Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

Involvement of phytochrome in stomatal movement: Effect of blue and red light

The effect of various experimental parameters upon the frequency of callus formation from cultured anthers of Oryza sativa has been investigated. Although certain medium components were found to be critical to callus formation, the concentration of these components had little effect upon the frequency of callus formation. The degree to which the callus formation frequency was influenced by cold pretreatment of the flowers was variable. Even though plants were grown under uniform conditions and flowers containing pollen in the microspore stage of development were selected for dissection, the frequency of callus formation varied nonrandomly between flowers. In experiments with populations of flowers in a physiological and developmental state favorable to callus formation 35% of plated anthers produced callus and at least 60% of these calluses gave rise to green plants.     

Influx of γ-Aminobutyric Acid and α-Aminoisobutyric Acid into Mouse Cerebrum Slices Incubated in a Pyruvate Medium with or without Added Glucose or Glucose Analogues Compared with Influx from a Glucose Medium

Concentrative influx of γ-aminobutyric acid (GABA) and α-aminoisobutyric acid (AIB) into incubated mouse cerebrum slices is decreased when pyruvate is substituted for glucose. Influx of GABA from pyruvate medium is not increased by presence of glucose, 2-deoxy-d -glucose (2-DOG), or 3-O-methyl-d -glucose (3-O-MeG). Influx of AIB is restored to the rate from glucose medium if 2-DOG is present initially, but is not restored if 2-DOG is added with AIB. Influx is not restored if 3-O-MeG is present initially, but is restored if 3-O-MeG is added with AIB. Influx is restored if glucose is present initially or is added with AIB.     

Chemically defined serum-free media for the cultivation of primary cells and their susceptibility to viruses

Summary Chemically defined media SFRE-199-1 for the growth and SFRE-199-2 for the maintenance of primary baboon kidney (Bak) cell cultures were formulated by supplementing medium M199 with insulin, sodium pyruvate, zinc sulfate, and increasing arginine-HCl, cysteine, cystine,l-glutamine,l-glutamic acid, glycine, histidine, tyrosine, and glucose to maximally active nontoxic concentrations. For prolonged maintenance of the cells, physiological pH control, and blocking of excessive lactic acid accumulation in the spent medium of the cell cultures, it was necessary to supplement the medium containing Earle's balanced salts withd-(+) galactose. The cells grew and were maintained equally well on glass or polystyrene surfaces. Selenium, when added to growth medium or substituted for insulin and zinc sulfate, did not stimulate cell growth. Electron microscopy showed that numerous dense particles, approximately 250 to 400 ? in diameter, with the appearance of glycogen, were found throughout the cytoplasm in the cells grown in SFRE-199-1 and maintained in SFRE-199-2. Echovirus types 1 to 3, poliovirus types 1 to 3, coxsackievirus types B2, B4, B5,Herpesvirus hominis type 1, simian herpesvirusH. simiae and SA8, and simian adenovirus SV34 when titrated in primary Bak cells and grown and maintained in SFRE-199-1 and 2, respectively, developed titers comparable to those obtained in conventionally grown and maintained cells. This study was supported in part by National Institute of Health Grant RR00361 and World Health Organization Grant V4/181/38. This laboratory serves as the NIH/WHO Collaborating Center for Reference and Research in Simian Viruses.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.     

Attachment and Short-Term Maintenance of Motility and Viability of Entamoeba Histolytica In A Defined Medium*

Organic requirements for attachment to glass, elongation, and motility of Entamoeba histolytica, have been determined. the trophozoite, which has been grown axenically only in highly complex media with reduced oxygen tensions, remains rounded and detached when placed in a Tris-HCl buffered solution containing NaCI, KCI, MgCI₂, and CaCI₂. A maintenance medium in which the amebae could attach to glass, elongate, and remain motile and viable for 12 to 24 h was devised with the addition of cysteine, ascorbic acid, bovine serum albumin, and the vitamin solution of medium NCTC #107. Tris-HCI was the most effective buffer tested and the optimal pH was 6.9 to 7.0. Survival, but not attachment, of the amebae was decreased at osmolalities ranging between 110 and 180 milliosmoles/kg, whereas both functions were decreased above ～260 milliosmoles/kg. Bovine serum albumin, the most effective of the proteins tested, and the vitamin solution helped maintain attachment of some ameba strains, but were not required by other strains. the requirements for cysteine and ascorbic acid were absolute and highly specific. During incubation in the maintenance medium, cell volumes decreased. Sensitivity of the organisms to agglutination by concanavalin A, wheat germ agglutinin, soybean agglutinin and fucose binding protein remained unchanged.     

Lack of expression on cultured human T-lymphocytes of a T-cell antigen shared by the molt-4 cell line and normal human thymocytes

Summary Four hematopoietic cell lines (CCRF-CEM, HSB-2, MOLT-4, and RPMI-8402), derived from acute lymphoblastic leukemia and expressing T-cell surface markers (T-HCL), were studied with two specific anti-T-cell sera. The sera were raised in rabbits against human thymocytes (anti-HTY) and against T-cells cultured in the presence of conditioned medium derived from lymphocytes stimulated with PHA (anti-CTC). Both sera were absorbed to obtain a T-cell specific pattern of reaction and were further absorbed with normal peripheral blood lymphocytes or with each of the four T-HCL. The anti-HTY sera absorbed with CEM, 8402, and HSB-2 still reacted with MOLT-4. A similar pattern of reactivity was found only with the anti-CTC absorbed with 8402, whereas, after absorptions with the other cell lines, this antiserum was unreactive against MOLT-4. After absorption with normal peripheral blood lymphocytes, anti-HTY still reacted with thymocytes and MOLT-4 but was negative on CTC. In contrast, anti-CTC absorbed with peripheral blood lymphocytes (PBL) was negative on thymocytes and MOLT-4 but still reacted against CTC. Our data confirm the existence of a T-cell antigen (probably an early T-cell differentiation antigen) shared between thymus and MOLT-4. This antigen is not expressed on CTC, although these cells express an antigenic pattern more complex than PBL. Antisera to CTC represents a source of anti-T-cell sera free of contamination with antibodies to early thymus-related antigens but containing other T-cell-related specificities. Supported in part by Naval Medical Research and Development Command, Research Task No. ZF51.524.013.1025, and National Cancer Institute Contract No. Y01-CB-00319. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.     

A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.