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A Mizutani

Publications and source records attributed to A Mizutani.

At least 19 recordsLinked to original sources
PubMed2001

Activated protein C prevents endotoxin-induced hypotension in rats by inhibiting excessive production of nitric oxide.

BACKGROUND: Excessive production of nitric oxide (NO) by the inducible isoform of NO synthase (iNOS) is critically involved in endotoxin (ET)-induced hypotension. Tumor necrosis factor-alpha (TNF-alpha) plays an important role in induction of iNOS. Because activated protein C (APC), a physiological anticoagulant, inhibits TNF-alpha production, it might prevent hypotension by inhibiting excessive production of NO. In this study, we examined this possibility using a rat model of septic shock. METHODS AND RESULTS: Intravenous administration of APC prevented both ET-induced hypotension and the increases in plasma levels of NO(2)(-)/NO(3)(-). The hypotension was also inhibited when APC was administered 30 minutes after ET administration. APC inhibited the increases in lung levels of iNOS activity by inhibiting expression of iNOS mRNA in animals given ET. APC significantly inhibited the increases in lung tissue levels of TNF-alpha and expression of TNF-alpha mRNA in animals given ET. Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor DIP-APC, an active site-blocked APC, showed any effect on these ET-induced changes. Both inhibition of TNF-alpha production by leukocytopenia and treatment with anti-rat TNF-alpha antibody produced effects similar to those induced by APC. Aminoguanidine, a selective inhibitor of iNOS, inhibited both the hypotension and the increases in plasma levels of NO(2)(-)/NO(3)(-) in this animal model. CONCLUSIONS: These observations strongly suggest that APC inhibits iNOS induction by decreasing TNF-alpha production, leading to the prevention of ET-induced hypotension. Furthermore, such effects of APC were not dependent on its anticoagulant effects but rather on its serine protease activity.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Purification and characterization of carbaryl hydrolase from Arthrobacter sp. RC100.

A carbaryl hydrolase was purified to homogeneity from Arthrobacter sp. strain RC100 by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, and gel filtration chromatographies. The native enzyme had a molecular mass of 100 kDa and was composed of two identical subunits with molecular masses of 51 kDa. The hydrolase activity was strongly inhibited by DIFP, PMSF, Hg(2+) and paraoxon but not by EDTA. The optimum pH and temperature for the enzyme activity were 9.0 and 50 degrees C, respectively. The enzyme hydrolyzed four N-methylcarbamate insecticides (carbaryl, xylylcarb, metolcarb and XMC), but was not able to hydrolyze fenobucarb, propoxur, and isoprocarb.

Arthrobacter↗
PubMed2001

Titration emulation: a computer-assisted technique that simplifies the quantification of anti-dsDNA antibodies using the Crithidia luciliae assay.

Titers of anti-double-stranded (ds) DNA antibodies in sera from patients with systemic lupus erythematosus (SLE) using the Crithidia luciliae assay method were compared by conventional titration vs the titration emulation method (ImageTiter) to evaluate whether the latter assay can replace manual titration. Titers by the two methods were identical or within one dilution in 98% (41/42) of samples. A single sample showed a two-dilution difference. Titration emulation showed a tendency to under-estimate the titer of high titer anti-dsDNA samples, although the difference was small. Titration emulation is a suitable alternative to the conventional titration method, offering an accurate and cost-effective approach to quantification of anti-dsDNA antibodies.

Animals↗
PubMed2001

Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and beta-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 was expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.

3T3 Cells↗
PubMed2001

Nonpolarizing guided-mode resonant grating filter for oblique incidence.

A new type of guided-mode resonant grating filter is described. The filter is independent of polarization state for oblique incidence. The filter has a crossed grating structure, and the plane of incidence on the filter contains the symmetric axis of the grating structure. Theoretical considerations and numerical calculations using two-dimensional rigorous coupled-wave analysis show that a rhombic lattice structure is suitable to such filters. In this configuration an incident light wave is diffracted into the waveguide and is divided into two propagation modes whose directions are symmetric with respect to the plane of incidence. In particular, when the propagation directions of the two modes are perpendicular to each other, the fill factor of grating structure can be approximately 50%. The filter was designed for an incident angle of 45 degrees. Tolerances of setting errors and fabrication errors for this filter were estimated by numerical calculations.

Journal Article↗
PubMed2000

Gene structure and promoter for Crad2 encoding mouse cis-retinol/3alpha-hydroxysterol short-chain dehydrogenase isozyme.

Cis-retinol/androgen dehydrogenase type 2 (CRAD2) has been shown to catalyze the dehydrogenation of retinols, including 9-cis retinol, and also to exhibit 3alpha- and 17beta- hydroxysteroid dehydrogenase activities. To examine the function of this enzyme and regulation of its gene, the Crad2 gene was cloned from a mouse genomic DNA library and characterized. The complete mouse CRAD2-coding region was found in four exons spanning an approximately 5kb region. The nucleotide sequences of the exons encoding 316 amino acids were identical to those of the previously reported mouse Crad2 cDNA. Primer extension analysis and RNase protection assay were used to map the major transcription initiation sites to the positions lying 87 and 89 base pairs upstream of the ATG translation start codon. The region proximal to the initiation sites exhibited the absence of both TATAA and CAAT boxes. This region had hepatocyte nuclear factor binding sites, consistent with its predominant expression in the liver. Computer analysis of an approximately 7.5kb 5'-flanking region also suggested the presence of binding sites for AP-1, SREBP1, HSF2, c-Rel, c-Myc, CREBP, GATA, Ets, E2F, and Oct-1, suggesting that various factors including retinoic acid, cholesterol, various kinds of stress, the cell cycle, and cyclic AMP may regulate the expression of this gene. Fluorescence in-situ hybridization analysis showed that Crad2 is located at the terminus of mouse chromosome 10, an area that corresponds to band 10D3, suggesting that RDH-related SDRs may be located together in the cluster locus. Northern blot hybridization and RT-PCR analysis demonstrated that CRAD2 was expressed not in early embryonic stages, and not in embryonic stem cells, but instead in the gastrointestinal tract during later embryonic development and adult stage. In conclusion, we have presented the first complete structural analysis, including that of the promoter and chromosomal location, of a member of the retinol/androgen dehydrogenase subfamily of the group of the short-chain dehydrogenase/reductase (SDR) isozymes. Our findings will provide the basis for in-vitro or in-vivo studies concerning the regulation of retinol and androgen metabolism and enable determination of the mechanism of diseases related to retinol, retinal, retinoic acid, and androgen.

Alcohol Oxidoreductases↗
PubMed2000

Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation.

We examined whether activated protein C (APC) reduces ischemia/reperfusion (I/R)-induced renal injury by inhibiting leukocyte activation. In a rat model, intravenous administration of APC markedly reduced I/R-induced renal dysfunction and histological changes, whereas intravenous administration of dansyl glutamylglycylarginyl chloromethyl ketone-treated factor Xa (DEGR-FXa; active-site-blocked factor Xa), heparin or diisopropyl fluorophosphate-treated APC (DIP-APC; inactive derivative of ARC) had no effect. Furthermore, APC significantly inhibited the I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability, whereas neither DEGR-FXa, heparin, nor DIP-APC produced such effects. Renal I/R-induced increases in plasma levels of fibrin degradation products were significantly inhibited by APC, DEGR-FXa, and heparin. These observations suggest that APC reduces I/R-induced renal injury independently of its anticoagulant effects but in a manner dependent on its serine protease activity. Renal levels of tumor necrosis factor-alpha (TNF-alpha), rat interleukin-8, and myeloperoxidase were significantly increased after renal I/R. These increases were significantly inhibited by APC but not by DEGR-FXa, heparin, or DIP-APC. Leukocytopenia produced effects similar to those of APC. These findings strongly suggest that APC protects against I/R-induced renal injury not by inhibiting coagulation abnormalities but by inhibiting activation of leukocytes that play an important role in I/R-induced renal injury. Inhibition of leukocyte activation by APC could be explained by the inhibitory activity of TNF-alpha. (Blood. 2000;95:3781-3787)

Amino Acid Chloromethyl Ketones↗
PubMed2000

SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms.

Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.

Amino Acid Sequence↗
PubMed2000

KL-6 as a novel marker for activities of interstitial pneumonia in connective tissue diseases.

The objective of this study was to determine the role of serum KL-6 levels as a marker for the activity of interstitial pneumonia in patients with connective tissue diseases. The serum concentrations of KL-6, a glycoprotein produced mainly by pulmonary type II epithelial cells, were measured in 21 patients with connective tissue disease. The activity of interstitial pneumonia was compared with the associated serum KL-6 concentrations. Serum KL-6 concentrations in patients with interstitial pneumonia were significantly higher than those in the controls. Among patients with active interstitial pneumonia, serum KL-6 concentrations following the treatment (after improvement) were significantly lower than the pretreatment values. The extent of the pulmonary fibrosis correlated positively with the serum KL-6 concentrations during the inactive phase of the interstitial pneumonia. These results suggest that sequential measurement of serum KL-6 levels is a new and useful means for the evaluation of interstitial pneumonia in patients with connective tissue diseases.

Antigens↗
PubMed2000

[A case of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) related glomerulonephritis associated with systemic sclerosis treated by steroid pulse therapy: a case report].

A 57-year-old woman had been diagnosed as systemic sclerosis (SSc) with Raynaud's phenomenon, acrosclerosis and polyarthritis since 1995. She admitted to our hospital in July 1996 because of general fatigue, hemosputa and progressive renal insufficiency. On admission, the blood pressure was normal and laboratory findings showed elevation of the serum creatinin level and a high titer of the myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) (> 1000 EU). The renal biopsy revealed crescentic glomerulonephritis. Both renal insufficiency and high titers of MPO-ANCA improved remarkably after methylpredonisolone pulse therapy. This case was suggestive of elucidating the pathogenesis of SSc and MPO-ANCA related glomerulonephritis.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1999

Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

Actins↗
PubMed1999

Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth.

Synaptotagmin I (or II), a possible Ca(2+)-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+ and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca(2+)-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I-V, X, and XI) and the ubiquitous type (synaptotagmin VI-IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca(2+)-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca(2+)-dependent synaptotagmin activation is also crucial for neurite outgrowth.

Amino Acid Sequence↗
PubMed1999

Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1.

Ascorbate-enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-gamma. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of I kappa B alpha by Western blotting, activation of the nuclear factor kappa B (NF-kappa B) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effect on the onset of either I kappa B alpha degradation or the nuclear translocation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The prolonged degradation of I kappa B alpha caused by ascorbate in LPS- and IFN-gamma-activated cells paralleled elevated NF-kappa B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascorbate exerted no effect on the expression of I kappa B alpha and ubiquitin genes in the activated cells. Ascorbate could modulate NF-kappa B DNA binding activity in response to combined LPS and IFN-gamma activation, which increases NO production in activated macrophages.

Animals↗
PubMed1998

Transcriptional activation of the alternative oxidase gene of the fungus Magnaporthe grisea by a respiratory-inhibiting fungicide and hydrogen peroxide.

Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.

Amino Acid Sequence↗
PubMed1998

A synthetic ceramide analog (L-PDMP) up-regulates neuronal function.

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its enantiomer, L-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by D-PDMP and facilitation by L-PDMP. Up-regulation of synaptic activity by L-PDMP could be correlated with the slow but robust activation of p42 mitogen-activated protein kinase. Treatment with L-PDMP after transient forebrain ischemia in rats ameliorated the deficit of a well-learned spatial memory by an 8-arm maze task, suggesting a new potential therapeutic approach for neurodegenerative disorders.

Animals↗
PubMed1998

Ascorbate-dependent enhancement of nitric oxide formation in activated macrophages.

Macrophages activated with bacterial lipopolysaccharide (LPS) and cytokines produce nitric oxide through the induction of iNOS gene expression. Ascorbate increased NOx (nitrite and nitrate) formation by approximately 40% in a mouse macrophage-like cell line, J774.1, activated with LPS and interferon-gamma. Ascorbate alone exhibited no inductive activity toward NO formation. N(G)-Monomethyl-L-arginine inhibited nitrite formation in cells activated in the presence or absence of ascorbate. Northern and Western blotting analyses showed that both iNOS mRNA and protein steady-state levels were increased approximately twofold in cells activated in the presence of ascorbate compared to in cells activated only with the inducers. These data suggest that ascorbate increased NO production by increasing the amount of iNOS in the activated macrophages.

Animals↗