In Vitro最新文献

Na+,K+-ATPase, HCO3(-)-ATPase, Ca2+,Mg2+,-ATPase, Ca2+-ATPase, and alkaline phosphatase activities were measured in cultures of osteoblastlike cells treated with fluoride and cortisol separately and in combinations. Low concentrations of cortisol increased HCO3- -ATPase (10(-11) to 10(-18) M cortisol) and alkaline phosphatase (10(-11) to 10(-9) M cortisol) activities, but higher cortisol concentrations reduced these activities. Na+,K+-ATPase, Ca2+,Mg2+-ATPase, and Ca2+-ATPase activities tended only to be reduced by cortisol. Fluoride (10(-6) and 5 X 10(-6) M) increased HCO3(-)-ATPase and alkaline phosphatase activities, but these activities were similar to controls in the presence of 10(-5) M fluoride. Ca2+,Mg2+-ATPase activity was decreased and Na+,K+-ATPase activity was increased as the concentration of fluoride increased (10(-6) to 10(-5) M). Preliminary experiments with fluoride indicated that lower concentrations (10(-7) M) were without effect. Cortisol concentrations of 10(-9) and 10(-8) M were chosen for studies with combinations of cortisol and fluoride because the effects of these concentrations on alkaline phosphatase activity were opposite, i.e. 10(-9) M increased whereas 10(-8) M decreased activity. Fluoride concentrations of 10(-6), 5 X 10(-6), and 10(-5) M were chosen because a peak of alkaline phosphatase activity occurred at 5 X 10(-6) M fluoride. Higher (10(-4) M) and lower (10(-7) M) fluoride concentrations were without effect. The effects of combinations of cortisol and fluoride depend on the enzyme activity measured. Fluoride (10(-6) M) combined with cortisol (10(-9) M) produced a peak of Na+,K+-ATPase activity. The increased activity obtained with all concentrations of fluoride alone was preserved when fluoride was combined with 10(-8) M cortisol, although the activity tended to be reduced at 5 X 10(-6) and 10(-5) M fluoride. HCO3(-)-ATPase activity was increased by fluoride combined with 10(-8) M cortisol and decreased by fluoride combined with 10(-9) M cortisol compared to the activities obtained with fluoride alone. The decrease in Ca2+,Mg2+-ATPase activity caused by fluoride alone was prevented by 10(-9) and enhanced by 10(-8) M cortisol, although all treatments produced the same activity at 10(-5) M fluoride. Ca2+-ATPase activity tended to be increased by combinations of fluoride and cortisol, but significantly so only at 10(-5) M fluoride in combinations with 10(-8) and 10(-9) M cortisol.(ABSTRACT TRUNCATED AT 400 WORDS)

We have investigated the potential for using cyclosporin A to increase the efficiency with which Epstein-Barr virus-transformed human lymphoblast lines can be prepared. Use of this immunosuppressive drug has permitted the development of a procedure with success rates exceeding 95% despite the processing of very large numbers of samples.

Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis.

Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3--ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.

Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B.

Glucose-6-phosphatase activity decreases whereas gamma glutamyltranspeptidase activity increases during hepatocarcinogenesis and the maintenance of hepatocytes in primary culture. This report describes the effect of culture conditions that are known to preserve hepatic glucose-6-phosphatase activity on gamma glutamyltranspeptidase activity. The results indicate that the regulation of glucose-6-phosphatase and gamma glutamyltranspeptidase activities is not coordinated in primary cultures of hepatocytes.

All cells examined from the non-B, non-T acute lymphoblastic leukemia cell line, NALL-1, stained positive both for terminal deoxynucleotidyl transferase and for common ALL antigen. In addition, peroxidase activity was detected by light microscopy in 55 to 75% of cells and peroxidase-positive granules were detected ultrastructurally in greater than 80% of cells. Peroxidase activity in NALL-1 may result from derepression of peroxidase genes or clonal proliferation of a biphenotypic precursor cell.

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.

As part of a study on X chromosomes, metaphase cell synchrony and chromosome isolation methods were developed for the opossum (Didelphis virginiana) kidney epithelial cell line (OK). The cell synchrony yielded large amounts of metaphase cells using a relatively simple method in which a key feature was a calcium- and magnesium-free balanced salt wash. A neutral pH chromosome isolation method was developed for the kidney epithelial cells, because they were somewhat difficult to disrupt fully by other methods. FACS IV flow microfluorometric analysis of OK chromosomes confirms a clear difference between the sizes of opossum X chromosomes and autosomes.

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.