Can nucleotides prevent Cu-induced oxidative damage?

Cu-induced oxidative damage is associated with cancer, diabetes, neurodegenerative and age related diseases. The quest for Cu-chelators as potential antioxidants spans the past decades. Yet, biocompatible Cu-chelators that do not alter the normal metal-ion homeostasis are still lacking. Here, we explored the potential of natural and synthetic nucleotides and inorganic phosphates as inhibitors of the Cu(I)/(II)-induced ()OH formation via either the Fenton or Haber-Weiss mechanisms. For this purpose, we studied by ESR the modulation of Cu-induced ()OH production, from the decomposition of H(2)O(2), by nucleotides and phosphates. ATP inhibited both Cu(I) and Cu(II) catalyzed reactions (IC(50) 0.11 and 0.04mM, respectively). Likewise, adenosine 5'-beta,gamma-methylene triphosphate (AMP-PCP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), ADP and tripolyphosphate were identified as good inhibitors. However, AMP and adenosine were poor inhibitors in the Cu(I)-H(2)O(2) system, IC(50) ca. 1.2mM, and radical enhancers in the Cu(II)-H(2)O(2) system. The best antioxidant was adenosine 5'-[beta,gamma-imino] triphosphate (AMP-PNP) (IC(50) 0.05mM at Cu(I)-H(2)O(2) system) which was 15 times more active than the known antioxidant Trolox. ATP and analogues inhibit Cu-induced ()OH formation through an ion chelation rather than a scavenging mechanism. Two phosphate groups are required for making active Fenton-reaction inhibitors. Nucleotides and phosphates triggered a biphasic modulation of the Haber-Weiss reaction, but a monophasic inhibition of the Fenton reaction. We conclude that nucleotides at sub mM concentrations can prevent Cu-induced OH radical formation from H(2)O(2), and hence may possibly prevent oxidative damage.     

S-Adenosyl-l-methionine activates the cardiac ryanodine receptor

S-Adenosyl-l-methionine (SAM) is the biological methyl-group donor for the enzymatic methylation of numerous substrates including proteins. SAM has been reported to activate smooth muscle derived ryanodine receptor calcium release channels. Therefore, we examined the effects of SAM on the cardiac isoform of the ryanodine receptor (RyR2). SAM increased cardiac sarcoplasmic reticulum [³H]ryanodine binding in a concentration-dependent manner by increasing the affinity of RyR2 for ryanodine. Activation occurred at physiologically relevant concentrations. SAM, which contains an adenosine moiety, enhanced ryanodine binding in the absence but not in the presence of an ATP analogue. S-Adenosyl-l-homocysteine (SAH) is the product of the loss of the methyl-group from SAM and inhibits methylation reactions. SAH did not activate RyR2 but did inhibit SAM-induced RyR2 activation. SAH did not alter adenine nucleotide activation of RyR2. These data suggest SAM activates RyR2 via a site that interacts with, but is distinct from, the adenine nucleotide binding site.     

Nucleotide-Dependent Isomerization of Glutamate Dehydrogenase in Relation to Total RNA Contents of Peanut

The physiological function of glutamate dehydrogenase (GDH) was investigated by treating germinating peanut (Arachis hypogaea L.) seeds with nucleoside triphosphate (NTP) solutions in order to alter the isoenzyme distribution patterns. The free nucleosides and nucleotides of the GTP-treated peanut were the highest [8.7 μmol g⁻¹(f.m.)], and they decreased through the ATP-treated peanut [5.8 μmol g⁻¹(f.m.)], and CTP-treated peanut [5.5 μmol g⁻¹(f.m.)], to the UTP-treated peanut [4.1 μmol g⁻¹(f.m.)]. The combination of 4 NTPs induced 20 % higher content of Pi [173 nmol g⁻¹(f.m.)] than in the control, but the combined ATP+UTP treatment induced the lowest (93.0 nmol g⁻¹(f.m.)] Pi. The 4 NTP treatment also induced the highest number of GDH isoenzymes (28) followed by the purine NTP treatments (15 to 20), but the pyrimidine NTP treatments and the combined purine + pyrimidine NTP treatments induced the lowest numbers (<15) of isoenzymes. The deamination/amination ratios were generally higher in the UTP (0.11), and CTP (0.06) treated peanuts than in the GTP (0.04), and ATP (0.07) treated peanuts. There were mutual relationships between higher numbers of GDH isoenzymes present in the GTP-, and ATP-treated peanuts and higher RNA (236.5 and 239.4 μg g⁻¹, respectively) contents on one hand, and between the lower numbers of isoenzymes in the CTP-, and UTP-treated peanuts and lower RNA (162.0 and 152.5 μg g⁻¹, respectively) contents. The recurrent relationships of the effects of the NTP treatments of peanut were UTP > ATP > CTP > GTP. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human bone cell cultures: a new model for studying the mechanism of action of calcitonin

An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells. Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol. These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor.     

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.     

Odorant-induced kinetics of Ca<Superscript>2+</Superscript>, NADH,and oxidized flavoproteins in frog olfactory mucosa

A study was made of the odorant-induced changes in the fluorescence of the Ca²⁺-chlortetracycline-membrane complex, NADH, and oxidized flavoproteins in the frog olfactory epithelium. Cineole and vanillin induce faster changes than camphor and pentanol. The different kinetics of NADH and membrane calcium evoked by these odorants are attributed to the heterogeneity of the molecular mechanisms involved in olfactory signal transduction. By contrast, ammonia and β-mercaptoethanol permeate the olfactory cells and without second messengers inhibit the mitochondrial respiratory chain and suppress the motility of olfactory cilia.     

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06–0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24–128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6–10% of UTP left, and 12–36% CTP), followed by DCL and PF, which were almost similar (6–16% UTP and 12–27% CTP), while PALA was the least active compound (10–70% UTP and 13–68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31–72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.