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.     

Ketomethylureas. A new class of angiotensin converting enzyme inhibitors

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term "ketomethylureas", is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE with I50 values in the nM range.     

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.     

Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of X-rays

The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.     

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.     

Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods

We have utilized monoclonal antibody against BrdUrd to detect sister-chromatid exchanges in CHO cells. This technique allows detection of SCEs at very low levels of BrdUrd incorporation. At incorporation level of 0.5%, a frequency of about 2 SCEs/cell/cycle was found. In a UV-sensitive mutant (43-3B) which has an increased spontaneous frequency of SCEs, it is found that this increase is due to incorporated BrdUrd. In MMS- and MMC-treated cells, an influence of BrdUrd on the frequencies of induced SCEs was found only when high concentrations of mutagens were employed.     

Induction of sister-chromatid exchanges by restriction endonucleases

Restriction endonucleases Cfo 1, Pvu II, Sma I, Hpa II, Taq I and Hae III were tested for their ability to induce SCEs in CHO cells. The results indicate that the DNA double-strand breaks induced during S-phase by these enzymes lead to an increase in the frequencies of SCEs.     

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.     

Effects of pre-treatment with sodium butyrate on the frequencies of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes

The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal aberrations were studied in human peripheral blood lymphocytes. Unstimulated (G0) lymphocytes were pre-treated with sodium butyrate (5 mM) for 24 h, X-irradiated and then stimulated to pass through the cell cycle. Cells in their first post-radiation metaphase were scored for chromosomal aberrations. In parallel biochemical experiments nucleoid sedimentation technique was used to examine the induction and repair of DNA-strand breaks. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. We therefore suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G0 lymphocytes.