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Biomedical subjects

H Ochiai

Publications and source records attributed to H Ochiai.

At least 19 recordsLinked to original sources
PubMed1992

In vitro reconstitution of an erythropoietin gene transcription system using its 5'-flanking sequence and a nuclear extract from anemic kidney.

We have developed an in vitro transcription system for the erythropoietin (Epo) gene. This system uses a plasmid carrying 0.2 kb of 5'-flanking sequence from the human Epo gene, rNTPs and a nuclear extract from mouse kidney. The transcribed RNA was assayed by primer extension with an end-labeled primer complementary to the sequence of the plasmid, dNTPs and reverse transcriptase. The primer extension product corresponding to the transcript was detected on a sequencing gel. The in vitro promoter activity of the Epo 5'-flanking sequence was observed with a nuclear extract from anemic kidney but not with that from normal kidney.

Anemia↗
PubMed1992

Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis.

Cytochrome b558 is an important constituent of the superoxide-generating system in neutrophils and B lymphocytes. In this paper, the topology of the cytochrome in human neutrophil membrane was studied using antibodies raised in rabbits against synthetic peptides corresponding to various regions in the large and small subunits of the cytochrome. The antibodies recognized the cytochrome subunits in immunoblots and the cytochrome in situ. An antibody raised against residues 150-172 in the large subunit (anti-L123) bound to intact neutrophils, indicating that this region is exposed to the outside of the cells. In contrast, antibodies raised against any of the carboxyl-terminal regions of the large and small subunits (anti-LC and anti-SC, respectively) or the amino-terminal region of the small subunit (anti-SN), bound to neutrophils only after the cells were made permeable by freezing and thawing. The region close to the carboxyl terminus of the large subunit was digested by extracellularly added papain and, as a result, an 18-kDa carboxyl-terminal fragment was detected. Thus the carboxyl-terminal region of the large subunit is cytoplasmic and/or buried in the membrane, and the region around residues 369-398 is exposed on the cell surface. In contrast to the large subunits, the small subunit in neutrophils was resistant to any of the proteinases tested, although the subunit in membrane or Triton-solubilized preparation was digestible with papain. These results indicate that the large subunit of cytochrome b558 is a transmembrane protein with at least two regions exposed on the cell surface and that the carboxyl terminus of this subunit and both termini of the small subunit are exposed to the cytoplasmic side.

Amino Acid Sequence↗
PubMed1992

Infection enhancement of influenza A NWS virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody.

Antibody-dependent enhancement (ADE) of influenza A NWS virus infection was investigated in primary murine macrophages (M phi) using anti-hemagglutinin(HA) monoclonal antibody (mAB). Contrary to previous reports of abortive influenza virus infection in primary M phi, this study demonstrated that the NWS virus replicated productively in both resident peritoneal M phi and thioglycolate-elicited peritoneal M phi providing cleavage of the HA was achieved by trypsin; 5 micrograms/ml of trypsin was the optimum concentration for the induction of infectivity. Under multiple-cycle growth conditions in the presence of mAB at various concentrations in trypsin-containing media, ADE was demonstrated in both M phi in the presence of subneutralizing concentrations of mAB. Flow cytometric analysis showed that the mechanism of virus entry into M phi could be through HA to specific virus receptors, or HA plus antibody to Fc receptors. These results indicate that ADE of the NWS virus infection actually occurs on Fc receptor-bearing primary murine M phi depending on the concentration of antibody in the presence of the appropriate protease for cleavage of viral HA.

Animals↗
PubMed1992

Intracardiac thrombus in murine Coxsackievirus B3 myocarditis.

We studied the appearance of intracardiac mural thrombi with time and the relationship between thrombosis and congestive heart failure (CHF) in murine coxsackievirus B3 (CB3) myocarditis. Four- to six-week-old C3H/He mice were inoculated intraperitoneally with CB3 and were observed for 90 days. Mice were sacrificed periodically on days 4, 8, 14, 30, and 90. Among 129 mice with myocarditis, 35 (27.1%) developed CHF and 40 (31.0%) demonstrated thrombi after day 8. The total incidence of thrombosis was significantly higher in mice with CHF (71.4%; 25/35) than in those without CHF (16.0%; 15/94) (P less than 0.001). The present study suggests that CB6 myocarditis carries a significant risk of thromboembolism, and that CHF is a risk factor for the appearance of thrombi.

Animals↗
PubMed1992

Magnetic resonance imaging of a malignant transformation of an intracranial cellular blue nevus. A case report.

As a follow-up to a case previously reported, a rare case of malignant transformation of cellular blue nevus (CBN) in the central nervous system preoperatively diagnosed by magnetic resonance imaging (MRI) is reported. On MRI, the malignant portion of the nevus was slightly hyperintense on both T1- and T2-weighted images. In contrast, the benign portion with a great deal of melanin was hyperintense on T1-weighted image and hypointense on T2-weighted image. MRI was useful and indispensable for detecting the malignant transformation of CBN.

Brain Neoplasms↗
PubMed1992

Comparison of antiviral assay methods using cell-free and cell-associated varicella-zoster virus.

Assay methods for varicella-zoster virus (VZV) susceptibility to acyclovir (ACV) of VZV were compared by using cell-free (CF) and cell-associated (CA) virus of 6 x plaque-purified VZV. The 50% effective doses (ED50) of ACV, as required to reduce virus plaque formation by 50%, were about 8 times higher for CA virus than for CF virus. Also, the ED50 of 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU) for CA-VZV was higher than for CF-VZV, and fresh clinical isolates of VZV gave higher ACV ED50 values than CF virus. CA virus prepared at various times after CF virus infection showed a gradual increase of the ACV ED50 with time, ranging from the ED50 for CF virus to that for CA virus.

Acyclovir↗
PubMed1992

Murine cytomegalovirus DNA polymerase: purification, characterization and role in the antiviral activity of acyclovir.

Murine cytomegalovirus (MCMV) neither induces a viral thymidine kinase (TK) nor enhances the activity of a cellular TK. Nevertheless, MCMV is highly susceptible to 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV). The cellular TK is neither responsible for phosphorylation of ACV nor its anti-MCMV activity. This is clear from the findings that little ACV triphosphate is formed in MCMV-infected mouse embryo fibroblasts (MEF) and that the replication of MCMV is inhibited equally well by ACV in TK+ and TK- cells. Even if trace amounts of ACV triphosphate would be formed by enzymes other than TK, and ACV triphosphate would be responsible for the anti-MCMV activity of ACV, then the MCMV DNA polymerase ought to be highly sensitive to ACV triphosphate. To examine this possibility, the MCMV DNA polymerase was partially purified and characterized. The apparent Ki value of the MCMV DNA polymerase for ACV triphosphate indicates that the sensitivity of the MCMV DNA polymerase to ACV triphosphate is equivalent to that of the HSV DNA polymerase. Therefore, the trace amounts of ACV triphosphate that are formed in MCMV-infected MEF seem to be insufficient to inhibit MCMV DNA polymerase and may not play a key role in the anti-MCMV activity of ACV.

Acyclovir↗
PubMed1992

GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining.

A double immunocytochemical method combining the preembedding PAP technique and the postembedding immunogold technique was used to examine interactions between GABAergic and serotonergic neurons in the same tissue sections of the dorsal raphe nucleus of the rat. A large number of immunogold stained GABAergic axon terminals were found to be presynaptic to strongly PAP immunostained serotonergic perikarya and dendrites. The types of synapses were mostly symmetrical although a few asymmetrical ones were also found. No axo-axonic synapse between the GABAergic axon terminals and the serotonergic neuronal profiles was found. These results suggest that GABAergic neurons could modulate serotonergic neurons in the dorsal raphe nucleus through synaptic relations.

Animals↗
PubMed1992

Effects of polyethylene glycol conjugated superoxide dismutase on coxsackievirus B3 myocarditis in mice.

OBJECTIVE: The aim was to examine the role of oxygen derived free radicals in the development of myocarditis by investigating the effects of polyethylene glycol conjugated superoxide dismutase (PEG-SOD), a potent scavenger of oxygen free radicals, upon coxsackievirus B3 (CB3) myocarditis. METHODS: Two week old male C3H/He mice were inoculated intraperitoneally with 10(3) plaque forming units of CB3. PEG-SOD, 1 x 10(3), 5 x 10(3), 1 x 10(4), and 1 x 10(5) U.kg-1 x d-1, was given subcutaneously daily on days 0 to 14. Treated groups were compared to the infected control. RESULTS: The survival rate of the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was lower than that of the infected control group (40% v 78%, p < 0.01). The survival rates of the other treated groups did not differ significantly from the infected control. The myocardial calcification score in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was higher than in the infected control group on d 7, when myocardial virus titres did not differ significantly among the five groups. The scores for myocardial cellular infiltration and myocardial necrosis in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups were significantly lower than in the infected control on d 14, when myocardial viruses were not detected in the five groups. However, the myocardial necrosis and myocardial calcification scores in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group were higher than in the infected control. CONCLUSIONS: The improvement of cardiac pathology in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups seems to have resulted not from the reduction in myocardial virus titres but from inhibition of generation of oxygen free radicals. The mechanism of the impaired survival and aggravation of cardiac pathology in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group is unknown. The results suggest that oxygen free radicals may be involved in the pathogenesis and development of CB3 myocarditis and that appropriate dosages of PEG-SOD have therapeutic potential for clinical CB3 myocarditis, although caution must be paid to the treatment window.

Animals↗
PubMed1992

Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus.

We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Study on proteoglycans having low-sulfated chondroitin 4-sulfate in human urine and serum.

State analysis of low-sulfated chondroitin 4-sulfate (LSC) in human urine and serum was performed by the use of high performance liquid chromatography and Western blot analysis. It was revealed that the most amount of LSC in urine is present as urinary trypsin inhibitor and a small amount (about 10% of total LSC) is as an LSC chain. The LSC in serum is mainly present as a proteoglycan such as inter-alpha-trypsin inhibitor (ITI), with a molecular weight of 212 kDa, but a small amount of LSC-proteoglycans having molecular weights of 128 and 38 kDa were also observed on SDS-PAGE. Those two compounds may be fragments of ITI, or one of the compounds (128 kDa) may be pre-alpha-trypsin inhibitor which was found by Enghild et al. (J. Biol. Chem., 264, 15975 (1989)).

Blotting, Western↗
PubMed1992

Pituitary metastasis of thyroid follicular adenocarcinoma--case report.

A rare case of pituitary metastasis of thyroid follicular adenocarcinoma occurred in a 62-year-old female manifesting as left retro-orbital pain and diplopia. Computed tomography and magnetic resonance imaging revealed a tumor in the pituitary fossa extending to the left cavernous sinus, sphenoid sinus, and prepontine cistern, destroying the upper portion of the clivus. An asymptomatic thyroid mass, probably malignant, was also found. She also had an incidental small meningioma in the posterior fossa. The pituitary tumor was partially removed, and the thyroid and posterior fossa tumors were totally removed in two operations. Both pituitary and thyroid tumors were verified to be follicular adenocarcinoma. Postoperatively, she developed panhypopituitarism for which cortisol and thyroxine replacement therapy was necessary. Three years after first therapy, she was alive but her symptoms did not improve.

Adenocarcinoma↗
PubMed1992

The effects of FK-506, a novel and potent immunosuppressant, upon murine Coxsackievirus B3 myocarditis.

To test the therapeutic efficacy of immunosuppression with FK-506 upon coxsackievirus B3 myocarditis, C3H/He mice were inoculated with coxsackievirus B3, and the effects of FK-506 were compared to those of cyclosporine. FK-506 (2.5 mg/kg/day) or cyclosporine (25 mg/kg/day) was administered s.c. daily on days 0 to 14 (experiment I) and on days 14 to 28 (experiment II). In experiment I, the survival rate of the FK-506 or cyclosporine-treated group was significantly lower compared with that of the untreated, control group. However, the score of myocardial cellular infiltration in both treated groups was lower compared to the control. On day 14, myocardial virus was not detected in the control group, but was present in both treated groups. Serum neutralizing antibody titers on day 14 in FK-506 group were lower than in the control group. In experiment II, survival rate did not differ significantly among the three groups. Serum-neutralizing antibody titers on day 21 in FK-506 group were lower than in the control. Histologically, marked cellular depletion in the thymus and spleen was evident in FK-506 groups; in cyclosporine groups, it was only evident in the thymus. Thus, FK-506 induced immunosuppression in coxsackievirus B3 myocarditis, associated with a high mortality, notwithstanding the reduction of myocardial cellular infiltration in the acute stage when immune mechanisms play a role in the pathogenesis of the disease. With respect to the dosage, the immunosuppressive action of FK-506 in vivo is at least 10-fold stronger compared to that of cyclosporine.

Animals↗
PubMed1991

Augmentation of murine lymphokine-activated killer cell cytotoxicity by beta-cyclodextrin-benzaldehyde.

We investigated the effect of beta-cyclodextrin-benzaldehyde (CDBA) on lymphokine-activated killer (LAK) cell activity of spleen cells from normal or RCT(+)H-2(+)-sarcoma-bearing C3H/He mice. CDBA augmented the induction of LAK cytotoxicity in vitro against RCT(+)H-2+ tumor cells by IL-2, whereas the culture with CDBA alone did not. In a LAK cytotoxicity assay in vitro, the augmentative effect of CDBA was strongly exerted against spleen cells originating from 2-week-tumor-bearing mice, rather than those from normal mice or mice that had born tumors for 5 weeks. Such an augmentative effect was not observed against other tumor cells (YAC-1, D-6, Colon-26 and EL-4 cells) non-specifically. When the intravenous adoptive transfer of LAK cells was carried out in the mice, LAK cells from tumor-bearing mice induced by combined culture with interleukin-2 (IL-2) and CDBA markedly inhibited the pulmonary metastases of RCT(+)H-2+ tumor, while neither LAK cells from the same tumor-bearing mice induced by only IL-2 nor those from normal mice inhibited the pulmonary metastasis. The majority of LAK cells induced either by IL-2 plus CDBA or by IL-2 alone were found to be Thy1.2+ and asialoGM1+ cells by flow-cytometric analysis, but no obvious phenotypical difference was observed between them. However, the most significant effect of CDBA might be the maintenance of the Lyt-2+ cell level in the spleen cells from tumor-bearing mice. These results suggested that the costimulation of spleen cells with IL-2 and CDBA might induce cytotoxic T cells specific for syngeneic tumor cells.

Animals↗
PubMed1991

Influence of trifluoperazine on the late stage of influenza virus infection in MDCK cells.

We investigated the influence of the anticalmodulin drug, trifluoperazine (TFP) on influenza virus growth in MDCK cells. The inhibitory effect of TFP on virus growth was observed even when TFP was added at a late stage of infection. This inhibitory effect was concentration-dependent in the concentration range of 20-35 microM. At 35 microM, TFP caused a complete alteration in the distribution pattern of hemagglutinin (HA), concomitant with a decrease in the appearance of HA on the cell surface. After removal of the drug, the HA gradually began to show a normal distribution pattern and reappeared on the cell surface. The time course of rearrangement of HA was in accord with that of the recovery of cell supernatant infectivity. Scanning electron microscopic study revealed that the drug did not cause accumulation of the progeny viruses on the cell surface. The drug effect on the virus growth was reversed by the simultaneous presence of purified calmodulin (CaM). These data suggest that TFP acts as a reversible inhibitor of influenza virus morphogenesis, but not budding, by disturbing cellular CaM and/or CaM-dependent functions.

Animals↗
PubMed1991

Immunological mechanisms in experimental coxsackievirus B3 myocarditis in mice.

To address unresolved questions, experimental models of viral myocarditis may be of great value. In this study, immunological mechanisms of myocardial damage in coxsackievirus B3 myocarditis in mice were investigated. The results showed that susceptibility to viral infection is primarily determined by the genetic background of the host, that the severity of myocarditis depends not upon B cells but upon T cells, and that antigen-specific T cells play a pivotal role in the pathogenesis of acute coxsackievirus B3 myocarditis.

Acute Disease↗