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Biomedical subjects

K Osada

Publications and source records attributed to K Osada.

At least 55 records · Page 3Linked to original sources
PubMed1995

Decreased integrin alpha 2, but normal response to TGF-beta in scleroderma fibroblasts.

The distribution and amount of integrin alpha2 were studied in cultured fibroblasts from normal subjects and scleroderma patients by immunofluorescence and immunoblotting using a monoclonal antibody against the human integrin alpha2 subunit. Integrin alpha2 was concentrated at the perinuclear regions in a dot-like pattern in normal fibroblasts on the glass coverslips until the 14th day after planting, and the staining pattern of integrin alpha2 was gradually changed to a dispersed dot-like pattern by the 19th day as examined by immunofluorescence microscopy by using the anti-integrin alpha2 antibody. No difference was observed in the distribution patterns between normal and scleroderma fibroblasts. By immunoblotting study, the amount of integrin alpha2 in scleroderma fibroblasts (n = 10) was less than that of normal fibroblasts (n = 10) (P < 0.01) in both cytosol and cytoskeleton-associated fractions. Furthermore, transforming growth factor beta (TGF-beta) increased the amount of integrin alpha2 in both normal fibroblasts and scleroderma cells by 33%. The total amount of integrin alpha2 in TGF-beta-stimulated scleroderma fibroblasts was less than that in TGF-beta-stimulated normal fibroblasts. These findings suggest that the amount of integrin alpha2, a collagen receptor, is reduced in scleroderma fibroblasts, but the integrin alpha2 production by TGF-beta stimulation is not impaired in scleroderma fibroblasts.

Adult↗
PubMed1995

Pharmacologic evidence for involvement of phospholipase C in pemphigus IgG-induced inositol 1,4,5-trisphosphate generation, intracellular calcium increase, and plasminogen activator secretion in DJM-1 cells, a squamous cell carcinoma line.

The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (IP3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3, PA secretion, and cell-cell detachment in DJM-1 cells. [Ca+2]i and IP3 contents were determined with or without 30-min pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxymethylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell-cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++]i and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 microM), but not with U73343 (10 microM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 caused no effects on cell viability and IgG binding to the cell surface. These results suggest that phospholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.

Calcium↗
PubMed1995

Pemphigus IgG, but not bullous pemphigoid IgG, causes a transient increase in intracellular calcium and inositol 1,4,5-triphosphate in DJM-1 cells, a squamous cell carcinoma line.

It is still unclear what kinds of mechanisms are involved in blister formation after antibodies bind to the antigens in pemphigus and bullous pemphigoid. The effects of IgGs from pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid sera on intracellular calcium concentration ([Ca++]i) and inositol 1,4,5-trisphosphate were examined in a human squamous cell carcinoma cell line (DJM-1 cells) and in cultured human keratinocytes to clarify whether signal transduction via calcium is involved. IgGs were purified with protein A affinity column from the sera of five pemphigus vulgaris patients, three pemphigus foliaceus patients, eight bullous pemphigoid patients, and 14 normal volunteers. Keratinocytes were cultured in Eagle's minimum essential medium containing 1.8 mM Ca++ and loaded with fura-2/AM, followed by addition of the IgGs. Subsequently, [Ca++]i was determined by measuring the fluorescence ratio (F340/F360) with videomicroscopy. Pemphigus IgGs (seven of eight cases) induced a rapid and transient increase in [Ca++]i in both the cells, whereas a [Ca++]i increase was caused by very few IgGs from bullous pemphigoid (one of eight cases) and normal sera (two of 14 cases). The pemphigus IgG-induced transient [Ca++]i increase was not affected by chelating extracellular Ca++ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid. In addition, monoclonal antibodies acid. In addition, monoclonal antibodies against 180-kD and 230-kD antigens did not exert this change. Pemphigus IgGs that caused a [Ca++]i increase induced rapid and transient production of inositol 1,4,5-trisphosphate, peaking at 20 seconds. These findings suggest that IgG from pemphigus induces Ca++ mobilization by inositol 1,4,5-trisphosphate from internal stores, and that mechanisms of antibody-transmitted signaling in pemphigus may differ from those in bullous pemphigoid.

Adult↗
PubMed1995

Restricted usage of T cell receptor V alpha-V beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy.

Prolonged myocardial cell damage initiated by acute myocarditis is thought to be one of the most important etiology of dilated cardiomyopathy. To investigate the immunological mechanisms involved in the pathogenesis of dilated cardiomyopathy, we analyzed the phenotypes of infiltrating cells and examined the expression of perforin in infiltrating cells in the hearts of patients with dilated cardiomyopathy as well as acute myocarditis. We also examined the expression of HLA and intercellular adhesion molecule-1 (ICAM-1) in myocardial tissue of these patients. Furthermore, to evaluate the antigen specificity of infiltrating T cells and persistence of viral genomes in the myocardial tissue, we analyzed the expression of T cell receptor (TCR) V alpha and V beta genes as well as enterovirus genomes by PCR. We found infiltration of perforin-expressing killer cells and enhanced expression of HLA class I and ICAM-1 in the myocardial tissue. We also found that the repertoires of TCR V alpha as well as V beta gene transcripts were restricted, indicating that a specific antigen in the hearts was targeted. Because no enterovirus genomes were detected in all patients, it is strongly suggested that a cell-mediated autoimmune mechanism triggered by virus infection may play a critical role in the pathogenesis of dilated cardiomyopathy. However, we could not exclude the possibility that viruses other than enteroviruses could be pathogenic in these patients.

Acute Disease↗
PubMed1995

Enhancement of production of IgM and interferon-beta in human cell lines by poly-lysine.

The promotive effects of poly-cations on immunoglobulin production was investigated using human-human hybridoma cells. Among poly-cations tested, epsilon-poly-L-lysine with hydrochloride (approximately 4 kDa), which has been used as an antibacterial food additive, had the greatest activity in enhancing IgM production of human-human hybridoma HB4C5 cells without stimulating cell proliferation. Immunoglobulin production stimulatory (IPS) activity of epsilon-poly-lysine was not affected by trypsin digestion. It was stable below 60 degrees C but completely inactivated with heating at 100 degrees C for 30 min. epsilon-Poly-lysine also enhanced interferon-beta (IFN-beta) production of human osteosarcoma MG-63 cells, but this stimulatory effect was reduced by the trypsin digestion.

Amino Acid Sequence↗
PubMed1995

Ras amplification in BHK-21 cells produces a host cell line for further rapid establishment of recombinant protein hyper-producing cell lines.

To rapidly establish recombinant protein hyper-producing cell lines we introduced a reporter plasmid into BHK-21 cells that had been 'primed' by transfection and amplification of the ras oncogene. The reporter plasmid used carries the human interleukin-6 (hIL-6) gene which is under control of the human cytomegalovirus immediate early promoter. The primed BHK cell lines were shown to produce many stable hIL-6 hyper-producing cells achieving about 15 times higher productivity than the control BHK-21 cells.

Animals↗
PubMed1995

E1A and ras oncogenes synergistically enhance recombinant protein production under control of the cytomegalovirus promoter in BHK-21 cells.

The amplified ras oncogene greatly enhanced the production of recombinant human interleukin-6 (hIL-6) under control of the cytomegalovirus immediate early promoter (CMV promoter) in BHK-21 cells. When the adenovirus E1A oncogene was further transfected into the above mentioned ras-amplified hIL-6 hyperproducing BHK cells, the transfectants had about 10 times higher productivity than non-transfectants. However, the E1A gene alone did not enhance productivity. These results implicate a ras and E1A synergistic ability that acts to enhance hIL-6 production.

Adenoviridae↗
PubMed1995

Development of a protein-free medium with iron salts replacing transferrin for a human-human hybridoma.

Many protein-free media have been developed, because protein-free media are usually more economical than serum-free or serum-containing media and facilitate the purification of bioactive materials. We evaluated various iron salts and chelating agents replacing transferrin to develop a protein-free medium for a human-human hybridoma, HB4C5, and found out that ferric citrate was favorable for the production and the productivity of monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1995

[Neurilemmoma of intra-temporal facial nerve:case report].

A case of facial nerve neurilemmoma within the temporal bone is reported. The patient is a 47-year-old female complaining of 10 years' history of left facial palsy. CT and MRI examinations showed an enlarged mass of the tympanic cavity protruding into the petrous bone, mastoid cavity and external auditory meatus. An open biopsy of the tumor at the external auditory meatus revealed neurilemmoma. The tumor was totally removed by a retroauricular approach. Five months after surgery, both the cross nerve graft using the peroneal nerve and the temporal muscle transfer, method modified by Johnson were performed for the functional recovery of facial palsy.

Cranial Nerve Neoplasms↗
PubMed1995

[Detection of chromosomal aberration using fluorescence in situ hybridization on breast cancer].

The relationship between interphase cytogenetics and prognostic factors, especially the grade of nuclear atypism, nuclear DNA content, histological lymph node metastasis and clinical data was examined in 48 primary breast cancer specimens (touch preparation). Using fluorescence in situ hybridization (FISH) with a chromosome-specific DNA probe, the copy number of pericentrometric sequences on chromosome 17 was examined within the interphase nuclei in touch preparations from the tumor. The copy number of chromosome 17 was correlated with the increase in the grade of nuclear atypism, tumor size, histological lymph node metastasis and nuclear DNA content. In the diploid type of nuclear DNA content, the copy number of chromosome 17 was correlated with the increase in the grade of nuclear atypism and histological lymph node metastasis. In conclusion, the numerical chromosomal aberrations detected by FISH were found in the DNA diploid cases by FCM. The detection of numerical chromosomal aberrations by FISH provide important information about the prognostic factors.

Breast Neoplasms↗
PubMed1995

Serial transesophageal echocardiography imaging of a postoperative aortic ring abscess: a case report.

Transesophageal echocardiography (TEE) was used for the serial observation of a postoperative perivalvular abscess of a prosthetic aortic valve in a 65-year-old man who underwent aortic valve replacement for aortic regurgitation. Five months later, TEE revealed an aortic ring abscess. Ascending aortic grafting (Bentall method) and translocation of the aortic prosthetic valve were conducted for progressive mechanical valve dysfunction. Postoperative TEE showed flow between the left ventricular outflow tract and the abscess, which was unrestricted 1 month later. Associated clinical improvement suggested that washout of the abscess was a critical factor. This case illustrates the usefulness of TEE for examining postoperative aortic ring abscess.

Abscess↗
PubMed1994

Decreased bradykinin binding sites in fibroblasts from progressive systemic scleroderma.

The numbers of bradykinin receptors (BK-R) in cultured dermal fibroblasts from patients with progressive systemic sclerosis (PSS) and from healthy controls were measured using a receptor binding assay. The numbers of BK-R were significantly fewer in PSS fibroblasts than in control fibroblasts (P < 0.02). However, no differences in affinity were observed in BK-R between PSS and control fibroblasts. The BK-R mRNA levels were determined in PSS and control fibroblasts by Northern blot hybridization using BK-R cDNA, but no significant differences were found. These findings suggest that the decrease in BK-R in PSS fibroblasts might occur during a posttranslational step.

Binding Sites↗
PubMed1994

Changes of monosaccharide availability of human hybridoma lead to alteration of biological properties of human monoclonal antibody.

The effect of glucose and other monosaccharide availability in culture medium on production of antibody by human hybridomas has been studied. Human hybridoma cells C5TN produce an anti lung cancer human monoclonal antibody, and the light chain is N-glycosylated at the variable region. When the cell line was grown in the presence of various concentrations of glucose, the antibodies produced changed their antigen-binding activities. Analysis of the light chains produced under these condition revealed that four molecular-mass variant light chains ranging from about 26 to 32 kDa were secreted. The twenty six-kDa species, which corresponds to a non-glycosylated form of the light chain, was recovered after enzymatic removal of all N-linked carbohydrate chains, indicating that the source of the heterogeneity of the light chain is due to the varied glycosylation. When the C5TN cells were cultured in medium containing either fructose, mannose or galactose instead of glucose, galactose elevated the antigen binding activity of the antibody more than the other sugars. These results suggest that change of glucose availability affects the antigen-binding activity of the antibody via the alteration of the glycosylation.

Antibodies, Monoclonal↗
PubMed1994

Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells.

In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that the ras product is able to transactivate the beta-actin, CMV and SR alpha promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with the ras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify the ras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.

Actins↗
PubMed1994

Lymphatic absorption of oxidized cholesterol in rats.

The absorption of cholesterol and of cholesterol oxidation products (oxidized cholesterols) was compared in lymph-cannulated rats. We found that the lymphatic absorption of an intragastrically administered, emulsified lipid meal containing 25 mg of cholesterol or 25 mg of oxidized cholesterols, within 24 h, was approximately 67 and 30%, respectively. The absorption rate of individual oxidized cholesterols differed considerably and was approximately 30% for 7 alpha-hydroxycholesterol, 42% for 7 beta-hydroxycholesterol, 32% for 5 beta-epoxycholesterol, 28% for 5 alpha-epoxycholesterol, 15% for cholestanetriol and 12% for 7-ketocholesterol. Moreover, cholesterol oxidation products delayed the absorption of oleic acid as triolein. Approximately 35 and 48% of cholesterol was recovered in chylomicrons (CM) and very low density lipoprotein (VLDL), respectively. In contrast, 54 and 40% of the oxidized cholesterols was recovered in CM and VLDL, respectively, although there was a significant difference in the distribution of individual oxidized cholesterols. The results of the present study indicate that oxidized cholesterols are absorbed to a lesser extent than is cholesterol, that they disturb fat absorption and that they distribute differently between lymphatic lipoproteins.

Absorption↗
PubMed1994

Etretinate administration reduces serum propeptide of type I procollagen level in patients with psoriasis.

The serum carboxyterminal propeptide of type I procollagen (PICP) level in 26 patients with psoriasis was significantly lower than in control subjects (124 +/- 47 and 224 +/- 78 ng/ml, respectively; P < 0.001). The patients were divided into two groups, those treated with etretinate and untreated patients. PICP levels in the treated group were significantly lower than those in the untreated group (P < 0.001), but there was no difference between the control and untreated groups. In addition, there was a negative correlation between PICP levels and the serum etretinate concentration in treated patients (r = -0.622, P < 0.05). There was no difference between procollagen type III aminoterminal propeptide (PIIIP) levels in patients and controls, nor was there any significant difference between etretinate-treated and untreated patients. In cell culture studies, etretinate dose-dependently (from 10(-9) to 10(-5) M) decreased the PICP concentration in the medium of fibroblasts from both healthy subjects and patients. In osteoblast cell culture, PICP levels were reduced only in a high concentration of etretinate (10(-5) M). However, no change was observed in preadipose cells. Our in vivo and in vitro observations indicated that psoriasis per se did not affect either serum PICP or PIIIP levels, but that etretinate had an inhibitory effect on collagen synthesis by fibroblasts. Hence, the administration of etretinate to psoriatic patients is, at least in part, responsible for the reduction of serum PICP levels in these patients.

Adult↗
PubMed1994

BE-23372M, a novel protein tyrosine kinase inhibitor. I. Producing organism, fermentation, isolation and biological activities.

BE-23372M, a novel protein tyrosine kinase inhibitor, was isolated from the culture broth of a fungus. The producing strain, F23372, was identified as Rhizoctonia solani, based on the cultural and morphological characteristics. The active principle was extracted from the mycelium with acetone and purified by solvent extraction, silica gel column chromatography and Sephadex LH-20 column chromatography. BE-23372M showed strong inhibitory activity against EGF receptor kinase with IC50 values of 0.02 and 0.03 microM on two different substrates, whereas IC50 values against protein kinase C and cAMP-dependent protein kinase were 4.5 and > 20 microM, respectively. The compound inhibited the growth of A431 human epidermoid carcinoma and MKN-7 human stomach cancer cell lines with IC50 values of 8 and 24 microM, respectively.

Amino Acid Sequence↗