Activated N-ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells

Activated mouse N-ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N-ras in a LTR-containing vector caused differentiation with a reduced efficiency, but normal N-ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF-resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following transfection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasia-promoting activity in other cells.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.     

Binucleate cells in the Ehrlich ascites tumor. Action of 5-fluorouracil

Time-dependent frequency distribution of binucleate cells (BC) was studied in Ehrlich ascites tumor (EAT) growing in mice. In animals that received no further treatment, the number of BC increased slowly from 2.6% to 16.5% of total cells within 8 days. In animals that were treated with different doses of 5-fluorouracil (FU) we found clearly higher numbers of BC. The number of BC increased with tumor age. The increase observed after treatment was reached more quickly in animals that had received the highest FU dose. The final number of BC was also dependent on the age of the tumor at the time of FU injection.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Balearic insular isolation and large continental spread framed the phylogeography of the western Mediterranean Cheirolophus intybaceus s.l. (Asteraceae)

Recent Quaternary geological and climate events have shaped the evolutionary histories of plant species in the Mediterranean basin, one of the most important hotspots of biodiversity. Genetic analyses of the western Mediterranean Cheirolophus intybaceus s.l. (Asteraceae) based on AFLP were conducted to establish the relationships between its close species and populations, to reconstruct the phylogeography of the group and to analyse potential unidirectional versus bidirectional dispersals between the Ibero‐Provençal belt and the Balearic Islands. AFLP data revealed two main genetic groups, one constituted by the Balearic populations and Garraf (NE Iberia) and the other formed by the remaining mainland populations that were further sub‐structured into two geographically separated subgroups (SE + E Iberia and NE Iberia + SW France). Genetic diversity and spatial structure analyses suggested a mid‐Pleistocene scenario for the origin of C. intybaceus in southern Iberia, followed by dispersal to the north and a single colonisation event of the Balearic archipelago from the near Dianic NE Iberian area. This hypothesis was supported by paleogeographic data, which showed the existence of terrestrial connections between the continent and the islands during the Middle–Late Pleistocene marine regressions, whereas the more recent single back‐colonisation of the mainland from Mallorca might be explained by several hypotheses, such as long‐distance dispersal mediated by migratory marine birds or sea currents.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.