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Biomedical subjects

T Miyazawa

Publications and source records attributed to T Miyazawa.

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

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1.

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Animals↗
PubMed2001

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells.

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.

Animals↗
PubMed2001

Genetic loci controlling susceptibility to gamma-ray-induced thymic lymphoma.

BALB/c is a susceptible strain for the development of gamma-ray induced mouse thymic lymphoma whereas MSM shows resistance. Association analysis of 220 backcross mice between the two strains using 67 markers was carried out to identify loci involved in the control of susceptibility. The genotype of mice with lymphoma showed excess heterozygosity relative to MSM homozygosity at D2Mit15 and D4Mit12 and was skewed toward MSM-derived alleles at D5Mit5. The P values in Mantel-Cox test were 0.0048 (D2Mit15), 0.0034 (D4Mit12) and 0.0048 (D5Mit5), suggesting association at the three loci in the susceptibility. Cooperative effect on lymphomagenesis was also observed among the three loci. To obtain independent evidence for linkage at D4Mit12, we made partially congenic mice in which a D4Mit12 region in BALB/c was replaced by MSM-derived homolog. Examination for the lymphoma susceptibility in 78 progeny of the congenic mice confirmed the effect of the locus near D4Mit12 (P=0.0037). The result, together with the linkage analysis, shows that the locus near D4Mit12 is regarded as a confirmed linkage but the other two loci as marginally suggestive.

Alleles↗
PubMed2001

Abnormal uterus with polycysts, accumulation of uterine prostaglandins, and reduced fertility in mice heterozygous for acyl-CoA synthetase 4 deficiency.

Arachidonate released by various stimuli is rapidly reesterified into membrane phospholipids initiated by acyl-CoA synthetase (ACS) and subsequent acyl-transfer reactions. ACS4 is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Female mice heterozygous for ACS4 deficiency become pregnant less frequently and produce small litters with extremely low transmission of the disrupted alleles. Striking morphological changes, including extremely enlarged uteri and lumina filled with numerous proliferative cysts of various sizes, were detected in ACS4+/- females. Furthermore, marked accumulation of prostaglandins was seen in the uterus of the heterozygous females. These results indicate that ACS4 modulates female fertility and uterine prostaglandin production.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.

The mechanism of hepatocarcinogenesis in hepatitis C virus (HCV) infection is still undefined. One possibility is the involvement of oxidative stress, which can produce genetic mutations as well as gross chromosomal alterations and contribute to cancer development. We recently showed that after a long period, the core protein of HCV induces hepatocellular carcinoma (HCC) in transgenic mice with marked hepatic steatosis but without inflammation, indicating a direct involvement of HCV in hepatocarcinogenesis. To elucidate the biochemical events before the development of HCC, we examined several parameters of oxidative stress and redox homeostasis in a mouse model of HCV-associated HCC. For young mice ages 3-12 months, there was no significant difference in the levels of hydroperoxides of phosphatidylcholine (PCOOH) and phosphatidylethanolamine in liver tissue homogenates between transgenic and nontransgenic control mice. In contrast, the PCOOH level was increased by 180% in old core gene transgenic mice > 16 months old. Concurrently, there was a significant increase in the catalase activity, and there were decreases in the levels of total and reduced glutathione in the same mice. A direct in situ determination by chemiluminescence revealed an increase in hydroperoxide products by 170% even in young transgenic mice, suggesting that hydroperoxides were overproduced but immediately removed by an activated scavenger system in young mice. Electron microscopy revealed lipofuscin granules, secondary lysosomes carrying various cytoplasmic organelles, and disruption of the double membrane structure of mitochondria, and PCR analysis disclosed a deletion in mitochondrial DNA. Interestingly, alcohol caused a marked increase in the PCOOH level in transgenic mice, suggesting synergism between alcohol and HCV in hepatocarcinogenesis. The HCV core protein thus alters the oxidant/antioxidant state in the liver in the absence of inflammation and may thereby contribute to or facilitate, at least in part, the development of HCC in HCV infection.

Animals↗
PubMed2001

Reactivation of feline foamy virus from a chronically infected feline renal cell line by trichostatin A.

Although acute infection of feline foamy virus (FeFV) is normally highly cytopathogenic in Crandell feline kidney (CRFK) cells, a noncytopathic persistent infection was established in the cells after cocultivation of the initially infected cells with uninfected cells four times. To investigate reactivation of persistent infection, CRFK cells chronically infected with FeFV were treated with trichostatin A (TA), a histone deacetylase inhibitor. TA induced higher FeFV production from the Coleman strain carrier culture and also induced marked syncytium formation. In contrast, human foamy virus, which contains less homologous long terminal repeat (LTR) and putative internal promoter (IP) sequences, persistently infecting baby hamster kidney cells was not reactivated by TA. The Sammy-1 strain of FeFV, from which a part of the U3 region in the LTR is naturally deleted, showed less reactivation. The Coleman LTR promoter-based beta-Gal-expressing plasmid was activated in the persistently Coleman-infected cells in the presence of TA, whereas the Sammy-1 LTR was not activated. Furthermore, the amounts of Gag protein expressed did not change in the presence or absence of TA. Because the putative IP region was very similar between the two strains, the initiation by TA is relatively specific for LTR sequences, and, therefore, histone deacetylation is at least in part responsible for reactivation of FeFV from carrier cell culture.

Animals↗
PubMed2001

Allelic loss mapping and physical delineation of a region harboring a thymic lymphoma suppressor gene on mouse chromosome 16.

Our previous mapping of allelic loss in gamma-ray induced thymic lymphomas in F(1) hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.

Alleles↗
PubMed2001

Dwarfism and early death in mice lacking C-type natriuretic peptide.

Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc(-/-) mice). The Nppc(-/-) mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc(-/-) mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.

Animals↗
PubMed2001

Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry.

Tetrodotoxin (TTX), a powerful sodium channel blocker, usually exists as a mixture of its analogs (TTXs) in natural sources. Due to the structural variation, some analogs are difficult to detect using the postcolumn liquid chromatography-fluorescent detection (LC-FLD) system. Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) analysis of TTXs can be achieved by a combination of chromatography on a reversed-phase column with long carbon chains (C30) and the mobile phase containing an ion pair reagent (ammonium heptafluorobutyrate). The relationship between the amount of applied standard TTX and its peak area on the mass chromatogram (m/z 320) showed good linearity over a range of 50-1000 pmol. The detection limit for TTX in the selected ion monitoring (SIM) mode was estimated to be 0.7 pmol (signal to noise ratio: 2). The tandem mass spectrometry (MS/MS) scan for the fragment ions of eight TTXs arising from the molecular ions provided characteristic spectra, and the structures of the origins of the prominent fragment ions were proposed. The intense fragment ions of TTX and 11-deoxyTTX were applicable to LC/MS/MS operated in the selected reaction monitoring mode. This method might be useful for further identification of TTXs.

Animals↗
PubMed2001

The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells.

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.

Antioxidants↗
PubMed2001

Effects of substituent and temperature on enantioselectivity for lipase-catalyzed esterification of 2-(4-substituted phenoxy) propionic acids in organic solvents.

Substituent effects on the enantioselectivity for the lipase-catalyzed esterifications in organic solvents were studied by use of 2-(4-substituted phenoxy)propionic acids as the substrates with various substituents of H, F, Cl, CF(3), CH(3), CH(3)CH(2), and CH(3)O. The distinction in the behavior of their enantioselectivity was primarily responsible for the size effects of the substituents, although the substituents are far away from the stereocenter of the substrates. For the similar substituents in size, CH(3) and CF(3), however, their electronic effects played an important role in controlling the enantioselectivity. This variation of the enantioselectivity due to the electronic effects is also supported by the discussion based on the value of the Michaelis constant (K(m)) obtained. In addition, by raising the reaction temperature with enough water added to isopropyl ether as the reaction medium, the enantioselectivity is found to be dramatically enhanced for the substrate bearing CH(3)O group due to the strong electron-donating effect.

Candida↗
PubMed2001

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus.

We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats.

Animals↗
PubMed2001

Enhanced level of n-3 fatty acid in membrane phospholipids induces lipid peroxidation in rats fed dietary docosahexaenoic acid oil.

The effect of dietary docosahexaenoic acid (DHA, 22:6n-3) oil with different lipid types on lipid peroxidation was studied in rats. Each group of male Sprague-Dawley rats was pair fed 15% (w/w) of either DHA-triglycerides (DHA-TG), DHA-ethyl esters (DHA-EE) or DHA-phospholipids (DHA-PL) for up to 3 weeks. The palm oil (supplemented with 20% soybean oil) diet without DHA was fed as the control. Dietary DHA oils lowered plasma triglyceride concentrations in rats fed DHA-TG (by 30%), DHA-EE (by 45%) and DHA-PL (by 27%), compared to control. The incorporation of dietary DHA into plasma and liver phospholipids was more pronounced in the DHA-TG and DHA-EE group than in the DHA-PL group. However, DHA oil intake negatively influenced lipid peroxidation in both plasma and liver. Phospholipid peroxidation in plasma and liver was significantly higher than control in rats fed DHA-TG or DHA-EE, but not DHA-PL. These results are consistent with increased thiobarbituric acid reactive substances (TBARS) and decreased alpha-tocopherol levels in plasma and liver. In addition, liver microsomes from rats of each group were exposed to a mixture of chelated iron (Fe(3+)/ADP) and NADPH to determine the rate of peroxidative damage. During NADPH-dependent peroxidation of microsomes, the accumulation of phospholipid hydroperoxides, as well as TBARS, were elevated and alpha-tocopherol levels were significantly exhausted in DHA-TG and DHA-EE groups. During microsomal lipid peroxidation, there was a greater loss of n-3 fatty acids (mainly DHA) than of n-6 fatty acids, including arachidonic acid (20:4n-6). These results indicate that polyunsaturation of n-3 fatty acids is the most important target for lipid peroxidation. This suggests that the ingestion of large amounts of DHA oil enhances lipid peroxidation in the target membranes where greater amounts of n-3 fatty acids are incorporated, thereby increasing the peroxidizability and possibly accelerating the atherosclerotic process.

Animals↗
PubMed2001

Genetic analyses of feline foamy virus isolates from domestic and wild feline species in geographically distinct areas.

To know the genetic diversities and phylogenetic relationship among feline foamy virus (FeFV) isolates from domestic cats (Felis catus) and FeFV-related viruses from the Iriomote cats (Felis iriomotensis) and leopard cats (Felis bengalensis) in geographically distinct areas, we sequenced a partial gag-pol region of 17 strains and a partial env region of nine strains, and the U3 region of long terminal repeat of three strains of the viruses. FeFV-related viruses from the feral cats were quite similar to the FeFV from domestic cats in the sequenced regions. In the partial gag region, the identities of nucleotide sequences among the isolates were from 94 to 99%. In the partial env gene, the isolates were divided into two distinct genotypes (F17- and FUV-types) as reported by Winkler et al. (Virology 247 (1999) 144-151). More than 94% nucleotide identities were observed in the env region within a particular env genotype and about 75% nucleotide identities were noted between the two genotypes.

Amino Acid Sequence↗
PubMed2001

Evaluation of regional liver damage by magnetic resonance imaging with superparamagnetic iron oxide in rat liver.

The purpose of this study was to investigate whether regional liver damage could be detected by means of enhanced MR imaging with a superparamagnetic iron oxide (SH U 555A) in an ischemia-reperfusion model of rat liver. Ischemic liver damage was induced in the right lobe by vascular clamping for 0 (sham), 30 (I-30), 60 (I-60), and 90 minutes (I-90). There was no significant difference in relative enhancement (RE) between the ischemic and nonischemic lobes in the sham, I-30 and I-60 groups, while RE of the ischemic lobe was significantly lower than that of its nonischemic counterpart in the I-90 group as seen on SH U 555A enhanced proton density spin echo images (P < 0.05). Histological examination revealed that iron deposits were significantly smaller in the ischemic than the nonischemic lobe in the I-90 group (P < 0.05), although there was no significant difference in the number of Kupffer cells. Our results indicate that severe regional liver damage can be evaluated by MR imaging with SH U 555A.

Animals↗
PubMed2001

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.

Amino Acid Sequence↗
PubMed2001

Giant intracranial granular-cell tumor arising from the abducens.

We present a case of an adolescent with a giant intracranial granular-cell tumor, who presented with diplopia and worsening visual acuity. The tumor measured 8.2 cm at the maximum diameter and occupied the right middle cranial fossa, with intrasellar and suprasellar involvement. It was composed of solid nests of oval or polygonal eosinophilic cells with a focal pseudo-papillary configuration. Pituitary macroadenoma was suspected from examination of the intraoperative frozen section, but immunohistochemical and ultrastructural findings were consistent with granular-cell tumor, and they suggested the origin to be the peripheral nerve sheath. Considering that abducens palsy was the first clinical manifestation, the tumor was thought to arise from abducens. From histological findings (focal cellular spindling, mitotic activity and a relatively high Ki-67 labeling index [10.1%]) and according to Fanburg-Smith's criteria, the tumor was suspected to have low-grade malignant potential. Granular-cell tumor should be included in the differential diagnosis of parasellar tumors, even in young patients or patients with large lesions. Both immunohistochemical and electron microscopic studies are essential for diagnosis.

Abducens Nerve↗