Change in survival of cells in the first 2 generations after irradiation and exposure to inhibitors of repair synthesis

In experiments on asynchronous population of HeLa S3 cells a study was made of the possibility of assessing DNA lesions which remained unrepaired for a long period of time following gamma-irradiation: in generation "O" directly affected by radiation and in generation "I" following the irradiated one. The presence of DNA damages was estimated by the reduction in survival of exposed cells incubated with inhibitors of repair and replicative syntheses of DNA, namely, with arabinoside cytosine and hydroxyurea. A considerable enhancement of the radiation effect was noted with the inhibitors added 0-6 h after irradiation (generation "O"), and a marked increase in the cell death was registered with the preparations injected 24-30 h after exposure (generation "I"). It is assumed that minor residual lesions persist in the generation of cells, following the one directly affected by gamma-radiation, which have completed the first postirradiation mitosis.     

Long-term DNA damage and mammalian cell survival

On the basis of our own data and those reported in the literature we have made an attempt to follow the fate of the DNA lesions which remain unrepaired during a long period of time, and their possible role in the fate of irradiated cells. The presence of long-lived ("residual") damages is determined by the changes in survival of exposed cells treated, at different times after irradiation, with a mixture of arabinoside cytosine and hydroxyurea. It is shown that "residual" damages can probably exist in the exposed generation and be retained in that following the irradiated one, i.e. after the first mitosis. The nearest descendants of exposed cells (the 3d-5th generations) exhibit a 50% decrease in the rate of DNA synthesis and fall of their proliferative activity, as well as a decrease in the rate of reproduction of their remote descendants. The comparison of the results obtained with those reported by other authors enable us to assume that "residual" DNA lesions play an important role in the fate of exposed cells, that is, in reproductive death, radiation mutagenesis, and malignant transformations.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide.     

Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit.

A cDNA of rat brain encoding the GABAA receptor alpha 4 subunit has been cloned. Recombinant receptors composed of alpha 4, beta 2 and gamma 2 subunit bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine 'alcohol antagonist' [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The alpha 4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The alpha 4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.