Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.     

Dimethylnitrosamine metabolism: II. In vitro activation of dimethylnitrosamine by hepatic and renal tissues from crosses among BALB/c, DBA and C57BL mice

Crosses among BALB/c, C57BL and DBA mice were performed to investigate the genetic mechanisms involved in metabolism of DMN by renal and hepatic tissues. Liver S-9 fractions from parental strain DBA had the greatest potential to activate DMN and liver fractions from parental strain BALB/c had the lowest. No age or sex-related differences were observed within strain. Crossing of either C57BL or DBA to BALB/c mice resulted in F1 hybrids with liver microsomal enzymes that gave results similar to the BALB/c parental strain. There were no sex or age differences within crossbred strains in the potential of liver to activate DMN. In contrast male DBA and C57BL parental mice renal S-9 fractions did not differ significantly from each other but did differ significantly from male BALB/c renal fractions and from female and immature animals of all strains. Crossing of either DBA or C57BL mice with BALB/c mice resulted in male F1 hybrids whose renal S-9 fractions did not differ significantly from males of the parental BALB/c strain. In all instances, male renal S-9 fractions had a significantly greater potential to activate DMN than female or immature animals. F1 DBA X C57BL hybrids had renal S-9 fractions that did not differ significantly from the parental strains. These data suggest that the gene(s) for low DMN metabolism of BALB/c mice are apparently dominant over the genes from both DBA and C57BL. The exact genetic or physiological mechanism needs further elucidation.     

Effect of the exogenous lipids of complex media on the fatty acid composition of mycelial microorganisms

The effect of exogenous lipid sources on the composition of fatty acids was studied in actinomycetes of the Streptomyces genus and in fungi belonging to the genera Blakeslea, Cunninghamella and Penicillium. The following sources of exogenous lipids were used: soybean and maize flour, sunflower by-products, chicken droppings, maize extract, yeast extract, peptone, sperm whale fat, sunflower and palm oil. The composition of fatty acids in total extracted lipids of the studied mycelial microorganisms was shown to reflect two processes: lipid synthesis de novo and assimilation of exogenous fatty acids. This fact ought to be taken into account both in the chemotaxonomic interpretation of fatty acid composition and in practical recommendations for the utilization of microbial lipids. It is of particular interest to study the physiological role of exogenous lipid metabolism in the cells of microorganisms.     

Chromosomal damage induced in human lymphocytes by low doses of D-T neutrons

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

Mineral licks as a sodium source for Isle Royale moose

Summary Natural mineral licks and their use by moose (Alces alces) on Isle Royale National Park, Michigan, were studied during 1982–85. The distribution of known licks suggested that they occurred in association with glacial debris, primarily in the western portions of the island. Moose utilized mineral springs extensively during the spring-summer period, and at least 5 licks were used year-round. During summer, a pronounced diel pattern of moose visitation was apparent, with peak use occurring between 0400–0800 h. Although daytime lick use declined by late June, morning and evening use continued to be relatively high throughout the study period. Peak lick use coincided with leaf-emergence in spring. Moose continued to utilize mineral licks despite the availability of ponds containing aquatic plants. Sodium appeared to be the element attracting moose to licks where they ingest copious amounts of water. Observed sodium ingestion rates (0.35 g/min) at licks indicate that licks provide a more concentrated source of sodium compared to aquatic plants (0.023 g/min). Based on the data presented, we reject the conclusions of earlier workers that aquatic plants constitute the only significant source of sodium for Isle Royale moose.     

Analysis of the frequency of allelic exclusion of immunoglobulin light chain genes and molecular characteristics of immunoglobulins secreted by hybridomas with expression of both allelic genes

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-la/Igk-1b rat splenocytes has revealed that 9,8% of Ig kappa-chain genes are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of nine such clones demonstrated that in six cases only one L-chain allotype is present in IgM. Thus for the first time the high frequency of selective association of H and L chains was shown. Evidently this selectively may function as one of the allelic exclusion mechanisms at the Ig assembly stage.     

Chromosome-damaging effects of heraclenin in human lymphocytes in vitro

Heraclenin, a furocoumarin with an epoxide group in its side chain, was analyzed to see if it induced structural chromosome aberrations and sister-chromatid exchanges (SCEs) in human lymphocytes in vitro. The results were compared directly with those of imperatorin, which differs from heraclenin only in lacking an epoxide group. An equally strong clastogenic effect was found for both heraclenin and imperatorin: the number of metaphases with breaks was increased in both cases by approximately a factor of 6. Heraclenin produced a considerable dose-dependent increase in the SCE rate, i.e., by about 60 induced SCEs/metaphase, whereas imperatorin induced only about 4 SCEs/metaphase. The results are discussed with respect to the occurrence of structural aberrations, which are primarily due to the basic furocoumarin structure itself, whereas the large increase in the SCE rate produced by heraclenin is most probably significantly influenced by its epoxide group.     

DNA interaction with the antitumor agent thiophosphamide

DNA interaction with an alkylating antitumor drug N,N',N"-triethylenethiophosphoramide (thiotepa) in water-salt solutions at 37 degrees C has been studied by UV-spectroscopy, heat denaturation and electron microscopy methods. Changes of the DNA melting curve parameters provide information on the kinetics of alkylation. The dependence of the alkylation rate on DNA and thiotepa concentrations shows that the alkylation reaction is biomolecular. The increase of sodium chloride concentration from 10(-3) to 10(-1) M is accompanied by a drastic decrease of the alkylation rate. Thiotepa binding results in destabilization of the DNA secondary structure and formation of cross-links. An increased amount of bounded thiotepa results in DNA denaturation; prolonged alkylation causes breaks in the sugar-phosphate backbone. The results of the work are discussed in connection with the literature data on DNA interaction with thiotepa in vivo.