Biomedical subjects

H Okada

Publications and source records attributed to H Okada.

At least 631 records · Page 35Linked to original sources

PubMed1993

Stabilization of xylanase by random mutagenesis.

Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser-26 to Trp, Gly-38 to Asp and Thr-126 to Ser; N102, Gly-38 to Asp; N104, Gly-38 to Ser and Arg-48 to Lys; F1, Ser-12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Endo-1,4-beta Xylanases↗

PubMed1993

Rapid transformant selection by human complement using HRF20 (CD59) cDNA as a selection marker.

The 20 kDa homologous restriction factor (HRF20, CD59) prevents formation of membrane attack complexes of human complement. Transfection of the cDNA of HRF20 to heterologous cells, which are sensitive to human complement, thereby renders these cells resistant to human complement attack. This property can then be used as a selection marker of transfection. In addition, the procedure is short and can be performed within 8 h. Since only 1 h is required for incubation with complement, the damage to the transfected cells is limited compared with damage resulting from selection for drug resistance which requires 10 days or more of incubation in the presence of harmful agents.

Animals↗

PubMed1993

Molecular diversity of calpastatin in human erythroid cells.

According to differences in mobility on SDS-polyacrylamide gel electrophoresis, calpastatins (inhibitor proteins of the calcium-dependent proteinase calpain) are classified into the tissue type (100-120 kDa) and the erythrocyte type (70 kDa), which lacks the amino-terminal domains (domains L and 1). We investigated the molecular diversity of calpastatin in human hematopoietic cells by Western-blot analysis and by the reverse-transcription-polymerase-chain reaction method. While the mononuclear and polymorphonuclear cells in peripheral blood showed the tissue type (110 and 114 kDa), a cell line of erythroid cells (JK-1) showed both the tissue type (110 kDa) and the erythrocyte type (70 kDa) at approximately equal ratios. When the lysate of JK-1 cells was incubated in the presence of ATP, the 110-kDa form was degraded much faster than the 70-kDa form. In human erythrocytes, the 110-kDa form was identified as the tissue type by an antibody recognizing domain L, and this form was also present in addition to the predominant 70-kDA form. JK-1 cells, as well as nucleated cells in peripheral blood, contained calpastatin mRNA with exon-3-deleted. Glioblastoma and fibroblast cell lines expressed the nondeleted calpastatin mRNA in addition to the deletion type, and they showed bands corresponding to 117 kDa as well as 110 and 114 kDa. The 117-kDa band was detectable by an anti-exon 3 peptide antibody. These results suggest that diversity among the tissue type calpastatins is caused by both alternative splicing and post-translational processing whereas the apparent conversion from the tissue type to the erythrocyte type is caused by proteolytic processing.

Adenosine Triphosphate↗

PubMed1993

Evidence for estrogen synthesis in adenomyotic tissues.

OBJECTIVE: To investigate steroidogenesis in eutopic and ectopic endometrial tissues in adenomyosis. STUDY DESIGN: Aromatase and estrone sulfatase activities were determined by anion-exchange resin column chromatography, thin-layer chromatography, and cocrystallization. The effects of danazol on the activity of these enzymes were also examined. Moreover, localization of aromatase in eutopic and ectopic endometrial tissues was immunohistochemically examined with antihuman placental aromatase cytochrome P-450 antibody. RESULTS: Aromatase and estrone sulfatase activities were detected in ectopic endometrium. The activity of these enzymes was significantly suppressed by danazol. In addition, aromatase was immunohistochemically detected in glandular cells of eutopic and ectopic endometrial tissues. CONCLUSIONS: The results suggest that estrogen is synthesized in the eutopic and ectopic endometrial tissues of women with uterine adenomyosis and that it may affect the growth of adenomyosis. Danazol suppressed synthesis of estrogen in vitro.

Adult↗

PubMed1993

Enhancement of Fc epsilon RII/CD23 expression on U937 cells with opsonized zymosan: the requirement of a Fc gamma RI/CD64 mediated signal associated phagocytosis.

The kinetics of surface Fc epsilon RII/CD23 was tested on monocytic cell line U937 stimulated with opsonized zymosan. Zymosan opsonized with human serum enhanced not only the expression of surface Fc epsilon RII/CD23 but also Fc epsilon RII/CD23 mRNA detected by Northern blot and in situ hybridization techniques. This stimulation showed a marked synergism with IL-4 in the induction of Fc epsilon RII/CD23. Heat-inactivation of serum did not affect the inducibility of Fc epsilon RII/CD23 by opsonized zymosan, suggesting the involvement of serum substances other than complement. Zymosan treated with human gamma-globulin also induced Fc epsilon RII/CD23, indicating the possible involvement of Fc gamma receptors. The Fc epsilon RII/CD23 inducing effect of opsonized zymosan was partially blocked by pretreatment with heat-aggregated human gamma-globulin or an anti-Fc gamma RI monoclonal antibody but not by the anti-Fc gamma RII or Fc gamma RIII antibody. Our results showed the involvement of signals from Fc gamma receptor associated phagocytosis in the induction of Fc epsilon RII/CD23.

Blotting, Northern↗

PubMed1993

Excitation and inhibition of neuronal activity in the pontine micturition center by pelvic rectal and pudendal anal afferents in dogs.

To examine whether or not the pontine micturition center (PMC) is involved in the inhibition of the micturition reflex by pelvic rectal and pudendal anal afferents, neuronal activity in the PMC was observed during inhibition of this reflex in paralyzed decerebrate dogs. Discharge of pelvic vesical branches (VBs) waxed and waned at a rhythm of about 2 Hz during the micturition reflex, which was activated by continuous stimulation of the contralateral VBs. This rhythmic discharge was modulated by continuous stimulation of contralateral pelvic rectal branches (RBs) superimposed on the VB stimulation. The modulation was composed of three effects; initial inhibition, augmentation and late inhibition. However, not all of the three effects were obvious in some dogs. One-sixth of 118 neurons examined in the pontine area ventromedial to the locus ceruleus exhibited rhythmic burst firings which preceded the rhythmic discharge of VBs by about 150 ms. Therefore, these pontine neurons are assumed to be output neurons of the PMC. The rhythmic firings of pontine neurons were augmented during continuous RB stimulation independent of the inhibitory and/or augmentative effects of the RB stimulation on the reflex discharge of the VBs. In contrast, the rhythmic firings of the pontine neurons and the reflex discharge of VBs were inhibited by mechanical stimulation of the anal canal and perineal hairs. These results suggest that the PMC is involved in the inhibition of the micturition reflex produced via pudendal afferents but not in that produced by pelvic rectal afferents, and that pelvic and pudendal afferents project to the PMC through separate pathways.

Animals↗

PubMed1993

Elimination of HIV-infected cells by lymphocytes armed with a bifunctional antibody to gp120 of HIV and CD3.

The T-cell receptor (TCR) can acquire a new antigen binding site by treatment with a bifunctional antibody (BFA) prepared with mAb against a specified antigen and an epitope of the TCR. Lymphocytes armed with BFA directed to CD3 and an HIV antigen were able to eliminate all HIV antigen-positive cells during incubation with a mixture of HIV-infected and uninfected cells. HIV antigen-positive cells even from persistently infected cells were undetectable with immunofluorescence staining although HIV genes were detectable by polymerase chain reaction (PCR) amplification indicating that only dormantly infected or low producer cells, if any, survived. This suggests that HIV antigen-positive cells could be eliminated by administration of BFA-armed lymphocytes leaving HIV patients with only dormantly infected or low producer cells.

Antibodies, Bispecific↗

PubMed1993

HRF20/CD59 complement regulatory protein expression is phenotype-dependent and inducible by the hypomethylating agent 5-azacytidine on Burkitt's lymphoma cell lines.

We studied the expression of the 20-kDa homologous restriction factor (CD59/HRF20), a complement regulatory protein, on two subsets of blood derived B cells and on Burkitt's lymphoma lines. Both low-density (activated) and high-density (resting) B cell populations expressed high levels of CD59. CD59 was detectable, however, only on a minority of cells or not at all on three Epstein-Barr virus (EBV)-negative BL lines (BL41, BL28 and DG75) and on clones of an EBV-positive BL line (Mutu) that phenotypically resembled resting B lymphocytes. On the other hand, CD59 was detected at high or medium levels on Mutu cells which had a lymphoblastoid cell-like phenotype. Expression of CD59 was upregulated by 5-azacytidine, a drug inhibiting cytosine methylation, on CD59-negative cell lines. Induction was accompanied by a partial hypomethylation in the 5' region of CD59 coding sequences.

Antigens, CD↗

PubMed1993

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta.

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (> or = 20 cigarettes a day) than in normal and moderate smokers (< 20 cigarettes a day). At term, the mean aromatase activity and P-450arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.

Aromatase↗

PubMed1993

Estrogen productivity of endometrium and endometrial cancer tissue; influence of aromatase on proliferation of endometrial cancer cells.

Aromatase, estrone (E1) sulfatase and E1 sulfotransferase activities were examined in endometrium and endometrial cancer tissue preparations. Aromatase and E1 sulfatase activities in endometrial cancer tissues were found to be significantly higher than in normal endometrial tissues. However, E1 sulfotransferase activity did not differ between benign and malignant tissue. We also examined the effect of testosterone (T) on aromatase activity and tritiated thymidine uptake (DNA synthesis) in various cultured cervical or corpus endometrial cancer cell lines (OMC-4, HHUA, Ishikawa, HEC-59). The results demonstrated that only the HEC-59 cell line had high aromatase activity and increased its DNA synthesis in response to T. This increase of DNA synthesis by T was not suppressed by simultaneous addition of cyproterone acetate, but was by tamoxifen. These data suggest that in situ estrogen production in endometrial cancer tissue is biologically important and that aromatase in cancer cells may contribute partially to cell proliferation if androgen substrate is provided.

Aromatase↗

PubMed1993

Relationship between aromatase activity and steroid receptor levels in ovarian tumors from postmenopausal women.

Aromatase activity, as well as steroid receptors, exists in nonfunctional ovarian tumors. Steroid receptor status has been reported to be related to prognosis in ovarian cancer patients. We determined aromatase activity and progesterone receptor (PR) and estrogen receptor (ER) levels in 43 ovarian tumors obtained from postmenopausal women. Aromatase activity was detected in 35 tumors (81%), PR in 21 tumors (49%) and ER in 13 tumors (30%). Eighty-three percent (10/12) of mucinous cystadenoma tissues showed positive PR with high aromatase activity, while 93% (13/14) of malignant tumors showed negative PR and low aromatase activity. Aromatase activity was detected in 95% (20/21) of PR-positive tumors, being greater than in PR-negative tumors (P < 0.002). There was a positive correlation between aromatase activity and PR (rs = 0.49, P < 0.001). However, there was no correlation between aromatase activity and ER. In 17 patients (43%), the serum estradiol level was higher than 30 pg/ml and there was a positive correlation among estradiol, estrone, androstenedione and testosterone. However, serum steroid levels were not correlated with aromatase activity, PR or ER. Aminoglutethimide inhibited aromatase activity of benign and malignant ovarian tumors, uterine myoma, choriocarcinoma cells and purified human placental P-450arom in a similar manner. These results suggest that aromatase activity is correlated with PR in ovarian tumors of postmenopausal women. In addition to steroid receptor status, aromatase activity may be a useful prognostic factor in ovarian cancers.

Adult↗

PubMed1993

Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: a new system for aromatase inhibitor screening.

In our previous study we found that MCF-7 cells possess aromatase activity and stimulate estrogen receptor-mediated growth. The pathways through which androgens are converted to estrogens by aromatase and estrogens interact with estrogen receptors contribute significantly to growth stimulation. The administration of aromatase inhibitor results in suppression of growth stimulation by androgens. This system enabled us to assess directly the biological activities of aromatase inhibitors. Aromatase activity was inhibited in a dose-dependent manner by the addition of aminoglutethimide and CGS 16949A, competitive inhibitors, and of 14 alpha-hydroxy-4-androstene-3,6,17-trione and 4-hydroxy-androstenedione, mechanism-based inhibitors. After preincubation with mechanism-based inhibitors, aromatase activity was significantly suppressed, whereas after preincubation with competitive inhibitors, it was adversely increased. These effects were concentration- and time-dependent. Preincubation with competitive inhibitors resulted in augmentation of subsequent androgen stimulation of thymidine incorporation, while preincubation with mechanism-based inhibitors resulted in diminished stimulation by subsequent androgen administration. These results suggest that in MCF-7 cells competitive inhibitors adversely induce aromatase and accelerate the subsequent androgen stimulation of DNA synthesis. Suicide inhibitors are more effective than competitive inhibitors. This system will be useful for aromatase inhibitor screening.

Aminoglutethimide↗

PubMed1993

Dissociation of clinical manifestation and histopathology in the course of mesangial injury by anti-thymocyte antibody.

Intravenous administration of heterologous anti-thymocyte antibodies, which react with rat mesangial cells, induced minimal mesangiolysis (6 hours to 3 days) followed by mesangial hypercellularity. Moreover, autologous immunoglobulins appeared in the mesangium within 1 or 2 weeks. At the same time, electron microscopy demonstrated mesangial deposits. Accompanying these pathological alterations, significant proteinuria developed. To accelerate an acute autologous immune reaction, rats were preimmunized with rabbit IgG prior to the injection of anti-thymocyte antibodies. They revealed marked mesangiolytic lesions 3 days after injection with deposition of autologous immunoglobulins and C 3 in the mesangium. In contrast, there was no significant proteinuria. Thus, mild pathological alterations in the mesangium induced by heterologous and subsequent autologous immune reactions caused proteinuria, and accelerated immune injury to the mesangium produced severe mesangiolysis without proteinuria. Based on these findings, it is suggested that the time-lapse between heterologous and autologous immune reactions to the mesangium influences the pathological alterations and clinical manifestations occurring in mesangial injury by anti-thymocyte antibody.

Animals↗

PubMed1993

Antisense sequences of 20-kDa homologous restriction factor (HRF20) are found in C9 and the C8 beta chain of homologous complement.

We examined, on the basis of the fact that sense and antisense peptides have affinity for each other, whether any relationship exists between homologous restriction factor (HRF20), a membrane inhibitor of the terminal stage of homologous complement attack, and the antisense sequence of the terminal complement components C8 and C9. In this article, we demonstrate that there are two regions of C9 that contain antisense sequences to one continuous region of HRF20 and that this relationship exists between human HRF20 and human C9, but not mouse C9. We also found one region of the C8 beta chain that contains an antisense sequence to HRF20.

Amino Acid Sequence↗

PubMed1993

Serious adverse effects induced by simultaneous administration of two nonsteroidal anti-inflammatory drugs.

We have described the case of a 58-year-old woman in whom acute hemolytic anemia, renal failure, and granulocytopenia developed after intake of mefenamic acid and diclofenac. Results of direct and indirect Coombs' tests were negative, but the lymphocyte transformation test was positive for these two nonsteroidal antiinflammatory drugs (NSAIDs). We suggest that the immune complex mechanism could have induced these adverse reactions. Although these drugs are very popular, simultaneous administration should be avoided because serious adverse effects can potentially occur.

Acute Kidney Injury↗

PubMed1993

Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87.

The DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the kcat value of the P-EII enzyme (9.2 s-1) was approximately half that of the F-EII enzyme (19 s-1), and the P-EII enzyme had higher affinity toward this substrate (Km for P-EII, 0.6 mM; Km for F-EII, 15 mM). The P-EII enzyme had a temperature optimum of 48 degrees C, and a pH optimum of 7.5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.

Amidohydrolases↗

PubMed1993

Immunohistochemical demonstration of membrane cofactor protein (MCP) of complement in normal and diseased kidney tissues.

The immunohistochemically stained membrane cofactor protein of complement (MCP/CD46), one of the complement regulatory proteins, was up-regulated in some diseased kidney tissues. MCP in diseased kidneys was strongly concentrated along the glomerular capillary walls as well as in the mesangial regions, while MCP in normal kidneys was weakly detected in all glomerular structural cells and in the epithelial cells of tubules. Since the enhanced staining was noted in those areas where depositions of C3b/C3c occurred, ongoing complement reaction might be responsible for the up-regulation of MCP expression. MCP expression may be up-regulated by complement fragments generated during complement activation in glomerulonephritis. Furthermore, anti-MCP staining was stronger in intensity in patients with moderate to massive proteinuria, indicating that up-regulation of MCP expression could be directly correlated to the kidney damage.

Antigens, CD↗

PubMed1993

Pharmacokinetics and potential use of gentamicin in budgerigars (Melopsittacus undulatus).

The pharmacokinetics of gentamicin were evaluated in budgerigars. Intramuscular (i.m.) administration of 5 and 10 mg/kg dosages produced serum gentamicin concentrations of 17.3 and 37.0 micrograms/ml respectively, at 0.25 hr following injection, but concentrations declined with a terminal half-life of 0.53 h. Based on these results, a dosage regimen of 5 to 10 mg/kg of gentamicin, intramuscularly, every 8 hours for up to 3 days, was considered to be safe and could be used against many of the common bacterial infections in budgerigars. Serum AST, ALT, LDH, CK, creatinine and uric acid values were determined after i.m. injection of either gentamicin or physiological saline every 8 hours for up to 3 days, and tissue samples of various organs were examined histologically. The most prominent adverse effect found was muscular damage at the injection site. Measurement of AST, ALT and CK activity seemed to be a sensitive method to determine pathological changes at the injection site in the budgerigars.

Animals↗