Reciprocal potentiation of the antitumoral activities of FK866, an inhibitor of nicotinamide phosphoribosyltransferase, and etoposide or cisplatin in neuroblastoma cells.

NAD is an essential coenzyme involved in numerous metabolic pathways. Its principal role is in redox reactions, and as such it is not heavily "consumed" by cells. Yet a number of signaling pathways that bring about its consumption have recently emerged. This has brought about the hypothesis that the enzymes that lead to its biosynthesis may be targets for anticancer therapy. In particular, inhibition of the enzyme nicotinamide phosphoribosyl transferase has been shown to be an effective treatment in a number of preclinical studies, and two lead molecules [N-[4-(1-benzoyl-4-piperidinyl)butyl]-3-(3-pyridinyl)-2E-propenamide (FK866) and (E)-1-[6-(4-chlorophenoxy)hexyl]-2-cyano-3-(pyridin-4-yl)guanidine (CHS 828)] have now entered preclinical trials. Yet, the full potential of these drugs is still unclear. In the present study we have investigated the role of FK866 in neuroblastoma cell lines. We now confirm that FK866 alone in neuroblastoma cells induces autophagy, and its effects are potentiated by chloroquine and antagonized by 3-methyladenine or by down-regulating autophagy-related protein 7. Autophagy, in this model, seems to be crucial for FK866-induced cell death. On the other hand, a striking potentiation of the effects of cisplatin and etoposide is given by cotreatment of cells with ineffective concentrations of FK866 (1 nM). The effect of etoposide on DNA damage is potentiated by FK866 treatment, whereas the effect of FK866 on cytosolic NAD depletion is potentiated by etoposide. Even more strikingly, cotreatment with etoposide/cisplatin and FK866 unmasks an effect on mitochondrial NAD depletion.

Nicotinamide phosphoribosyltransferase­related signaling pathway in early Alzheimer's disease mouse models.

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system that is characterized by progressive cognitive dysfunction and which ultimately leads to dementia. Studies have shown that energy dysmetabolism contributes significantly to the pathogenesis of a variety of aging­associated diseases and degenerative diseases of the nervous system, including AD. One focus of research thus has been how to regulate the expression of nicotinamide phosphoribosyltransferase (NAMPT) to prevent against neurodegenerative diseases. Therefore, the present study used 6­month­old APPswe/PS1ΔE9 (APP/PS1) transgenic mice as early AD mouse models and sought to evaluate nicotinamide adenine dinucleotide (NAD+) and FK866 (a NAMPT inhibitor) treatment in APP/PS1 mice to study NAMPT dysmetabolism in the process of AD and elucidate the underlying mechanisms. As a result of this treatment, the expression of NAMPT decreased, the synthesis of ATP and NAD+ became insufficient and the NAD+/NADH ratio was reduced. The administration of NAD+ alleviated the spatial learning and memory of APP/PS1 mice and reduced senile plaques. Administration of NAD+ may also increase the expression of the key protein NAMPT and its related protein sirtuin 1 as well as the synthesis of NAD+. Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.

Delayed haemolytic activity by the freshwater puffer Tetraodon sp. toxin.

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

Interaction of palytoxin with red cells: structure-function studies.

Palytoxin (PTX), a potent toxin isolated from the marine soft coral Palythoa tuberculosa increases the cationic permeability of red cell membranes and inhibits the (Na+,K+)-activated ATPase, effects that are completely reversed by ouabain. It has also been shown to compete with ouabain for a binding site and it has been suggested that it binds to the Na+,K(+)-pump molecule in cells. In a search for analogues of PTX that would bind covalently and could thus be used to identify and characterize the binding site, we have used compounds which differed from PTX at one end or at both ends simultaneously. In order to determine whether these derivatives could be used to replace palytoxin, we tested their potency to induce an increased cation flux, complete with ouabain for its binding site, and inhibit the isolated, purified (Na+,K(+)-ATPase). The results obtained indicate that departures from the PTX structure at one end or at both ends simultaneously substantially decrease the ability of the compounds to increase the cationic permeability of red blood cells and to inhibit the (Na+,K(+)-ATPase). These actions were found to be completely reversed by ouabain, but the analogues are two to three orders of magnitude less potent than PTX in competing with ouabain for its binding site. These results suggest that both ends of the palytoxin molecule participate in the interactions of the toxin with its receptor and that modifications in these regions of the molecule produce significant alterations in its binding and subsequent action on red cell membranes.

A simple, sensitive method for detecting early peripheral nerve dysfunction in the rat following acrylamide treatment.

The "ocular zingerone test" was employed to detect early alterations in peripheral nerve function which were associated with acrylamide intoxication in the rat. Acrylamide treatment resulted in a dose-related prolongation of the behavioral response to ocular zingerone. Significant alterations in the zingerone response occurred prior to detectable alterations in peripheral sensory and motor nerve function. Acrylamide-induced prolongation of the zingerone response appears to be the result of functional alterations in cholinergic neurotransmission in the autonomic nervous system. It is concluded that the "ocular zingerone test" is a simple, sensitive technique with which the degree of acrylamide intoxication may be quantified in the rat.

Evaluation of the ability of d-penicillamine to protect rats against the neurotoxicity induced by zinc pyridinethione, acrylamide, 2,5-hexanedione and p-bromophenylacetylurea.

Dietary exposure of rats to three different concentrations of zinc pyridinethione (ZPT; 166, 332, 498 ppm) caused delayed onset failure in a treadmill test and, at the higher concentrations (332 and 498 ppm), death. Daily treatment with d-penicillamine (d-PEN) increased the latency period for treadmill failure and lethality. Comparable levels of toxicity were achieved only after d-PEN treated rats had consumed 2-3 times more ZPT than rats not treated with d-PEN. In contrast to ZPT, administration of d-PEN did not affect the onset of treadmill failure associated with acrylamide, p-bromophenylacetylurea or 2,5-hexanedione. Thus, d-PEN provided protection which was selective for ZPT.

Glutamate and aspartate do not modify the ataxic gait of acrylamide treated animals.

The purpose of this experiment was to examine the effects of intraventricular injections of glutamate and aspartate on the gait of animals rendered ataxic by the administration of acrylamide. Contrary to their previously reported corrective influence on ataxia induced by 3-acetyl pyridine, these amino acids did not modify the ataxic gait of acrylamide treated animals. This suggests that glutamate and aspartate can act in cerebellar but not in peripheral types of ataxia in animals.

Effect of transient receptor potential vanilloid 1 (TRPV1) receptor antagonist compounds SB705498, BCTC and AMG9810 in rat models of thermal hyperalgesia measured with an increasing-temperature water bath.

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.

Effect of oral CDP-choline on acrylamide-induced lesion.

Administration of low doses of cytidine diphosphate choline (CDP-choline, citicoline, Somazina) (50 mg/kg) to mice and rats has proved to be effective in preventing acrylamide-induced neurological syndrome. Likewise, the simultaneous dosing of both drugs, which causes an evident loss of weight in the animals, has demonstrated that an activation on the dopaminergic system is produced.

Acrylamide induced immunosuppression in rats and its modulation by 6-MFA, an interferon inducer.

In the present communication, we describe acrylamide (ACR) induced immunotoxicity and its modulation by an interferon inducer, the 6th mycelial fraction acetone (6-MFA) of Aspergillus ochraceus ATCC 28706. ACR administration to rats produced a significant decrease in the weight of spleen (p < 0.001), thymus (p < 0.001) and mesenteric lymph nodes (p < 0.05). A decrease in cellularity of spleen (p < 0.001), thymus (p < 0.001), bone marrow (p < 0.001) and circulating blood lymphocyte population (p < 0.001) was also recorded. ACR suppressed the humoral as well as cell mediated immunity as assessed by erythrocyte antibody complement (EAC)-rosettes (p < 0.001), hemagglutination titre (p < 0.001), PFC (p < 0.001) and the delayed type hypersensitivity response against sheep red blood cells (SRBC, p < 0.001). ACR treated immunosuppressed rats when treated with 6-MFA restored the circulating lymphocyte number to the normal level and a partial recovery in the weight of spleen and thymus. Potentiation of EAC-rosettes, hemagglutination titre, IgM-PFC and DTH response against SRBC was observed. It is concluded that 6-MFA ameliorate the ACR induced toxicity. This study may be of significance in prevention of ACR toxicity.

Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover.

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.