Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

Dissolved oxygen and fish behavior

Synopsis This essay reviews the behavioral responses of fish to reduced levels of dissolved oxygen from the perspective of optimization theory as used in contemporary behavioral ecology. A consideration of oxygen as a resource suggests that net oxygen gain per unit of energy expenditure will be the most useful currency for ecological models of breathing. In the process of oxygen uptake, fish always expend energy on perfusion, usually on ventilation and often on locomotion. These costs, and the risk of predation, will vary with oxygen availability and the type of behavioral response shown. The principal categories of behavioral response to reduced external availability of dissolved oxygen are (1) changes in activity, (2) increased use of air breathing, (3) increased use of aquatic surface respiration, and (4) vertical or horizontal habitat changes. Fish should choose whichever combination of responses minimizes the costs of meeting their oxygen demands. A small number of studies provides qualitative support for this prediction.     

Structure of two new aminophospholipids from Methanobacterium thermoautotrophicum.

The methanogenic bacterium Methanobacterium thermoautotrophicum (A.T.C.C. 29183) was shown to contain two new aminophospholipids. These are 2-aminoethyl phosphate ester of diphytanylglycerol diether and a sugar containing bisdiphytanyldiglycerol tetraether. The two aminophospholipids were stable to acid methanolysis except for the sugar on the bisdiphytanyldiglycerol tetraether. Strong acid (6 M-HCl) hydrolysed the alkyl ether and aminophosphate ester bonds. The structure of the phosphate linkage was demonstrated by 31P n.m.r., and the 2-ethanolamine structure was elucidated by 1H- and 13C-n.m.r. spectroscopy and by fast-atom-bombardment m.s.     

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.     

Spin-trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production

The spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to investigate oxy-radical production in post-ischemic rat hearts previously exposed to 20, 30, or 40 minutes of global ischemia. A hydroxyl spin adduct (DMPO-OH) was identified in coronary effluent during the initial seconds of reperfusion by Electron Spin Resonance (ESR) Spectroscopy. The intensity of the ESR signal in post-ischemic effluent increased as ischemic duration was prolonged; however, regardless of the duration of ischemia, maximal spin adduct detection occurred 3 minutes after initiation of reperfusion. Superoxide dismutase inhibited the formation of DMPO-OH, suggesting that superoxide anion was initially generated and is the principle source for the production on the hydroxyl adduct. Our investigations indicate that superoxide anion is produced during the early moments of reperfusion and that its production in the post-ischemic heart is related to the severity of ischemia.     

Template-determined,variable rate of RNA chain elongation

Qβ replicase polymerizes MDV-1 RNA at a markedly variable rate. Electrophoretic analyses of partially synthesized strands showed that a few of the elongation intermediates are much more abundant than others, reflecting a variable rate of chain elongation. Our data suggest that at a relatively small number of specific sites in the sequence of this RNA, the progress of the replicase is temporarily interrupted, and then resumes spontaneously, with a finite probability. Since the time spent between these pause sites is negligible compared with the time spent at pause sites, the mean time of chain elongation is well approximated by the sum of the mean times spent at each pause site.Nucleotide sequence analysis of the most prominent elongation intermediates indicated that they all have the potential to form a 3′ terminal hairpin structure. This suggests that the marked variability in the rate of chain elongation is due to the formation of terminal hairpins in the product strand, or the reformation of hairpins in the template strand. A survey of the literature shows that this phenomenon occurs with most, if not all, nucleic acid polymerases. Structure-induced pauses may play a role in the regulation of nucleic acid synthesis.     

Purification and characterization of mouse glucose 6-phosphate dehydrogenase

Summary Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP-and -2, 5-ADP-Sepharose columns and biospecific elution with NADP⁺ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD⁺ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP⁺ were determined to be 50 m and 10 m respectively. NADPH is a competitive inhibitor of NADP⁺ and has a K_i of 18 µm. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig.