Biomedical subjects

T Okigaki

Publications and source records attributed to T Okigaki.

At least 37 records · Page 2Linked to original sources

PubMed1989

Growth, morphology, function, and morphogenetic properties of rat renal glomerular epithelial cells in vitro: effects of retinyl acetate.

We report here that retinyl acetate (RA) regulates growth, morphology, function and cell organization in rat renal glomerular epithelial cells (SGE1). SGE1 cells are able to grow in a serum-free medium (DHFs medium) which is supplemented with insulin, transferrin, selenium, bovine serum albumin (BSA), linoleic acid and epidermal growth factor (EGF). When 0.1 and 1 micrograms/ml RA were added to the medium, the growth rates in the sparse culture were noticeably increased, compared to those in DHFs alone, whereas more than 10 micrograms/ml RA was cytotoxic to the cells. In the confluent culture, addition of 0.1, 1.0 and 10 micrograms/ml RA prolonged the cell survival. Since 10 micrograms/ml RA is not cytotoxic to the confluent culture, the cytotoxic action of RA seems to be dependent on cell density as well as RA dose. Ultrastructural observation revealed that RA treatment caused an increase of microvilli and alteration of cell shape, from flattened to columnar. Biochemical and immunological studies revealed that RA treatment increased the activity of r-glutamyl transpeptidase (GGT) and an amount of the membrane component with molecular mass (Mr) of 108,000 which is identical to one of nephritogenic antigens, Fx1A. By using fluorescence phalloidin stain, it was found that RA treatment increased content and organization of F-actin fibers. Furthermore, in collagen-embedding culture, RA induced 3-dimensional (3D) growth of SGE1 cells leading to the formation of organoids, cystic spheres with central lumen, in a serum-free condition; the addition of DHFs to collagen gel alone was ineffective for the 3D growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1988

Creatine kinase isoenzymes.

Analytical methods for and the clinical significance of ATP:creatine-N-phosphotransferase (EC 2.7.3.2, CK) isoenzymes have been described. The main assay methods for the isoenzymes are electrophoresis, ion-exchange chromatography and immunoinhibition. Electrophoresis is a time-consuming technique, but it is essential for the identification of CK isoenzymes. Ion-exchange chromatography is reliable for the determination of CK-MB and useful for the isolation and purification of CK isoenzymes. The disadvantages of this method are overlapping of isoenzymes with neighbouring fractions and the inevitable dilution of enzyme activity by the elution buffer. Immunoinhibition is a rapid, sensitive, specific and quantitative technique. However, mitochondrial CK and CK-immunoglobulin complex remain after the inhibition of the M subunit with the antibodies. Thus, when assessing the experimental results, it is necessary to be certain that a false positive for CK-MB has not been obtained. Since each technique has both merits and demerits, the technique most suited to the particular laboratory should be selected.

Animals↗

PubMed1988

Establishment and characterization of an epithelial cell line, SGE1, from isolated rat renal glomeruli.

An epithelial cell line, designated as SGE1, has been established from isolated rat renal glomeruli. SGE1 cells are able to grow continuously in a serum-free medium at similar growth rates to those in the medium containing serum. Quantitative studies of the cells in the serum-free condition demonstrated that SGE1 cells required a collagen type I or IV, fibronectin, or laminin-coated substratum for adhesion and growth, and among them, collagen type I and IV were most effective. Essential medium supplements for the adhesion and growth were epidermal growth factor and transferrin, respectively, and both effects were noticeably enhanced with linoleic acid. Morphological observation found that in a monolayer culture, SGE1 cells formed domes, and in a collagen embedding culture, they formed cystic spheres having features of a simple cuboidal epithelium, polarized formation of microvilli and tight junctions as well as a lateral cell membrane with cytoplasmic projections. In addition, SGE1 cells expressed Fx1A antigens, which are nephritogenic antigens on their microvilli.

Animals↗

PubMed1987

Enhancing effects of bovine serum albumin on cell injury in vitro induced with Fe-NTA.

Direct effects of ferric nitrilotriacetate (Fe-NTA) on normal rat liver epithelial cells (RL34) in serum-free culture were studied. More than 10 micrograms/ml iron of Fe-NTA was cytotoxic and the cytotoxicity was prevented by adding apotransferrin into culture medium. Also, bovine serum albumin (BSA), which is known to bind Fe-NTA, was found to promote the cytotoxicity, while fatty acids-free BSA (F-BSA) prevented it. This result indicates that fatty acids rather than albumin promote Fe-NTA cytotoxicity.

Animals↗

PubMed1987

Role of superoxide radicals in cytotoxic effects of Fe-NTA on cultured normal liver epithelial cells.

We studied the cytotoxic effects of ferric nitrilotriacetate (Fe-NTA) on normal rat liver epithelial cells (RL34) cultured in medium containing 10% fetal calf serum. Marked cytolysis was present in cells exposed to greater than or equal to 25 micrograms/ml iron of Fe-NTA, but not all the cells exposed to 50 micrograms/ml iron were lethally injured. The remaining cells showed anomalous growth, namely cell pile-up and aggregation. Superoxide dismutase inhibited this iron-induced cytotoxicity, whereas catalase, mannitol, dimethyl sulfoxide, and 1,4-diazabicyclo-[2.2.2.] octane did not. RL34 cells exposed to Fe-NTA actually produced a large amount of superoxide radicals (O2-.), whereas unexposed control cells produced none. Allopurinol inhibited O2-. production and prevented cell injury by Fe-NTA. These results show that the injury to cells produced by Fe-NTA depends on the generation of O2-., the source of which may be xanthine oxidase.

Animals↗

PubMed1987

Adhesion and growth of rat liver epithelial cells on an extracellular matrix with proteins from fibroblast conditioned medium.

We previously reported that proteins from serum-free conditioned medium (PCM) of rat embryo fibroblasts promote the adhesion of nonmalignant rat liver epithelial cells (RL34) seeded on a collagen substratum (Yamada, M. and Okigaki, T., Cell Biol. Int. Rep. 7, 1115(1983)). We now have compared the adhesion and growth of RL34 cells seeded on an extracellular matrix (RLECM), a product of RL34 cells, or on a collagen (type I) substratum with or without PCM in the medium. The adhesion rate for RL34 cells on RLECM was higher than on collagen, and once RL34 cells adhered to the collagen substratum they showed no growth, whether or not PCM was present. Cells that adhered to RLECM grew, and the growth rate was markedly higher in medium containing PCM than in medium without it. Immunological, enzymatic and chemical analyses revealed that the adhesion- and growth-promoting activities of RLECM involved trypsin-sensitive proteins other than collagens, fibronectin (FN) or laminin (LN), and that heparan sulfate proteoglycan may be a major component of RLECM.

Animals↗

PubMed1987

Adhesion-promoting factor from embryonic fibroblasts for normal liver epithelial cells.

In the present study we show that adhesion of normal rat liver epithelial cells (RL34) to substratum coated with type I collagen (collagen substratum) is promoted by a factor involved in 80% ammonium sulfate precipitated proteins from serum-free conditioned medium (PCM) of rat embryo fibroblasts. Adhesion of RL34 cells to collagen substratum was promoted dose dependently by whole PCM and the maximum effects on adhesion could be achieved by 200 micrograms/ml whole PCM. Kinetics studies with 100 micrograms/ml whole PCM showed that adhesion proceeded very slowly, taking 16 h to reach a plateau. Adhesion-promoting activity in whole PCM was sensitive to treatments with trypsin, acid, and heat but stable to dithiothreitol treatment. Further purification of whole PCM was performed using a combination of chromatography on blue Sepharose column, gel filtration column and heparin Sepharose column. The partially purified proteins, referred to as heparin PCM, are not bound or only weakly bound to heparin under physiological ion strength and pH, and the apparent molecular weight (Mr) range is estimated to be 40,000 to 60,000 from gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. When whole PCM or heparin PCM was used for coating on plastic or collagen substratum, they no longer exerted the promoting activity.

Animals↗

PubMed1986

Sandwich electroimmunofixation (SEIF) for the assessment of isoenzyme specificities of immunoglobulins isolated from enzyme-immunoglobulin complexes.

Sandwich electroimmunofixation (SEIF) was used to determine immunologic specificity for isoenzymes of immunoglobulins isolated from enzyme-immunoglobulin complexes. After electrophoretic separation of isoenzymes, isoenzyme-specific human immunoglobulins obtained from complexes and antihuman immunoglobulin antibodies were applied to the supporting medium and allowed to react. Complexes of isoenzyme-immunoglobulins-anti-immunoglobulin antibodies formed immunoprecipitates and were fixed in the supporting medium. After washout of the unreacted enzymes and proteins with buffer, the immunoprecipitates were stained. A comparison with control enzymograms allowed for a determination as to whether the immunoglobulins reacted with specific isoenzymes. When an immunologic reaction with more than 2 isoenzymes occurred, the specificity was quantitated by densitometry. SEIF, used to examine immunoglobulins isolated from aspartate aminotransferase-, lactate dehydrogenase-, alkaline phosphatase- and amylase-immunoglobulin complexes, was found to be a rapid and reliable technique. This approach showed that immunoglobulins differ in their specificities for various isoenzymes.

Alkaline Phosphatase↗

PubMed1986

The effect of conditioned medium on fetal human liver cells in primary culture.

A medium conditioned by rat embryo cultures (RCM) promoted the adhesion of liver cells from human fetuses to plastic dish. Colonies were also formed in primary cultures of the same cells in the presence of RCM. The majority of the colonies formed were composed of large polygonal cells with a few colonies composed of both clear epithelial-like cells and fibroblast-like cells. RCM was superior to the rat embryo feeder layer for promotion of colony formation of cells. A number of colonies were formed from fetal human livers when a conditioned medium from human hepatoma cells (HCM) was used, but most of the colonies formed were composed of fibroblast-like cells. The cells derived from the polygonal cell colonies, which were formed in the presence of RCM, have been passaged four times and they are still growing with albumin-producing capacity. The effect of RCM was reduced by various physico-chemical treatments.

Animals↗

PubMed1986

Strain specific responses of inbred rats on the severity of experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis with pulmonary hemorrhage was induced in rats of 4 inbred strains, F344/DuCrj, LEW/Crj, WKY/NCrj and SHR/NCrj, by the injection of a nephritogenic antigen from bovine glomerular basement membrane. The severity and clinical course of glomerulonephritis within the strains were very similar, but those between the strains were different. Among them, F344 rats demonstrated mild glomerulonephritis with petechial pulmonary hemorrhage. LEW rats showed more severe symptoms from pulmonary hemorrhage than F344 rats and had heavy, long-lasting proteinuria that caused nephrotic syndrome. WKY rats showed very severe glomerulonephritis with pulmonary hemorrhage, and had azotemia by 4 weeks after the injection. SHR rats had both severe glomerulonephritis and severe pulmonary hemorrhage. Investigation of the dose-effect relationship of the nephritogenic antigen in F344 and WKY rats revealed that the minimum dose required to induce nephritis in F344 and WKY rats was 10 micrograms and 3 micrograms respectively, and that the injection of more than a certain amount (i.e., 30 micrograms in F344, 10 micrograms in WKY) of the antigenic material induced almost the same severity of disease. These results indicate that genetic differences in experimental animals influence the severity of renal damage, suggesting that the use of inbred strains is necessary for adequate study of experimental glomerulonephritis.

Animals↗

PubMed1985

Comparison of enzymatic characteristics of creatine kinase BB from human healthy and tumor stomach tissues.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK) BB isoenzyme from stomach tumor tissue was partially purified and its characteristics were compared with those from healthy tissue. Molecular mass of tumor CK-BB was estimated to be 82 000 by polyacrylamide gel electrophoresis. Tumor CK-BB was separated into 2 main subbands around pH 4.5 and 11, minor subbands around pH 5-7.5 by agarose isoelectric focusing. The isoenzyme reacted with anti-human brain CK-BB antibodies and formed a hybrid, CK-MB, with CK-MM prepared from healthy human skeletal muscle. The above physicochemical and immunological characteristics of tumor CK-BB were the same as those of normal CK-BB from normal stomach tissue. Optimum pH of tumor CK-BB was more acidic than that of normal CK-BB. Affinity for creatine phosphate and heat sensitivity of tumor CK-BB were slightly lower than those of normal CK-BB. Tumor CK-BB was more stable after iodoacetamide and urea treatments.

Brain↗

PubMed1984

Mitochondrial creatine kinase as a tumor-associated marker.

Sera from 167 patients with malignant tumor were examined with regard to mitochondrial creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK). The CK (CKm) was detected in 66 cases (40%), in 42 of 96 men and in 24 of 71 women. Seventeen patients had hepatic carcinomas and 14 gastric carcinoma. CKm was also present in sera from patients with malignancy of the lung, the mammary gland etc. In the CKm positive group, about 85% had metastatic and/or infiltrating malignancy, and about 90% were undergoing chemotherapy. The figures resembled those in the CKm negative group. Mortality rates in the CKm positive group were 1.7 times higher than in the CKm negative group. CKm may be a marker of some biological factors leading to and/or resulting from a poor prognosis and not simply of primary or metastatic tumors. Most of the total serum CK activities in the CKm positive group were within normal limits. CKm activities in the gastric carcinoma group were higher than those in other groups, particularly in comparison to the liver carcinoma group.

Adult↗

PubMed1984

Isolation and characterization of nephritogenic antigen from bovine glomerular basement membrane.

A method for isolation of a potent nephritogenic antigen from bovine glomerular basement membrane has been established; the glomerular basement membrane was solubilized by trypsin digestion and fractionated successively by gel filtration on Ultrogel AcA-34, concanavalin A affinity chromatography and affinity chromatography on immobilized antibodies. The antigen thus prepared was found to be highly nephritogenic; it causes glomerulonephritis in rats by a single injection of 0.1 mg per individual. Amino acid and carbohydrate analyses revealed that the antigen is a glycoprotein which contains amino acids and sugars characteristic of collagen, namely, hydroxyproline, hydroxylysine, glycine, glucose and galactose, although the relative amounts of these amino acids and sugars are less than those found in Type IV collagen of glomerular basement membrane.

Amino Acids↗

PubMed1984

Growth factors.

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Animals↗

PubMed1984

Experimental autoimmune glomerulonephritis with pulmonary hemorrhage in rats. The dose-effect relationship of the nephritogenic antigen from bovine glomerular basement membrane.

Experimental autoimmune glomerulonephritis with pulmonary hemorrhage was induced in rats by a single injection of soluble antigen obtained from bovine glomerular basement membrane. Groups of rats were injected with various amounts of the nephritogenic antigen with Freund's complete adjuvant and killed 10 weeks later. Glomerulonephritis with linear deposition of IgG along the glomerular basement membrane developed in 7 of 10 rats injected with 10 micrograms of the antigen and all 20 rats of other 2 groups with 100 or 1,000 micrograms of the antigen. Pulmonary hemorrhage was observed in 21 of 27 rats with nephritis, and it was particularly severe in those injected with 1,000 micrograms. Glomerulonephritis with pulmonary hemorrhage and the linear deposition of IgG along with the glomerular basement membrane suggests that this model resembles human anti-glomerular basement membrane antibody-induced glomerulonephritis including Goodpasture's syndrome. The nephritic rats showed symptoms of nephrotic syndrome such as hypoalbuminemia, hypercholesterolemia and hypertriglyceremia, and the severities of these symptoms clearly depended on the degree of proteinuria.

Animals↗

PubMed1983

Promotion of epithelial cell adhesion on collagen by proteins from rat embryo fibroblasts.

We have found that the promoting effects of proteins from fibroblast conditioned medium (PCM) promote adhesion of rat liver epithelial cells (nonmalignant RL-34 cell line) seeded on collagen coated substratum. Since the effect was completely inhibited by treating the cells with cycloheximide the protein(s) are probably produced by the RL-34 cells. We also found that PCM-A fraction which is identical to fibronectin purified from whole PCM had no effect on RL-34 cell adhesion and that the adhesion was not inhibited even by adding affinity purified anti-FN antibody to the medium containing FN-free PCM (PCM-B fraction). This indicates that adhesion of rat liver epithelial cells on collagen substratum does not require FN, but requires the adhesion promoting factor(s) existing in PCM-B.

Animals↗

PubMed1977

Mechanism of crystallization of purified human midcycle cervical mucus.

The crystallization phenomenon of human midcycle cervical mucus was studied with purified mucin and sodium chloride. The drying of the purified mucin in saline was subjected to stress by the use of an air curtain, and crystallization (fern pattern) was found to follow the stress. Lithium chloride and calcium chloride were not compatible with the mucin for the formation of fernlike crystals. When a dilute solution of mucin dissolved in saline was dried, a radial crystallization pattern was observed to emanate from a single crystal in the center. Upon closer examination, the pattern was observed to emanate from the corners of the crystal.

Calcium Chloride↗

PubMed1977

Transformation of rat liver epithelial cells by Kirsten murine sarcoma virus.

We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.

Animals↗