Search PubMed⌕ Search

Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 55 records · Page 3Linked to original sources
PubMed1999

[Successful treatment of acute renal failure in a patient with essential mixed cryoglobulinemia using prednisolone and cryofiltration].

We report a case of acute renal failure associated with cryoglobulinemic glomerulonephritis. The patient, a 49-year-old woman, was referred to our hospital because of acute nephritic syndrome. After admission, she developed oliguria, and hemodialysis was instituted. Renal biopsy was performed and the specimens showed moderate endocapillary proliferation, large deposits filling the capillary lumen ("intraluminal thrombi"), and a double-contoured appearance, which are typical morphologic features of cryoglobulinemic glomerulonephritis. Immunoelectrophoresis showed a monoclonal increase of IgM kappa. On the basis of these findings, we diagnosed type II essential mixed cryoglobulinemia. Cryofiltration was performed with oral administration of prednisolone. Following the start of therapy, the patient's renal function gradually improved. Because of severe hypoproteinemia, cryofiltration was discontinued after three sessions. However, renal function recovered and was maintained with prednisolone only. This case shows that acute oliguric renal failure caused by cryoglobulinemic glomerulonephritis can be reversible if immunosuppressive therapy, together with plasmapheresis in more severe cases, is instituted promptly.

Acute Kidney Injury↗
PubMed1999

Heme-binding by Drosophila retinoid- and fatty acid-binding glycoprotein (RFABG), a member of the proapolipophorin gene family.

We previously have cloned and characterized a retinoid- and fatty acid-binding glycoprotein (RFABG) isolated from the heads of Drosophila melanogaster. The protein is composed of two glycosylated subunits (Mr = >200,000 and 70,000) and is a member of the proapolipophorin gene family. Spectral analysis of purified RFABG revealed an absolute absorbance peak at 405 nm, which is typical for a heme-containing protein. The aim of the present study was to characterize the heme-binding properties of RFABG. Upon saturation of the protein solution with carbon monoxide followed by dithionite reduction, a red shift of the Soret peak to 424 nm and the characteristic alpha- and beta- bands at 567 and 539 nm were observed. Native RFABG contains approximately 0.175 moles of heme (mol/mol) indicating that purified RFABG is primarily the apoprotein. Hemin-agarose affinity chromatography of the native RFABG followed by Western blot analysis showed a single immunoreactive band at 70 kDa, indicating that the heme-binding domain resides in the 70 kDa subunit. Although retinoid and fatty acid also bind to the 70 kDa subunit, no competition was observed when an excess of heme was added to a solution of retinoid or fatty acid bound to RFABG. Heme added to a solution of purified RFABG bound in a saturable manner with an affinity of 3.8 x 10(-7) m.Thus, the current study clearly demonstrates that retinoid- and fatty acid-binding glycoprotein is a novel heme-binding protein, which may be involved in the transport and/or metabolism of heme in Drosophila.

Animals↗
PubMed1999

Recurrence of IgA nephropathy 17 months after renal transplantation in the allograft transmitted thin basement membrane disease (TBMD) from donor.

Recurrence of IgA nephropathy (IgAN) following renal transplantation has been described in 40-50% of such patients and it usually has a good outcome. We present the case of a 20-yr-old woman with IgAN who developed end-stage renal failure in 1995. In November 1996, she received a kidney from a living-related donor and was treated with tacrolimus, azathioprine and steroids. Zero- and one-hour biopsies were performed, which revealed minor glomerular abnormalities in light microscopy, thin basement membrane disease (TBMD) in electron microscopy. Eight months later she developed microscopic hematuria and proteinuria; however, the graft function was normal. Renal biopsy revealed an IgAN that is thought to be due to recurrence of the original disease.

Adult↗
PubMed1998

Chemiluminescence assay for oxidatively modified myoglobin.

Treatment of myoglobin with H2O2 results in covalent alteration of the heme prosthetic group, in part, to protein-bound adducts. These protein-bound heme adducts are known to be redox active and are suspected to participate in oxidative tissue injury. In the course of our studies on the toxicological role of these heme adducts, we sought to develop a sensitive assay for their detection and quantitation. We have discovered that protein-bound heme adducts, due to their inherent peroxidase activity, can be detected with the use of enhanced chemiluminescence detection reagents, following SDS-PAGE and electroblotting. The assay is specific for protein-bound heme adducts as we have identified conditions where noncovalently bound hemes are completely dissociated from the protein during electrophoresis. Signal intensity was quantified by laser densitometry and found to be linear over a concentration range of 0.44-22 pmol of protein-bound heme adduct, which represented a 20-fold greater sensitivity than the currently available HPLC method. Moreover, we have identified tris(2-carboxyethyl)phosphine as a thiol reducing agent that does not interfere with the detection of the heme-mediated peroxidase activity. The current method may be utilized to identify heme-binding regions of proteins in addition to the detection of oxidatively modified myoglobin.

Animals↗
PubMed1998

Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death.

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.

Antibodies, Monoclonal↗
PubMed1998

Ultrastructural evaluation following catheterization of the fallopian tube with a hysteroscopic catheter.

PURPOSE: Our purpose was to assess the morphology and ultrastructural changes in the tubal epithelium following catheterization of the fallopian tube. METHODS: Fallopian tubes were obtained from 20 women who had undergone hysterectomies. Catheterization was performed in 20 tubes using a catheter developed for hysteroscopic tubal embryo transfer. The catheter has a 3-French diameter, tapering to 2 French (0.66 mm) at the tip portion. The 20 contralateral tubes served as controls and were not catheterized. Ultrastructural changes were examined by scanning electron microscopy and transmission electron microscopy. RESULTS: Scanning electron microscopy showed no transformation or defects of the tubal epithelium surface in catheterized or control tubes. Transmission electron microscopy showed no significant differences in the percentage of abnormal desmosomes and the percentage of basement membrane in ciliated and nonciliated cells between catheterized and noncatheterized tubes. No transformation or defects were observed in catheterized or noncatheterized tubes. CONCLUSIONS: These findings suggest that catheterization of the tube using a hysteroscopic catheter caused no acute damage to the tubal epithelium.

Basement Membrane↗
PubMed1998

Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1.

The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 microM, respectively. After incubation of P450 2B1 with 300 microM peroxynitrite, the heme moiety was not altered, but the apoprotein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactivite band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in the enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further demonstration of tyrosine nitration was confirmed by GC/MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards; approximately 0.97 mol of nitrotyrosine per mole of P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C, and the resulting peptides were separated by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160-225 of P450 2B1, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite resulted in the nitration of tyrosines at either residue 190 or 203 or at both residues of P450 2B1 concomitant with a loss of 2B1-dependent activity.

Animals↗
PubMed1998

Metabolism-based inactivation of penile nitric oxide synthase activity by guanabenz.

Guanabenz (Wytensin) was shown to inactivate nitric oxide synthase (NOS) activity in vitro and in vivo. In in vitro studies with the use of a cytosolic fraction from penile tissue, the inactivation was found to depend on NADPH, time, and the concentration of guanabenz. The L-, but not the D-, isomer of arginine could protect from the inactivation, suggesting an active site-directed event. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (Ki) and a pseudo first-order inactivation constant (kinact) of 38.5 microM and 0.179 min-1, respectively. In in vivo studies, guanabenz was shown to inhibit penile cytosolic NOS activity in a dose- and time-dependent manner. Treatment of rats with guanabenz (5 mg/kg/day) for 4 days caused a decrease of approximately one-half in the NOS activity of the penile cytosolic fraction with a concomitant loss in the amount of immunodetectable NOS protein. Treatment for 4 days at a dose of 0. 5 mg/kg/day showed a similar decrease in activity, whereas a dose of 0.05 mg/kg/day showed no effects. Due to the multitude of processes that are regulated by NO, the inactivation of NOS is a potential mechanism to be considered in a variety of biological effects associated with drugs.

Animals↗
PubMed1997

Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges superoxide (O2-) ions. We studied the regulation of MnSOD gene expression by irradiation and the mechanisms in human monocytic cell line THP-1. We found that irradiation induced expression of the MnSOD gene through the autocrine mechanism, involving the production of tumour necrosis factor (TNF). Irradiation increased TNF production in THP-1 cells, and TNF increased the levels of MnSOD transcripts. Supernatant from irradiated THP-1 cells induced the expression of MnSOD mRNA, and anti-TNF antibody blocked the induction of MnSOD mRNA. Irradiation also increased the levels of MnSOD mRNA in other myelocytic cell lines, HL60 and KG-1, and the ovarian cancer cell line SK-OV-3. Moreover, increased levels of MnSOD mRNA were observed in mature myeloid cells, including macrophages and granulocytes, as well as in immature cells. However, irradiation did not increase the level of MnSOD mRNA in THP-1 cells with prolonged exposure to PMA. We also found that irradiation increased the rate of MnSOD transcription, and irradiation stabilized MnSOD mRNA in THP-1 cells. Our results indicate that the endogenous production of TNF is required, at least in part, for the induction of MnSOD mRNA expression by irradiation in THP-1 cells, and the increased levels of MnSOD transcripts on irradiation occur through a pathway involving protein kinase C activation. Our results also indicate that the increase in MnSOD mRNA caused by irradiation is regulated by both transcriptional and post-transcriptional mechanisms.

Autocrine Communication↗
PubMed1997

Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: evidence for a novel oxidative pathway.

Treatment of metmyoglobin with H2O2 is known to lead to the crosslinking of an active site tyrosine residue to the heme [Catalano, C. E., Y. S. Choe, and P. R. Ortiz de Montellano (1989) J. Biol. Chem. 264, 10534-10541]. We have found in this study that this reaction also leads to an altered heme product not covalently bound to the protein. This product was characterized by visible absorption, infrared absorption, and mass and NMR spectrometry as an iron chlorin product formed from the saturation of the double bond between carbon atoms at positions 17 and 18 of pyrrole ring D with concomitant addition of a hydroxyl group on the carbon atom at position 18 and lactonization of the propionic acid to the carbon atom at position 17. Studies with the use of (18)O-labeled H2O2, O2, and H2O clearly indicate that the source of the added oxygen on the heme is water. Evidently, water adds regiospecifically to a cationic site formed on a carbon atom at position 18 after oxidation of the ferric heme prosthetic group with peroxide. Prolonged incubation of the reaction mixture containing the iron hydroxychlorin product led to the formation of an iron dihydroxychlorin product, presumably from a slow addition of water to the initial iron hydroxychlorin. The iron chlorin products characterized in this study are distinct from the meso-oxyheme species, which is thought to be formed during peroxide-mediated degradation of metmyoglobin, cytochrome P450, ferric heme, and model ferric hemes, and give further insight into the mechanism of H2O2-induced heme alterations.

Animals↗
PubMed1997

Preparation and characterization of the F (ab)2 fragments of an aromatase activity-suppressing monoclonal antibody.

The preparation and characterization of the Fab and F(ab')2 fragments of a murine monoclonal antibody specific for aromatase cytochrome P-450 and which is suppressive of estrogen biosynthesis are described. This monoclonal antibody, MAb3-2C2, was purified from murine ascites using protein A affinity chromatography and digested with immobilized papain to produce antibody fragments. The Fab and F(ab')2 fragments were then purified using protein A affinity chromatography and S-200 HR size exclusion chromatography. The Fab fragment was further purified using S-100 HR size exclusion chromatography. Both the Fab and F(ab')2 fragments of the MAb3-2C2 suppressed aromatase activity in a dose-dependent manner. While the F(ab')2 fragment (110 kDa) maintained potent suppressive activity, the Fab fragment (42 kDa) required a higher concentration to suppress aromatase activity as compared to the IgG.

Antibodies, Monoclonal↗
PubMed1997

Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium.

To determine whether local estrogen production takes place in endometriotic or adenomyotic tissues, in eutopic endometrium from patients with endometriosis or adenomyosis, and in normal endometrium, tissue specimens were examined by immunohistochemistry, catalytic activity, and mRNA expression for aromatase cytochrome P450 (P450arom). P450arom was immunohistochemically localized in the cytoplasm of glandular cells of endometriotic and adenomyotic tissues, and of eutopic endometrium from patients with the respective diseases, whereas estrogen receptors and progesterone receptors were localized in the nuclei of the glandular cells and stroma. Aromatase activity in the microsomal fraction of adenomyotic tissues was inhibited by the addition of danazol, aromatase inhibitors, and anti-human placental P450arom monoclonal antibody (mAb3-2C2) in a manner similar to such inhibition in other human tissues. Reverse transcription polymerase chain reaction and Southern blot analysis also revealed P450arom mRNA in these tissues. However, neither P450arom protein activity nor mRNA was detected in endometrial specimens obtained from normal menstruating women with cervical carcinoma in situ but without any other gynecological disease. These results suggest that at a local level, endometriotic and adenomyotic tissues produce estrogens, which may be involved in the tissue growth through interacting with the estrogen receptor.

Adult↗
PubMed1997

Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus.

American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March, 15 June, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggests that aromatase and oestrogen may play a role in re-initiating spermatogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Antibody responses against Em18 and Em16 serodiagnostic markers in alveolar and cystic echinococcosis patients from northwest China.

Western blot analysis was carried out in order to evaluate new serodiagnostic markers, Em18 and Em16, for differentiation of alveolar echinococcosis (AE) from cystic echinococcosis (CE) using 36 serum samples from hydatid patients from Xinjiang, China, where AE and CE are both endemic and one double infection case has been reported. All AE cases except one (5/6) who exhibited a calcified lesion and a single case of double infection showed antibody responses against Em18 and Em16. Some of CE patient sera (6/22) showed antibody response against Em16 except one who showed that against Em18. Analyses of IgG subclass responses against Em18 and Em16 were carried out using all serum samples showing antibody responses against Em18 and/or Em16 (seven CE, five AE, and one AE + CE) and additional samples of three CE and 22 AE from Sichuan, China. IgG4 was the most predominant antibody subclass. Em18 and Em16 were recognized by both IgG4 and IgG1 (in most cases) or by either IgG4 or IgG1 (in minor cases) or by IgG3 (in very rare cases). Neither Em18 nor Em16 was recognized by IgG2 antibodies. The usefulness of Em18 and Em16 as potential new markers for serological differentiation of human AE and CE, respectively, is discussed.

Antibody Formation↗
PubMed1997

Diverse function of aromatase and the N-terminal sequence deleted form.

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Aromatase↗