Halobacterial megaplasmids are negatively supercoiled

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal-field alternating gel electro-phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

<Superscript>15</Superscript>N natural abundance of fossil peat reflects the influence of animal-derived nitrogen on vegetation

'¹⁵N signatures of fossil peat were used to interpret past ecosystem processes on tectonically active subantarctic Macquarie Island. By comparing past vegetation reconstructed from the fossil record with present-day vegetation analogues, our evidence strongly suggests that changes in the '¹⁵N signatures of fossil peat at this location reflect mainly past changes in the proportion of plant nitrogen derived from animal sources. Associated with uplift above sea level over the past 8,500 years, fossil records in two peat deposits on the island chronicle a change from coastal vegetation with fur and elephant seal disturbance to the existing inland herbfield. Coupled with this change are synchronous changes in the '¹⁵N signatures of peat layers. At two sites ¹⁵N-enriched peat '¹⁵N signatures of up to +17‰ were associated with a high abundance of pollen of the nitrophile Callitriche antarctica (Callitrichaceae). At one site fossil seal hair was also associated with enriched peat '¹⁵N. Less ¹⁵N enriched '¹⁵N signatures (e.g. -1.9‰ to +3.9‰) were measured in peat layers which lacked animal associated C. antarctica and Acaena spp. Interpretation of a third peat profile indicates continual occupation of a ridge site by burrowing petrels for most of the Holocene. We suggest that ¹⁵N signatures of fossil peat remained relatively stable with time once deposited, providing a significant new tool for interpreting the palaeoecology.     

Role of Asp-9 and Glu-36 in the active site of the pneumococcal CPL1 lysozyme: an evolutionary perspective of lysozyme mechanism.

The role of carboxylic amino acids Asp-9 and Glu-36 in the activity of CPL1 lysozyme was investigated by site-directed mutagenesis. The enzymatic activity of the single mutants D9E, D9N, D9H, D9K, D9A, E36D, E36Q, E36K, and E36A and of the double mutant D9A-E36A was analyzed using a highly sensitive radioactive assay. All mutants but D6K showed detectable activities. Interestingly, the mutants E36D and E36Q retained 67% and 37% activity, respectively. Amino acid replacements at position 9 turned out to be more critical for activity than at position 36. In analogy to the mechanism described for hen egg-white lysozyme, where the proton donor play a central role, we propose that, in the CPL1 lysozyme, Asp-9 might act as the proton donor for activation of the substrate, and Glu-36 could help in the stabilization of the intermediate oxocarbocation. The residual activity of lysozyme mutants lacking one or two of the acidic amino acids may be explained by the participation of a water molecule as proton donor and/or to electrostatic contributions in the active center stabilizing the transition state of the reaction. Our results are in agreement with the hypothesis that enzymes have been optimized during evolution from an ancestral protein able to bind more tightly the transition state of the substrate than the substrate itself, by the acquisition of amino acids serving a function in catalysis.