Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons

In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.     

Mapping of the plasmid (pLQ3) from Achromobacter and cloning of its cephalosporinase gene in Escherichia coli

We have constructed a physical map of the plasmid pLQ3 which was originally isolated from Achromobacter and which codes for a beta-lactamase. The enzyme specified by pLQ3 is expressed in Escherichia coli and is unusual in that it is a cephalosporinase, an enzyme usually coded for by chromosome. Plasmid pLQ3 is 12.4 kb in length and has a unique Bam HI site and two BglII sites. From a BamHI + BglII double digest of pLQ3, we have constructed a "shortened" plasmid, pLQ10, in which a 2.96-kb fragment is deleted. We have constructed a clone, pLQ22, in which a 3.27-kb fragment of pLQ3, carrying the beta-lactamase gene, is inserted into the BamHI site of pACYC184. By "comparative mapping" of single and multiple digests of each of these plasmids, we have been able to locate the cleavage sites for PstI, which makes seven cuts in pLQ3.     

Necrotic core in EMT6/Ro tumour spheroids: Is it caused by an ATP deficit?

Although commonly related to nutrient deprivation, the cause of the formation of the necrotic core in the multicellular tumour spheroids is still a controversial issue. We propose a simple model for the cell ATP production that assumes glucose and lactate as the only fuel substrates, and describes the main reactions occurring in the glycolytic and the oxidative pathways. Under the key assumption that cell death occurs when ATP production falls to a critical level, we formulate a multiscale model that integrates the energy metabolism at the cellular level with the diffusive transport of the metabolites in the spheroid mass. The model has been tested by predicting the measurements of the necrotic radius obtained by Freyer and Sutherland (1986a) in EMT6/Ro spheroids under different concentrations of glucose and oxygen in the culture medium. The results appear to be in agreement with the hypothesis that necrosis is caused by ATP deficit.     

Design and evaluation of xanthine based adenosine receptor antagonists: Potential hypoxia targeted immunotherapies

Molecular modeling techniques were applied to the design, synthesis and optimization of a new series of xanthine based adenosine A_2A receptor antagonists. The optimized lead compound was converted to a PEG derivative and a functional in vitro bioassay used to confirm efficacy. Additionally, the PEGylated version showed enhanced aqueous solubility and was inert to photoisomerization, a known limitation of existing antagonists of this class.     

Kinetic,thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37°C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37°C and the inhibition constants are 42.8 and 96.8 μM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27°C and uncompetitive at 37°C with inhibition constants of 56.4 and 30.0 μM, at respectively. The binding constant and enthalpy of binding, at 27°C are 45 μM, ? 64.5 kJ/mol and 61 μM, ? 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Adenosine Transport in HPRT Deficient Lymphocytes from Lesch‐Nyhan Disease Patients

We have analysed adenosine transport and [³H] NBTI binding in peripheral blood lymphocytes obtained from Lesch‐Nyhan patients, in basal conditions and following 24 h incubation with hypoxanthine. We found that adenosine transport and [³H] NBTI Binding were significantly decreased in PBL‐LN with respect to PBL‐C in basal conditions. Following 25 µM hypoxanthine incubation, adenosine transport is decreased in PBL‐LN with respect to basal transport, however, [³H] NBTI binding in PBL‐LN was not decreased following hypoxanthine incubation.     

AMPD1 C34T Mutation Selectively Affects AMP-Deaminase Activity in the Human Heart

Possession of the nonsense mutation in AMPD1 C34T gene has been linked to improved survival in patients with heart failure, possibly by promoting the formation of adenosine. This mutation is known to decrease the activity of AMP-deaminase in skeletal muscle. We have found that the AMPD1 mutation decreases the activity of AMP-deaminase in the heart without changing the activity of any other enzymes of adenine nucleotide metabolism. Protective mechanism of this mutation may be thus induced by local cardiac metabolic changes.     

Metabolism of Adenosine in Human Colorectal Tumour

The aim of this work is to analyse the activities of the enzymes metabolising adenosine in fragments of neoplastic and normal‐appearing mucosa, surrounding the tumour in 20 patients affected by colorectal cancer. The results show that the activities of the enzymes are markedly higher in tumour in comparison to normal mucosa to coope with the accelerated purine metabolism in cancerous tissues.     

Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin‐ and of pannexin‐injected oocytes; ArA also blocked K⁺‐induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye‐release and, evidently, ATP‐release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non‐metabolizable analog eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 621–631, 2013