Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.     

Factors affecting the photoproduction of ammonia from dinitrogen and water by the cyanobacterium Anabaena sp. strain ATCC 33047

Synthesis of ammonia from dinitrogen and water by suspensions of Anabaena sp. Strain ATCC 33047 treated with the glutamine synthetase inhibitor L-methionine-D,L-sulfoximine is strictly dependent on light. Under otherwise optimal conditions, the yield of ammonia production is influenced by irradiance, as well as by the density, depth, and turbulence of the cell suspension. The interaction among these factors seems to determine the actual amount of light available to each single cell or filament in the suspension for the photoproduction process. Under convenient illumination, the limiting factor in the synthesis of ammonia seems to be the cellular nitrogenase activity level, but under limiting light conditions the limiting factor could, however, be the assimilatory power required for nitrogen fixation. Photosynthetic ammonia production from atmospheric nitrogen and water can operate with an efficiency of ca. 10% of its theoretical maximum, representing a remarkable process for the conversion of light energy into chemical energy.     

A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

The polyene antibiotic amphotericin B acts as a Ca2+ ionophore in sterol-containing liposomes

Amphotericin B (AmB) was shown to induce a Ca2+ influx across ergosterol- and cholesterol-containing large unilamellar liposomes, by following spectrophotometrically the formation of the Arsenazo III-Ca2+ complex. At equivalent antibiotic concentrations the Ca2+ influx was much more extensive through ergosterol-containing membranes (almost 100% with 1 microM AmB, 160 microM lipid) than through cholesterol-containing membranes (below 0.5 microM the influx of Ca2+ was negligible). In the presence of ergosterol-containing membranes the initial rate of Ca2+ influx had the same linear dependence on the ratio antibiotic/lipid whatever the lipid concentration, which was not the case in cholesterol-containing membranes. These results suggest that the channels responsible for the AmB-induced Ca2+ permeability across cholesterol- and ergosterol-containing liposomes have different structures.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.