of the 13 cases showing a weak clonal shift. The study of antibody binding capacity, which was calculated by the mean fluorescence intensity of the test sample on the basis of a calibration curve using standard beads, showed that cases with more than 150 sites/cell could be positive. Thy-1 positivity in pre-B ALL was not associated with the expression of B-cell differentiation antigens. Thy-1 expression was significantly higher in pre-B ALL cases with karyotypic abnormalities than in those with normal karyotype (p = 0.0071). The t(9;22) abnormality was found in 18 of the 25 Thy-1+ cases. Simultaneous expression of transcriptional factors, GATA-2 and SCL, was frequently detected in the Thy-1+ cases. bcr-abl and GATA-2 are thought to play important roles in the proliferation of immature hematopoietic cells. Indeed, cell-cycle analysis showed that the cell population in the S/G2/M phase of the present Thy-1+ cases was less than that in the Thy-1- cases (p = 0.001770). Our data suggest that Thy-1 expression indicates the proliferative status of the leukemia cells, not their phenotypic immaturity.

Adolescent↗