A flow‐injection chemiluminescent method for the evaluation of the antioxidant activity of 5′‐nucleotides

In the present work, a simple chemiluminescence (CL) method coupled with flow‐injection analysis for the evaluation of antioxidant activity of 5′‐nucleotides (5′‐AMP, 5′‐CMP, 5′‐GMP, 5′‐UMP) was proposed. It is based on inhibition effect of the studied substances on CL emission of luminol–potassium ferricyanide–pyrogallol. Experiments were performed to evaluate the nature of the inhibition by 5′‐nucleotides of the CL reaction and their antioxidant activities. Based on the experimental results, it was observed that 5′‐nucleotides are available antioxidants that could effectively scavenge superoxide anion free radicals in a concentration‐dependent way. This will provide a basis for further development of the use of nucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

The purine metabolism of human erythrocytes

This review summarizes currently available information about a crucial part of erythrocyte metabolism, that is, purine nucleotide conversions and their relationships with other conversion pathways. We describe the cellular resynthesis, interconversion, and degradation of purine compounds, and also the regulatory mechanisms in the conversion pathways. We also mention purine metabolism disorders and their clinical consequences. The literature is fragmentary because studies have concentrated only on selected aspects of purine metabolism; hence the need for a synthetic approach. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 581–591.     

Purification and Some Characteristics of an ACTH-Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane-bound protein (B-50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X-100 in 75 mM-KCl. Using column chromatography over DEAE-cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B-50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B-50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B-50 after 60 min. The inhibition by ACTH_{1_24} of the B-50 protein kinase in ASP 55–80 was dose-dependent; the half-maximal effective concentration was 5 × 10⁻⁶ M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor-phins had no effect on the B-50 protein kinase. The B-50 protein kinase required both magnesium and calcium for optimal activity. Using two-dimensional electrophoresis on polyacrylamide slab gels the B-50 protein kinase and the B-50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B-50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B-50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B-50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.     

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.     

Presynaptic cGMP sets synaptic strength in the striatum and is important for motor learning

The striatum is a subcortical brain region responsible for the initiation and termination of voluntary movements. Striatal spiny projection neurons receive major excitatory synaptic input from neocortex and thalamus, and cyclic nucleotides have long been known to play important roles in striatal function. Yet, the precise mechanism of action is unclear. Here, we combine optogenetic stimulation, 2‐photon imaging, and genetically encoded scavengers to dissect the regulation of striatal synapses in mice. Our data show that excitatory striatal inputs are tonically depressed by phosphodiesterases (PDEs), in particular PDE1. Blocking PDE activity boosts presynaptic calcium entry and glutamate release, leading to strongly increased synaptic transmission. Although PDE1 degrades both cAMP and cGMP, we uncover that the concentration of cGMP, not cAMP, controls the gain of striatal inputs. Disturbing this gain control mechanism in vivo impairs motor skill learning in mice. The tight dependence of striatal excitatory synapses on PDE1 and cGMP offers a new perspective on the molecular mechanisms regulating striatal activity.     

Glutathione depletion associated with rose bengal-photosensitized mortality in the housefly,Musca domestica

Glutathione, pyridine nucleotides, and lipid peroxides were measured in adult houseflies following various regimens of dye treatment and light exposure. Comparisons were made between dark control and light control flies to judge the effect of light exposure alone; between dark control and dark, dye-treated flies to evaluate the effects of dye-feeding in the dark; and between dark, dye-treated and light, dye-treated flies to measure the effect of photodynamic action. No significant effect was observed in levels of NAD⁺, NADH, or NADP⁺. However, a decrease (～ 16.7%) in NADPH during photodynamic treatment was measured. Relatively small inductions of glutathione were observed in light controls and dark, dye-treated flies. Depletion of both GSH and total glutathione (the sum of GSH and GSSG, expressed as GSH equivalents) occurred in light, dye-treated flies as compared to dark, dye-treated flies. Depletion of NADPH, when related to GSH depletion, suggested that GSH is being utilized to conjugate some products of photooxidation or that it is being directly oxidized to GSSG. However, the observation of a reduction in total glutathione also suggests that a fraction of GSH is being either oxidized to a product other than GSSG or irreversibly conjugated. No significant effects from photodynamic treatment on peroxidative potential or lipid hydroperoxides were observed.     

Regulation of glycolysis by adenosine diphosphate in Pisum sativum

The role of ADP in controlling glycolysis has been examined in a soluble extract of germinating pea seeds. A shortage of ADP appears to retard glycolysis principally by restricting the conversion of phosphopyruvate to pyruvate rather than by restricting formation of phosphoglycerate. Upon addition of ADP to the extract there is an immediate decrease in the concentration of phosphopyruvate accompanied by an increase in pyruvate. Apparently the pyruvate-kinase step shows the most marked response to fluctuations in ADP availability. The glycolytic response to ADP depends on the concentration of ATP magnesium ions. The relation of magnesium-ion availability to adenine-nucleotide control of glycolysis is discussed.     

Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi) in the rhesus monkey

Webster H. K., Haut M. J., Martin L. K. and Hildebrandt P. K. 1982. Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi in the rhesus monkey. International Journal for Parasitology12: 75–79. Blood levels of purine and pyrimidine nucleotides were determined during synchronous infection by Plasmodium knowlesi in rhesus monkeys. Infected monkeys followed over 2–3 intraerythrocytic cycles showed variations in nucleotide pool levels characteristic of the predominant schizogonic growth stage. These changes in nucleotide levels as described for ‘ring’-stage, trophozoite growth and schizogony indicate a cyclically varying relationship between nucleotide concentrations and a specific stage of parasite development during the blood-phase of malaria infection.     

Ca2+ - and Nitric Oxide-Dependent Stimulation of Cyclic GMP Synthesis in Neuronal Cell Line Induced by P2-Purinergic/Pyrimidinergic Receptor

Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ～1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′-(γ-thio)triphosphate > UTP = 2-methylthio-ATP > ADP ? CTP, AMP, α,β-methylene-ATP, 2′- and 3′-O-(4-benzoylbenzoyl)ATP. The EC₅₀ of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P_2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC₅₀ = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (IC₅₀ = 20–30 µM). Pretreatment of cells with the Ca²⁺ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca²⁺ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC₅₀ for nitroarginine = 1 µM) or by hemoglobin (IC₅₀ = 2 µM). This indicates that ATP/UTP via a P₂-receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca²⁺ level, mostly due to release of Ca²⁺ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross-desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ～40% only. This indicates a heterologous cross-desensitization predominantly in one direction (bradykinin ? ATP).