Mutant cell lines resistant to azetidine carboxylic acid: Quantitative and qualitative differences in pyrroline-5-carboxylate synthase activity

Mutant Chinese hamster lung fibroblasts were selected that are resistant to the proline analog L-azetidine-2-carboxylic acid. Resistance in the two mutant cell lines is associated with two distinct alterations in pyrroline-5-carboxylate synthase, the enzyme that catalyzes the proline biosynthetic step leading from glutamic acid to pyrroline-5-carboxylate. In one mutant cell line, pyrroline-5-carboxylate synthase specific activity is increased 30-fold over the level in control cells. In the other mutant line, pyrroline-5-carboxylate synthase activity is not increased, but the enzyme has become insensitive to inhibition by ornithine and proline.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

Hormonal requirements for the induction of cytochrome P-450 in hepatocytes cultured in a serum-free medium

Drug mediated induction of cytochrome P450 was studied in cultures of hepatocytes that had never been cultured in the presence of serum. Propylisopropylacetamide induced a five-fold increase in cytochrome P450, approximating $\text{in ovo}$ induced levels, when triiodothyronine and/or dexamethasone were included in the culture medium. Insulin was apparently not required for this induction. Cytochrome P450, free of cytochrome oxidase, could be fully recovered from cell homogenates in a 8700g supernatant, by use of a buffer containing 0.2% Emulgen.     

Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture

Summary An improved basal medium is presented that requires only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response. Supported by the Medical Research Service of the Veterans Administration.     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8–20 h period.Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types, even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstantial evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Defined nutrient medium for the in vitro maintenance ofXenopus laevis oocytes

Summary A procedure is described for the isolation and culture of large numbers of follicle cell-freeXenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenance of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morphological and functional characteristics of oocytes for periods up to 3 weeks. Research supported by grant BMS 74-18790 from the National Science Foundation to JJE. Operated by Union Carbide Corporation for the U.S.E.R.D.A.     

Light-Induced Body Movement of Euglena gracilis Coupled to Flagellar Photophobic Responses by Mechanical Stimulation*†

SYNOPSIS. In low viscosity media, Euglena gracilis strain Z responds to a sudden change in light intensity by a cessation of forward movement, followed by a reorientation of the locomotor flagellum which results in turning of the cell around the lateral axis (photophobic response). At a viscosity interface between low [～ 1 cP (centipoise)] and high (4000 cP) media, the cells exhibit avoidance responses or become immobilized in the higher viscosity medium. Upon changing the light intensity, free swimming cells have photophobic responses, while immobilized ones undergo body contractions. For cells immersed in media of varying viscosity, the delay between light stimulation and body contraction (transduction time) is shortest at high viscosities. From 500 to 2000 cP, where the cells are capable of both movement and light-induced body contractions, there is a logarithmic dependence of the transduction time on the viscosity. The transduction time does not vary appreciably with the intensity of the primary light stimulus within a range of 0.14-1.13 kW/m².     

Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus

The amino acid incorporation and -aminoisobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8-and 10-bacteria, while no difference was observed in phospholipid composition.Abbreviations AIB Amino-isobutyric acid     

Stimuli for cuticle formation and ecdysis in vitro of the infective larva of Anisakis sp. (Nematoda: Ascaridoidea)

Third-stage larvae of the genus Anisakis from the fish Leionura atun (Trichiuroidei: Perciformes) form a new cuticle and moult in vitro in about 72 h. If the culture medium is Krebs-Ringer under 5% carbon dioxide in air at 37°C, relatively few moult and survival is poor. But more moult and survival is enhanced if worms are incubated in tissue culture medium 199, even if the gas phase is air, although they moult more quickly if it contains 5% carbon dioxide. In both Krebs-Ringer and 199 the benefits of high concentrations of carbon dioxide only accrue if the gas is present during the first 40 h of incubation. Worms do not feed in these media until they have moulted.