Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Release of microbial contamination from fractured solids

Estimation of the probability of release of microbial contamination from the interior of solids upon fracture due to impact is essential to the formulation of planetary quarantine and spacecraft sterilization requirements. A model system was designed in which known concentrations of bacterial spores were incorporated in methyl methacrylate plastic. Pieces of plastic were fractured in a uniform manner exposing interior surface areas of consistent and measurable size. Known surface areas were incubated in sets of 20 culture tubes containing liquid growth medium. The subsequent occurrence of visible growth expressed as percent of tubes positive was interpreted as an estimate of the probability of release of at least one viable micro-organism.From these experiments probability of release as a function of microbial concentration in plastic was estimated for exposed interior surface areas of 30.6, 61.2, 91.8 or 122.4 mm². Good agreement of the empirical results with a theoretical mathematical model of the probability of release of contamination from solids was demonstrated. Analysis of the data using the maximum likelihood procedure provided a means of calculating a proportionality constant representing the effective thickness of the exposed area and the characteristics of the recovery procedure.     

Isolation and characterization of isoprene mutants of Escherichia coli.

Isoprenoid compounds are found in all organisms. In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis. Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis. This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E. coli. Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells. The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content. In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC 5.3.3.2), farnesyldiphosphate synthetase (EC 2.5.1.1), and higher prenyl transferases.     

Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate-ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts

The effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate-ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50-500 microM H2O2 for 20 min or 100 microM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250-500 microM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50-500 microM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant-related DNA damage is a function of the type of oxidative stress used and may be cell-specific.     

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study.

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.