Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

Root Rot of Trifolium subterraneum Induced by Fusarium solani

A fungus disease of Trifolium subterraneum, previously unknown in South Africa, was identified as Fusarium root rot caused by F. solani (Mart.) Sacc. Diseased plants were severely stunted. The characteristic internal root discolouration observed in the field, is described. The disease occurred mainly between Swellendam and Humansdorp in the southern and south-eastern Cape Province. The subterranean clover fields inspected were between 95 and 100 % infected with the root rot disease. T. repens and T. pratense were also susceptible.     

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.     

In-vivo and in-vitro steroidogenic activity of post-ovulatory ovarian follicles of the rabbit

Pseudopregnancy was induced in rabbits by injection of 50 i.u. hCG into the lateral ear vein. Blood was collected on Days 0, 1, 2 and 6 after hCG from the lateral ear vein and, in some studies, from the ovarian vein as well. Ovaries were collected on Days 0, 1, 2 or 6 after hCG injection, and follicles (greater than 0.8 mm diam.) were obtained by microdissection. Concentrations of oestradiol-17 beta, testosterone and progesterone in blood and follicular homogenates were measured by RIA. Follicular steroidogenesis was increased significantly on Day 6 after ovulation, at the time when the corpus luteum transforms into an oestrogen-dependent tissue. The ability of developing post-ovulatory follicles to secrete steroids in vitro in response to a gonadotrophic stimulus also increased over this same time interval. Follicular oestradiol production on Days 1 and 2 of pseudopregnancy may synergize with the post-ovulatory secretion of FSH to promote further follicular growth in the pseudopregnant rabbit.     

Differentiation of newborn rat adipocyte precursors in defined serum-free medium

Summary Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment. This work was supported by grants from the Juvenile Diabetes Foundation, File #185221 and from the National Institutes of Health 1 PO1 CA37589. Editor’s Statement This paper extends to primary cultures the serum-free methods previously applied to studies of adipocyte differentiation in established lines. The observation that serum can block differentiation in this system suggests the existence of previously unrecognized circulating plasma or platelet factors affecting adipocyte differentiation, and the model developed provides an assay for the identification of these factors.     

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

The processing and collaborative assay of a reference endotoxin

A preparation of Escherichia coli bacterial endotoxin, the latest of successive lots drawn from bulk material which has been studied in laboratory tests and in animals and humans for suitability as a reference endotoxin, has been filled and lyophilized in a large number of vials. Details of its characterization, including stability studies, are given. A collaborative assay was conducted by 14 laboratories using gelation end-points with Limulus amebocyte lysates. Approximate continuity of the unit of potency with the existing national unit was achieved. The lot was made from the single final bulk but had to be freeze-dried in five sublimators. An assessment was therefore made for possible heterogeneity. The results indicate that the lot can be used as a large homogeneous quantity. The advantages of using it widely as a standard for endotoxins are discussed.