Ionizing-radiation-induced damage in the DNA of cultured human cells. Identification of 8,5-cyclo-2-deoxyguanosine.

Epstein-Barr-virus-transformed peripheral-blood B-lymphocytes were gamma-irradiated at 0 degree C at doses from 10 to 100 Gy. The cells were immediately lysed and the DNA was isolated. Subsequently, the DNA was hydrolysed to 2'-deoxyribonucleosides with a mixture of DNAase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was dried, trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. The (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyguanosine were observed in a ratio of 1:3, and their formation was dose-dependent. It was possible to detect and characterize one such lesion in approx. 4 X 10(4) guanine nucleotide subunits of DNA.     

The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.     

Factors affecting malate dehydrogenase activity in freezing-thawing processes

Malate dehydrogenases from several sources show different behaviour when frozen-thawed in 100 mM sodium phosphate buffer, pH 7.4, containing chaotropic ions. The effects produced by the addition of various metabolites, protein concentration and buffer medium used on the loss of activity induced by the freezing-thawing process are reported. The major part of the loss of activity is caused by the formation of "wrong" aggregates of high mol. wt.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

Influence of α-interferon therapy on blood lymphoid cells

Summary The influence of natural -interferon (-IFN) therapy (3×10⁶ units i.m. daily) on blood lymphoid cells was studied in 20 patients with gynecological neoplasias (7 patients with condylomata accuminata and 13 patients with ovarian carcinoma). There was a statistically significant increase in the intracellular levels of 2'–5'oligoadenylate synthese 1 day after the first injection of IFN and with few exceptions this activity remained increased during 3 months of treatment. In most of the patients, the capacity of blood lymphoid cells to produce IgA, IgG, and IgM following stimulation with pokeweed mitogen was decreased 1 day after the first injection of IFN and with few exceptions it remained low during 6 months of IFN therapy. In most patients there was a decrease in the capacity of lymphoid cells to act as stimulator or responder cells in a mixed lymphocyte culture during IFN therapy. The -IFN therapy had no major influence on the response of lymphoid cells to mitogens. We conclude, that neither this nor our previous studies on the influence of IFN therapy on immunological functions have given support to the hypothesis that the antitumor action of IFN is mediated by the immune system.     

Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products

The organisation and expression of the rpl22, rps3, rpl16 and rpl14 genes, which belong to the S10- and spc-like operons of spinach chloroplasts, have been studied. Northern experiments and nuclease S1 mapping show that the two operon-like groups of genes are cotranscribed. It is demonstrated that the intron-containing rpl16 gene is spliced in vivo. Based on amino acid composition and protein sequence data, the products of the rpl22, rpl16 and rpl14 genes are identified respectively as the spinach chloroplast ribosomal proteins CS-L13, CS-L24 and CS-L29. The rpl22 gene product is a 5S rRNA binding protein and therefore is distinguishable from the homologous Escherichia coli L22 ribosomal protein.     

Maintenance of low cl concentrations in mesophyll cells of leaf blades of barley seedlings exposed to salt stress

The concentrations of vacuolar Na⁺ and Cl⁻ in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl⁻ in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl⁻ in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl⁻ in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl⁻ in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na⁺ in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl⁻ (and to a lesser extent Na⁺) and suggest that the site of regulation of Cl⁻ concentration in the barley leaf is located in the mesophyll cells of the blade.