Biomedical subjects

M Ozawa

Publications and source records attributed to M Ozawa.

At least 73 records · Page 4Linked to original sources

PubMed1998

Stimulatory effect of calcitonin gene-related peptide on adrenocorticotropin release from rat anterior pituitary cells.

In the present study, we examined the direct regulatory effect of rat calcitonin gene-related peptide (CGRP) on adrenocorticotropin (ACTH) release from rat cultured anterior pituitary cells. CGRP significantly increased ACTH release at concentrations of 10(-8)-10(-11) M. The ACTH release was gradually increased by CGRP concentrations lower than 10(-10) M, and was decreased at concentrations higher than 10(-9) M, presenting a bell-shaped dose-response curve. As well as having an additive effect on corticotropin-releasing factor-induced ACTH release, CGRP stimulated the accumulation of intracellular cAMP. The CGRP-induced ACTH release was inhibited by a protein kinase A inhibitor, suggesting that its stimulatory effect on the ACTH release was mediated via an adenylate-cyclase-protein kinase system. CGRP-like immunoreactive nerve fibers have been reported to innervate the anterior pituitary, so that the stimulatory effect of CGRP on the ACTH release suggests that this peptide may be involved in neural regulation of hormone secretion in the anterior pituitary.

Adrenocorticotropic Hormone↗

PubMed1998

Release of monocyte chemoattractants by polymorphonuclear leukocytes stimulated by their adhesion to stratum corneum opsonized via complement activation, measured with a human acute monocytic leukemic cell line, THP-1.

Stratum corneum (SC) exposed to living tissues, induces inflammation characterized by the formation of mixed cell granulomas consisting of infiltrative polymorphonuclear leukocytes (PMNs) and monocytes/macrophages. In this study, to clarify the mechanism for the later monocyte accumulation in SC-induced granulomas, we evaluated monocyte chemotactic activity induced by PMNs treated with serum-opsonized SC by using a human acute monocytic leukemic cell line, THP-1. When the supernatant was obtained from a PMN suspension cultured with opsonized plantar SC, higher THP-1 chemotactic activity was detected as compared with that cultured with non-opsonized SC. Although some concentrations of the chemokines, MIP-1alpha and MIP-1beta, were detected in supernatants obtained from the PMN suspensions cultured with plantar SC than in the control suspensions of PMN alone, their production by PMN was not influenced by the opsonization procedure. In contrast, MCP-1 was found to be secreted from PMN suspensions constitutively, showing no correlation to this THP-1 chemotactic activity. Moreover, HPLC analysis of PMN suspensions indicated that factors with far higher molecular weight values than these chemokines are involved in the chemotaxis of THP-1 cells.

Cell Adhesion↗

PubMed1998

Changes in iron and ferritin in anemic calves infected with Theileria sergenti.

Changes in iron and ferritin in calves infected with Theileria sergenti were investigated to elucidate iron metabolism in animals with extravascular hemolytic anemia. During severe anemia, serum iron was remarkably elevated while the total iron-binding capacity remained relatively unchanged or decreased slightly in the infected calves, resulting in elevated transferrin saturation. The serum ferritin concentration gradually increased with the progress of anemia. The erythrocyte ferritin content drastically increased when mean corpuscular volume was elevated. The concentration of non-heme iron and ferritin in the liver, spleen, and bone marrow of the infected calves was markedly higher than that in the respective tissues of the control animals. In particular, the liver of the anemic calves was found to contain 23 and 35 times as much non-heme iron and ferritin, respectively, as that of the non-anemic healthy cattle. The liver type (L) to heart type (H) subunit ratio of liver ferritin was significantly higher in the protozoa-infected than in the non-infected cattle. On the other hand, the L/H ratio of marrow ferritin was significantly reduced by the anemia. These results indicate that the anemic calves infected with T. sergenti apparently present symptoms of iron overload.

Anemia, Hemolytic↗

PubMed1998

[DNA and RNA contents of megakaryocytes using acridine orange staining].

We developed a new microfluorometric method to measure the DNA and RNA contents of individual megakaryocytes using acridine orange (AO) staining in human bone marrow smears. Some bone marrow smears were fixed with ethanol and treated with pretreatment solution and then stained with an AO staining solution. The DNA content was assayed by measuring the green fluorescence and the RNA content was assayed by measuring the red fluorescence under B excitation with microfluorometer using peripheral lymphocytes as the control. The ploidy peak was shown to be 16N in all of 5 normal controls, but it was 8N in the patient with refractory anemia with excess of blasts in transformation (RAEB-T). There was not difference in the RNA content/DNA content ratios (RI) between each ploidy in the normal controls. The RI of the patient with RAEB-T was lower than that of the normal controls. The measurement of the DNA-RNA contents may be useful as a new megakaryocytic parameter.

Acridine Orange↗

PubMed1998

The inhibitory effects of pravastatin on natural killer cell activity in vivo and on cytotoxic T lymphocyte activity in vitro.

BACKGROUND: We have reported that heart transplant recipients treated with pravastatin demonstrate decreases in the incidence of clinically severe acute rejection episodes, the incidence and progression of transplant coronary vasculopathy, and natural killer cytotoxicity. These patients also exhibited a significant improvement in 1-year allograft survival. Because of these clinical findings suggesting an immunosuppressive effect of pravastatin unique to transplant recipients and the unclear role of natural killer cells in allograft rejection, we postulated that pravastatin may exert its immunomodulatory effect by acting with cyclosporine to alter T lymphocyte function. METHODS: Twenty patients randomized into an ongoing trial of pravastatin after heart transplantation were monitored serially for natural killer cell cytotoxicity. In a separate experiment, lymphocytes isolated from normal volunteers were treated with various combinations of pravastatin and cyclosporine and tested for cytotoxic T lymphocyte toxicity in a one-way mixed lymphocyte reaction. RESULTS: Pravastatin-treated heart transplant recipients exhibited a decrease in natural killer cell cytotoxicity (9.8% mean natural killer cell cytotoxicity vs 22.1% in the control group, p < 0.01). In the one-way mixed lymphocyte reaction with blood obtained from control subjects, there was a synergistic inhibition of cytotoxic T lymphocyte activity when the cells were cultured in a combination of pravastatin and cyclosporine (20.3% mean cytotoxicity of target cells vs 41.4% in the control group, p < 0.01). CONCLUSIONS: Pravastatin exerts an immunosuppressive effect in heart transplant recipients as expressed by a reduction in rejection and natural killer cell cytotoxicity. Pravastatin and cyclosporine act synergistically to reduce cytotoxic T lymphocyte activity. This synergistic effect of pravastatin and cyclosporine may explain why this immunosuppressive effect is unique to transplant recipients.

Adjuvants, Immunologic↗

PubMed1998

[A case of congenital myopathy with the pathologic transformation from fiber type disproportion to type 1 fiber predominance myopathy].

Many patients with a severe infantile form of congenital myopathies have respiratory and feeding difficulties from early infancy. We experienced a male patient who required an artificial ventilation soon after birth and showed marked generalized muscle weakness involving the facial muscles. He had skull deformity and mild mental retardation at the age of one year and 10 months. The first muscle biopsy at the age of 1 month showed small caliber fibers and an increase in the number of undifferentiated type 2C fibers, indicating muscle fiber immaturity. Since type 1 fibers were smaller than type 2 fibers, he was tentatively diagnosed as having congenital fiber type disproportion myopathy. The muscle biopsied findings improved significantly in the second biopsy at 1 year of age which showed type 1 fiber predominance but no cytoarchitectural abnormalities. Accordingly, he was diagnosed as having congenital myopathy with type 1 fiber predominance. A severe neonatal form of congenital myopathies may show striking muscle fiber immaturity in the early infancy. This may later develop into characteristic morphologic findings such as the formation of nemalines and cores, and abnormal fiber type distribution.

Biopsy↗

PubMed1998

[Late phase II trial of oral etoposide administered for 21 consecutive days in patients with cervical cancer. ETP 21 Study Group--Cervical Cancer Group].

We conducted a multi-site late phase II trial of oral etoposide administered for 21 consecutive days in patients with cervical cancer in cooperation with 32 institutes. Fifty mg/body of oral etoposide was administered daily for 21 consecutive days. Treatment cycles were to be repeated at 4- to 5-week intervals. Eighty patients were enrolled and 70 patients were evaluated. The overall response rate (95% CI), including one complete response patient and 18 partial response patients, was 27.1% (19/70). The most commonly observed toxicity was myelosuppression such as leukopenia, neutropenia, hemoglobin decrease and thrombocytopenia. Other adverse effects were gastrointestinal toxicities such as anorexia, nausea, stomatitis and vomiting, as well as fatigue and alopecia. These adverse effects were well tolerated and controlled with medications. From these results we concluded oral etoposide administered for 21 consecutive days was an effective drug against cervical cancer.

Adenocarcinoma↗

PubMed1997

Identification of the domain of alpha-catenin involved in its association with beta-catenin and plakoglobin (gamma-catenin).

alpha-Catenin is a 102-kDa protein exhibiting homology to vincuin, and it forms complexes with cadherins or the tumor-suppressor gene product adenomatous polyposis coli through binding to beta-catenin or plakoglobin (gamma-catenin). The incorporation of alpha-catenin into the cadherin-catenin complexes is a prerequisite for expression of the cell-adhesive activity of cadherins. Using an in vitro assay system involving bacterially expressed proteins, we localized a region in alpha-catenin required for molecular interaction with beta-catenin and plakoglobin. Analysis of various truncated alpha-catenin molecules revealed that amino-terminal residues 48-163 are able to bind to beta-catenin and plakoglobin. Consistent with the observation that beta-catenin and plakoglobin bind to the same region of alpha-catenin, beta-catenin competed with the binding of plakoglobin to alpha-catenin and vice versa. Under the conditions used, beta-catenin bound to alpha-catenin with higher affinity than did plakoglobin. Scatchard analysis indicated that the affinity of the interaction between alpha-catenin and beta-catenin or that between alpha-catenin and plakoglobin was moderately strong (Kd = 3. 8 x 10(-8) and 7.7 x 10(-8), respectively). When transfected into L cells expressing E-cadherin, the amino-terminal region of alpha-catenin (from residue 1 to 226) formed complexes with beta-catenin supporting the in vitro binding experiment results.

Binding Sites↗

PubMed1997

Sphingosine induces apoptosis in androgen-independent human prostatic carcinoma DU-145 cells by suppression of bcl-X(L) gene expression.

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL-60 cells [Ohta et al. Cancer Res. 1995;55:691-697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down-regulation of bcl-2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177-180]. In this study, we examined the sphingosine-induced apoptosis of the androgen-independent human prostatic carcinoma cell line DU-145, which expresses bcl-X(L) and Bax but not bcl-2, and found that treatment of DU-145 cells with sphingosine suppressed bcl-X(L) in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl-X(L) or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1-phosphate, failed to induce apoptosis. These results indicate that, in DU-145 cells, sphingosine, but not its metabolites, induces apoptosis through down-regulation of bcl-X(L), independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl-2 or bcl-X(L) activity in these cells.

Androgens↗

PubMed1997

Metanestin, a glycoprotein with metastasis-associated expression in transitional cell carcinoma of the urinary bladder.

A 60-kDa glycoprotein named metanestin was identified by molecular cloning. The glycoprotein had a twice-repeated motif of Pro-Gly-Pro-Gly and carried metastasis-associated carbohydrate epitopes. The antibody to the protein portion of metanestin strongly reacted with lymph-node metastasis of transitional-cell carcinoma of the urinary bladder, but the reactivity to the primary tumor was generally weaker and the normal urothelium was scarcely reactive. Thus both metanestin and the carbohydrate on it showed metastasis-associated expression. In addition, we identified a 100-kDa glycoprotein with a 4-times-repeated motif of Pro-Ala-Pro-Ala. The antibody to the 100-kDa glycoprotein showed reactivity similar to that to metanestin.

Amino Acid Sequence↗

PubMed1997

Myoclonus epilepsy associated with ragged-red fibers: a G-to-A mutation at nucleotide pair 8363 in mitochondrial tRNA(Lys) in two families.

In addition to well-known mutations at nucleotide pair 8344 and 8356 in mitochondrial DNA in patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), we found a new G-to-A point mutation at nucleotide 8363 in two Japanese families. The probands had the typical clinical characteristics of MERRF. Since the 8363 mutation was present in a heteroplasmic state, and seen in none of 92 patients with other mitochondrial diseases or 50 normal individuals, this mutation is thought to be disease-related and probably specific to MERRF. As seen in muscle biopsies with the previous two mutations, focal cytochrome c oxidase (CCO) deficiency was the most characteristic finding. With single fiber analysis, the CCO-negative fibers contained a higher percentage of mutant DNA (88.4 +/- 6.6%) than CCO-positive fibers (65.1 +/- 8.0%). These findings suggest that mutations in tRNA(Lys) coding region are related to the MERRF phenotype and are responsible for the reduced CCO activity.

Adolescent↗

PubMed1997

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically.

We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat antimouse IgG antibodies. They were then stained with 4',6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P = 0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients.

Bone Marrow Examination↗

PubMed1997

Recurrent pain attacks in a 3-year-old patient with myoclonus epilepsy associated with ragged-red fibers (MERRF): a single-photon emission computed tomographic (SPECT) and electrophysiological study.

We reported a 3-year-old girl with myoclonus epilepsy associated with ragged-red fibers (MERRF) who was afflicted with recurrent pain attacks and allodynia on the right side of the body. Although magnetic resonance imaging showed normal intensity in the thalamus, single-photon emission computed tomography (SPECT) revealed hypoperfusion in the thalamus. Somatosensory evoked potentials showed delayed early cortical responses, particularly on right median nerve stimulation. The parenteral administration of cytochrome c with flavin mononucleotide and thiamine diphosphate abolished the intolerable pain. This clinical improvement was objectively supported by the results of SPECT and neurophysiological findings. These observations suggested that a dysfunction of the thalamus was responsible for her pain and that cytochrome c therapy was of benefit for this symptom.

Child, Preschool↗

PubMed1997

Central administration of PACAP stimulates gastric secretion mediated through the vagal pathway in anesthetized rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide that was originally isolated from ovine hypothalamic tissue. The peptide has two amidated forms, PACAP38 and PACAP27. In this study, we examined the effects of centrally administered PACAP38 and PACAP27 on gastric secretion in anesthetized rats. Centrally administered PACAP stimulated gastric acid and pepsin secretion in a dose-dependent manner. PACAP38 was 1.5-2 times more potent than PACAP27 on gastric secretion. By contrast, intravenously administered PACAP38 had no effect on basal or pentagastrin-stimulated gastric secretion. PACAP6-38, a PACAP antagonist, by itself at high doses also stimulated gastric and pepsin secretion, but at lower doses had no effect. Centrally administered PACAP6-38 at a dose that had no effect on gastric secretion, atropine pretreatment, or vagotomy pretreatment, suppressed the stimulatory effect of PACAP38. It is concluded that centrally administered PACAP may have a regulatory effect on gastric secretion through PACAP receptors and the vagal pathway.

Anesthesia↗

PubMed1997

Adrenal adenoma with bilateral adrenocortical nodular change in a patient with Cushing's syndrome.

We report a 57-year-old male patient with adrenocorticotrophin (ACTH)-independent Cushing's syndrome and long-standing hypertension. Both abdominal computed tomographic scan and magnetic resonance imaging revealed bilateral adrenal enlargement with the presence of a tumour in the left adrenal. Marked uptake of 131I-6 beta-iodomethyl-19-norcholesterol was observed only in the left adrenal gland. Left adrenalectomy and biopsy of the right adrenal gland were subsequently performed. Histological examination revealed the presence of an adrenocortical adenoma in the left adrenal with multiple adrenocortical nodules in both left and right non-neoplastic adrenals. Peri- and intraadrenal arteries and arterioles demonstrated marked arteriosclerotic vascular changes. Immunoreactivity for several steroidogenic enzymes was present in the tumour and markedly diminished in the non-neoplastic adrenals. This patient with Cushing's adenoma is considered to have developed adrenocortical nodules in the nonneoplastic adrenal possibly as a result of localized compensatory overgrowth of adrenocortical cells in response to ischaemic changes due to arteriopathy. When examining patients with Cushing's syndrome and bilateral adrenal enlargement, the possibility of concomitant adenoma and adrenocortical nodule formation should also be considered in the differential diagnosis

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Determination of interferon-alpha2 allele composition in the genomic DNA from healthy volunteers and leukemic patients in Japan.

The three interferon-alpha2 (IFN-alpha2) sequences identified to date differ from each other in just two nucleotide positions, both of which result in changes in amino acids. Thus, the mature IFN-alpha2a protein product is characterized by a lysine residue at position 23 (AAA) and a histidine at position 34 (CAA), IFN-alpha2b has an arginine at position 23 (AGA) and histidine at position 34 (CAT), and IFN-alpha2c has arginine residues at both positions 23 (AGA) and 34 (CGT). These nucleotide variations in the DNA sequence can be distinguished by selective restriction enzyme analysis. We studied the distributions of the three IFN-alpha2 variants by analyzing chromosomal DNA from 103 Japanese volunteers and 33 patients with hematologic disorders. Fragments of 238 bp and 617 bp of the IFN-alpha2 gene containing codons 23 and 34 were amplified by PCR using specific primers, and the PCR products were analyzed with specific restriction nucleases to identify the IFN-alpha2 variant sequences. Only IFN-alpha2b gene was detected in normal volunteers, and no IFN-alpha2a gene was detected in Japanese subjects. However, IFN-alpha2c was detected in 4 of 33 (12.1%) patients with leukemia.

Adult↗

PubMed1997

Identification of the Ca(2+)-binding domains in reticulocalbin, an endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs.

Reticulocalbin (RCN) is a member of the EF-hand Ca(2+)-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca(2+)-binding capability and adopted new functions. To identify the domains involved in Ca(2+)-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system. 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca(2+)-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.

Amino Acid Sequence↗

PubMed1997

C3 production of cultured human epidermal keratinocytes is enhanced by IFNgamma and TNFalpha through different pathways.

We investigated the regulation of C3 production by human cultured epidermal keratinocytes by enzyme-linked immunosorbent assay. The results showed that IFNgamma and TNFalpha enhanced the synthesis of C3 by epidermal keratinocytes in a concentration-dependent manner. Moreover, a protein kinase C (PKC) inhibitor blocked C3 production, whereas PMA enhanced it. There was a synergistic effect between IFNgamma and TNFalpha. In experiments to investigate the role of protein tyrosine kinase (PTK) in C3 production, we found that treatment with herbimycin A, a specific inhibitor for the c-Src-related PTK, caused significant enhancement of the C3 production induced by IFNgamma or TNFalpha, suggesting that c-Src-type PTK(s) provides a negative signal to C3 production. Each competitive inhibitor of PTK, genistein or tyrphostin, substantially increased the C3 production by IFNgamma at lower concentrations, although each agent had little effect on TNFalpha-associated production of C3 at the same concentrations. The data show that pro-inflammatory cytokines IFNgamma and TNFalpha synergistically augment C3 production by epidermal keratinocytes by different pathways.

Cells, Cultured↗