Release of extracellular purines from plant roots and effect on ion fluxes

Extracellular purine nucleotides appear capable of regulating plant development, defence and stress responses by acting in part as agonists of plasma membrane calcium channels. Factors stimulating ATP release include wounding, osmotic stress and elicitors. Here we show that exogenous abscisic acid and L-glutamate can also cause ATP accumulation around Arabidopsis thaliana roots. Release of ADP from root epidermis would trigger ionotropic receptor-like activity in the plasma membrane, resulting in transient elevation of cytosolic free calcium. Root epidermal protoplasts (expressing aequorin as a cytosolic free calcium reporter) can support an extracellular ADP-induced cytosolic calcium elevation in the presence of an extracellular reductant. This confirms that ADP could elicit calcium-based responses distinct to those of ATP, which have been shown previously to involve production of extracellular reactive oxygen species.     

Characterisation of mitochondrial fractions isolated from four genotypes of wheat coleoptiles

Mitochondrial fractions were isolated at various stages of germination from wheat coleoptiles of 4 different genotypes: WRH, Capitole, male-sterile Capitole (MSC) and F₁ hybrid obtained by crossing MSC with WRH. The ADP: O values were the highest in F₁ and in MSC and decreased during germination. The oxygen consumption was weaker in F₁ and in WRH than in the other genotypes. Quantitative differences in polypeptides exist between the different genotypes. On germination, increase in the polypeptides of high MW was more rapid in F₁ than in the other genotypes. The fatty acids of the mitochondria of F₁ and MSC are characterized by an abundance of linoleic acid. The content of this fatty acid decreased during germination more rapidly in F₁ and in MSC than in the others. The cytoplasm of Triticum timopheevi has a marked influence on mitochondria of MSC and F₁. This cytoplasm confers better oxidative properties, which can be correlated with changes in their biochemical constituents. However the F₁ hybrid differs from the other genotypes in many biochemical features.     

The effect of adenine nucleotides on the rate and fate of glutamine utilization by incubated mitochondria isolated from rat mesenteric lymph nodes

Summary The rate of glutamine utilization by isolated lymphocyte mitochondria is 21.4 nmol/min per mg protein, of which 72% is converted to glutamate. Addition of ATP, ADP or AMP increased the rate of glutamine utilization by 60%. Evidence is presented that this is due to a stimulation of oxoglutarate dehydrogenase activity by ADP: this may account for the stimulation of glutamine utilization by concanavalin A in lymphocyte.     

Feeding behaviour in response to blood fractions and chemical phagostimulants in the black-fly, Simulium venustum

ABSTRACT. Wild-caught black-flies ( Simulium venustum Say complex) were presented with diets at 37°C in an artificial feeding apparatus. Washed human red cells resuspended 1:1 in Ringer solution were potent phagostimulants, causing 89% of flies to gorge. Whole plasma was more potent (32% gorging) than platelet-poor plasma (2%). The ED₅₀ for red cells was 3.5%. Although ADP, contained in high-concentration in platelets, was confirmed as a more potent phagostimulant than ATP (ED₅₀ of 5πM V. 12μM), red cells were clearly more phagostimulatory than platelets, and with a potency more than adequate to trigger gorging in vivo. A high response to the ATP analogues, β, γ-methylene ATP and adenine phosphosulphate, supports the view that the phosphate chain is relatively unimportant in determining nucleotide potency to simuliids. The compounds phytic acid and 2,3-disphosphoglycerate, potent stimulants to Rhodnius prolixus , produced only moderate and no response, respectively at 1 mM; 5-hydroxytryptamine, another major constituent of platelets, also produced only a moderate response. Only flies caught while showing a characteristic probing behaviour would subsequently probe and feed in vitro; this 'blood-feeding mode' rapidly disappeared in the absence of stimuli eliciting actual probing, but for flies in this state a small temperature rise was sufficient stimulus for probing.     

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux (“ATP paradoxon”). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.     

Monitoring ADP/ATP ratio in yeast cells using the fluorescent-protein reporter PercevalHR

PercevalHR (Perceval High Resolution) is an artificially designed fluorescent protein, which changes its excitation spectrum based on the ADP/ATP ratio of the environment. Here we demonstrated that PercevalHR can be used for monitoring energy status of Saccharomyces cerevisiae cells, which are affected by diauxic shift and mitochondria inhibition, in a non-invasive and non-destructive manner.     

The metabolic integrity of Fasciola hepatica during in vitro maintenance

Levels of metabolic intermediates and end products in F. hepatica after 24 and 48 h in Hédon-Fleig salt solution with added glucose were compared with levels obtained immediately on removal from the host. Glycogen levels dropped initially, probably due to the expulsion of eggs; thereafter they remained constant. Internal glucose concentrations increased as the parasites equilibrated with the medium. Other changes in internal pool sizes were consistent with regulation to the in vitro conditions. ATP levels increased; ATP/ADP ratios were maintained. Comparisons of mass action ratios and equilibrium constants suggest that hexokinase, pyruvate kinase and phosphofructokinase are regulatory. Output of excretory products approached linearity; from the calculated regressions the proportions of lactate, acetate and propionate were 1: 2: 4. The implications for metabolic regulation in F. hepatica are briefly discussed, and it is concluded that, for at least 48 h in vitro, energy metabolism is not adversely affected.     

The Effect of 3-Aminobenzamide on Thymidine Incorporation in Ultra-Violet Irradiated Chick Pigment Epithelium Cells

The addition of 3-aminobenzamide (3-AB) to cultures of chick embryo pigmented epithelium rescues these cells after high doses of ultraviolet treatment. The addition of 3-AB prevents cells from losing pre-formed protein and DNA and stimulates thymidine incorporation by the cells after ultraviolet irradiation. Since 3-AB is an inhibitor of poly (ADP) ribosylation, these observations support the conclusion that death of these cells after ultra-violet irradiation depends upon poly (ADP) ribosylation and may be an apoptotic response.     

RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1

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