Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Conjugal transfer of recombinant transposon Tn916 from Escherichia coli to Bacillus stearothermophilus.

A gene for thermostable amylase has been inserted at the BstXI site of Tn916. Mating experiments demonstrated that unlike Tn916, the recombinant transposon, designated Tn916A, could transfer from Escherichia coli to Bacillus stearothermophilus BR219 in broth matings, resulting in chromosomal integration of the transposon and expression of the amylase at significant levels.     

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.     

Cytostatic drug activity in plasma, a bioassay for detecting mutagenicity of directly and indirectly acting chemicals, an evaluation of 20 chemicals

We have utilized in vivo drug metabolism for detecting the mutagenicity of known indirectly-acting chemical mutagens. Exponentially growing Chinese hamster ovary (CHO) cells were incubated with plasma derived from treated rats containing active metabolites of the test chemicals. The genotoxicity was assessed by the 18 of tested chemical mutagens. Plasma from rats treated with known non-mutagens did not increase the frequencies of SCEs. The results indicate that this method could be useful for the demonstration of genotoxicity of chemicals which need metabolic activation to be effective, and especially those which are not effective, when in vitro activation conditions (S9 mixture) are used.     

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Heterochromatin distribution and chiasma localization in the grasshopper Bryodema tuberculata (fabr.) (Acrididae)

The problem of extreme localisation of chiasmata in the grasshopper species Bryodema tuberculata has been reinvestigated, using C-banding, Q-banding and benzimidazol techniques. These techniques reveal the precise localisation of heterochromatin in different chromosomes. Single or double heterochromatic blocks are present near the centromeric regions, except in chromosomes 5 and 11, which have larger blocks. These two chromosomes possess a distal chiasma while the other autosomes have a proximal chiasma. The results with regard to the distribution of chiasmata, in relation to the localisation of heterochromatin, as well as the existence of a short arm, are compared with the earlier observations of White, and discussed briefly.     

Chromosome breakage induced by alkyl-alkane-sulfonates under different physical treatment conditions

A detailed study of chromosome breakage induced by three alkane sulfonates, which differ in chemical structure, functionality and reaction mechanism has been made in barley under different treatment conditions of temperature and hydrogen ion concentration. This study has indicated that, (i) the frequency and the types of chromosome breakage, at mitosis and meiosis, indicate certain qualitative and quantitative differences between the biological action of these three chemicals, (ii) the temperature of the treatment solution profoundly influences the frequency of chromosome breakage — a high frequency of breakage is observed at higher temperature, (iii) the effect of pH, though not very significant, is evident from the production of appreciably low frequency of breakage at alkaline pH for a given dose of chemical, (iv) by appropriate manipulation of treatment conditions, increase or decrease in the frequency of chromosome breakage can be accomplished to a considerable extent, (v) the qualitative and quantitative differences observed with respect to chromosoma breakage reflect the differences in the mode of biological action of these agents.