Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases.

Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one. Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes. At the other central positions, uracil was eliminated with different initial velocities. Single stranded phi X 174 DNA fragments were used to study the influence of the sequence. Cytosine bases were deaminated to give uracil by bisulfite treatment. It was shown that the initial excision velocity of two vicinal uracil residues was decreased. The same observation was made for two uracils separated by one base. A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.     

The influence of mineral salts on fecundity of the water flea (Daphnia magna) and the implications on toxicity testing of industrial wastewater

The effects of mineral salts constituting water hardness on fecundity ofDaphnia magna were assessed. Of the salts tested, increased concentrations of NaHCO₃ and MgSO₄ had no effect on fecundity, CaSO₄ significantly increased fecundity, and KCl significantly reduced fecundity. The number of offspring produced per daphnid was correlative to the CaSO₄ concentration at CaSO₄ concentrations between 91 and 2100 mg/ℓ. The effects of CaSO₄ on daphnid fecundity could influence the interpretive outcome of industrial wastewater toxicity tests using this species when the waste and dilution waters contain different concentrations of CaSO₄. It is recommended that when performing these tests, dilution water be sampled at the intake site of the industry's water source, thus assuring initial comparability of the waste and dilution waters. The CaSO₄ content of the water prior to and after industrial use should be determined to identify any alterations of CaSO₄ concentration during use. Identification of CaSO₄ concentration differences can aid in the interpretation of effects associated with the wastewater.     

Kinetic properties of Na(+) -H(+) antiport in Escherichia coli membrane vesicles: Effects of imposed electrical potential, proton gradient, and internal pH

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.     

Cellobiose metabolism in Erwinia: genetic study

Summary The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism. In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb^- specific mutants. When introduced into wild-type E. coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes. From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three--glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

Stimulation par la lumière rouge lointain de la synthèse de la δ-aminolévulinate déshydratase des cotylédons de radis

Stimulation of de novo synthesis of δ-aminolevulinate dehydralasc of radishes grown under far-red light .
Density labelling studies of δ-aminolevulinate dehydratase (ALAD) in cotyledons of radish ( Raphanus sativus L. cv. Longue Rave Saumonée) seedlings demonstrate that far-red light stimulates de novo synthesis of ALAD and that the turn-over of this enzyme is very poor. Cycloheximide reduces considerably both the increase of ALAD activity and the incorporation of deuterium in ALAD, which indicates that ALAD synthesis depends upon cytoplasmic ribosomes.     

Use of microcalorimetry for the characterization of marine metabolic activity at the water-sediment interface

Metabolic processes occurring at the sea-water-sediment interface were studied using a circulation flow microcalorimeter. A methodology was developed to characterize rapid and global changes in metabolism and energy flow, not easily detectable with reductionist approaches. Sea water was pumped continuously, 5–10 mm above the sediment, in experimental microcosms; a 100-μm filter prevented passage of meiofauna. This “circulating interface” was taken through the microcalorimeter and from there to an oxygen electrode, and was returned to the microcosm. The microcosms were experimentally eutrophicated using peptone (4 mg·ml ^?1). The relationship between heat production and oxygen tension in the circulating interface has been compared with ATP production, ¹⁴CO₂ and [¹⁴C]particulate matter turnovers. Initial heat steady-state production rises to a peak of 130 to 180 μW·ml^?1 in 6 to 8 h after peptone treatment. The microcalorimetric peak is closely correlated with ¹⁴CO₂ turnover and partially correlated with micro-events on the pO₂ curve. ATP concentration and particulate-¹⁴C turnover increase constantly and then stabilize, with the establishment of a new heat production steady state. The approach provides an indication of the temporal behaviour of complex mixtures of microorganisms and ciliates at the water-sediment interface, and gives holistic measurements of energy flow after induced perturbation (eutrophication) of the ecosystem. Although many problems remain to be solved in this field, it is shown here that flow microcalorimetric measurements can be used to monitor the effects of addition of reagents like pollutants and nutrients.     

Resistance to Disruption of Multilamellar Fragments of Central Nervous System Myelin

Abstract: Single-bilayer vesicles of myelin are desirable for studying myelin development and metabolism. Accordingly, our interest was drawn to a procedure for ves-iculating myelin (Steck et al., Biochim. Biophys. Acta 509, 397–408, 1978). We used X-ray diffraction analysis to examine these putative vesicle preparations because much larger amounts of material can be surveyed by this method than by electron microscopy. The sharpness (width) of the rings in the X-ray diffraction pattern varies inversely with the number of bilayers per multilayer structure. We therefore expected to see the diffuse diffraction pattern characteristic of single bilayers. Diffraction patterns were recorded from isolated rat brain myelin before and after the vesiculation procedure. Both patterns showed sharp rings, indicating numerous multilayered structures. Average values ranging from 7 to 10 bilayers per multilayer were calculated in both cases. This procedure did produce a small fraction of single-bilayer structures, which were isolated by differential centnfu gation; however, these accounted for only about 1% of the total myelin present. The diffraction pattern of this material showed the diffuse band typical of single-bilayer structures, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated it had the same protein composition as in normal myelin. Similar results were also obtained using either fresh or frozen bovine brain myelin. Variations of the published vesiculation procedure (incubation in 0.1 M NaCl or in buffers containing glycerol; disruption by sonication or use of a Tissumizer) also were not effective in breaking down the multilamellar fragments into thinner structures. We conclude that the multilamellar fragments of isolated CNS myelin resist disruption into single-bilayer structures.     

Leukotriene B3, leukotriene B4 and leukotriene B5; binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.