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C Moncada

Publications and source records attributed to C Moncada.

15 recordsLinked to original sources
PubMed2003

Mutations in mitochondrial aldehyde dehydrogenase (ALDH2) change cofactor affinity and segregate with voluntary alcohol consumption in rats.

Genetic factors influence alcohol consumption and alcoholism. A number of groups have bred alcohol drinker and non drinker rat strains, but genetic determinants remain unknown. The University of Chile rat lines UChA (low drinkers) and UChB (high drinkers) display differences in the relative K(m) for NAD+ of mitochondrial aldehyde dehydrogenase (ALDH2) but no V(max) differences. The relative K(m) differences may be due to mitochondrial changes or to genetic differences coding for ALDH2. We investigated whether there are differences in the coding regions of ALDH2 cDNA in these lines and whether the Aldh2 genotype predicts the phenotype of alcohol consumption and the K(m) of ALDH2 for NAD+. Liver cDNA was prepared, and the Aldh2 transcript was amplified, cloned and sequenced. Genotyping was conducted by DNA amplification and restriction enzyme digestion. When compared to Aldh21 of Sprague-Dawley, 94% of the UChA (low drinker) rats (n = 61), presented a mutation that changes Gln67 to Arg in the mature enzyme (allele referred to as Aldh22). In UChB (high drinker) rats (n = 69), 58% presented the Aldh21 allele, while 42% presented the Gln67Arg change plus a second mutation that changed Glu479 to Lys (allele Aldh23). The Aldh22 allele was absent in high drinker rats. Rats of different Aldh2 genotypes displayed marked phenotypic differences in both ethanol consumption (g/kg/day; means +/- SE): (Aldh21/Aldh21) = 5.7 +/- 0.2, (Aldh22/Aldh22) = 0.9 +/- 0.2 and (Aldh23/Aldh23) = 4.6 +/- 0.2; and K(m)s for NAD+ of 43 +/- 3 microm, 132 +/- 13 microm and 41 +/- 2 microm, respectively (Aldh22 versus Aldh21 or Aldh23; P < 0.0001 for both phenotypes). Overall, the data show that alleles of Aldh2 strongly segregate with the phenotype of ethanol consumption and the relative K(m) for NAD+ of ALDH2. Bases mutated suggest that non drinker Aldh22 is ancestral with regard to the coding changes in either Aldh21 or Aldh23, variants which would allow ethanol consumption and may provide an evolutionary advantage by promoting calorie intake from fermented products along with carbohydrates.

Aldehyde Dehydrogenase↗
PubMed2001

[Carbohydrate deficient transferrin, gammaglutamyl transferase and mean corpuscular volume in the evaluation of recent alcohol intake in excessive drinkers].

BACKGROUND: There are no reliable markers to detect heavy drinking or as a tool to control abstinence compliance in alcoholic treatments. The Mean Corpuscular Volume (MCV), and the gammaglutamyl transpeptidase (GGT), are widely used although their predictive value is somewhat limited due to their low specificity. On the other hand, the Carbohydrate-deficient transferrin (CDT) described in the eighties is highly specific and would be of value in early detection of problem drinking. AIM: To compare the sensitivity and specificity of CDT, GGT, and MCV in order to evaluate their single and combined use as markers for detection of heavy drinking behaviour. PATIENTS AND METHODS: CDT, GGT, and MCV values were determined in blood samples from (a) alcoholics (drinking more than 100 9 alcohol/day; n = 47) and (b) healthy volunteers, teetotalers from the Church of Saints of Later Days (n = 34). At the time of sampling alcoholics were presently drinking or had been abstinent for no more than six weeks. ROC curves were used to determine the best cut-off point for each marker. RESULTS: Sensitivity was found to be similar for all three markers. Specificity was found higher for GGT (90.9%) and CDT (91.0%). The combined use of MCV, GGT and CDT, that is, when at least one of the markers is altered, was shown to detect 83% of the patients. No correlation was observed between the markers and the level of alcohol intake. CONCLUSIONS: CDT could be of value as a marker to detect heavy drinking when used with GGT and MCV values combined. CDT is particularly higher in drinking alcoholics and remains significantly high for at least six weeks after they stop drinking.

Adult↗
PubMed2001

Protein binding of alpha-hydroxyethyl free radicals.

BACKGROUND: The auto-oxidation of ethanol is likely to proceed via the initial formation of hydroxyethyl radicals (HERs), the one-electron oxidation product. In the laboratory, HERs can be generated by the Fenton reaction (H2O2+ Fe+2) in the presence of ethanol. We report studies on the binding of HERs to serum albumin, generated under Fenton and non-Fenton conditions. METHODS: The generation of HER was determined by electron paramagnetic resonance spectroscopy. The formation of ethanol-derived protein adducts was determined by 14C-ethanol incorporation into serum albumin and by the binding of antibodies raised against HER adducts. RESULTS: We report that serum albumin, used as a model protein, is an effective trapping agent of HERs. In addition, HER radicals covalently bind to albumin to form acid stable adducts. Unexpectedly, we found that under aerobic conditions, the incubation of 50 mM ethanol and phosphate buffer (which contains iron traces) in the absence of the Fenton reagent yields HER radicals as shown by electron paramagnetic resonance spectroscopy and the formation of acid stable protein adducts that are recognized by antibodies raised against HER radical adducts. CONCLUSIONS: Proteins (serum albumin used as a model) are avid trapping agents of HER. There are minimal requirements for the generation of HER, because in the presence of oxygen and a phosphate buffer that contains traces of iron, ethanol readily generates HERs. Thus, HER production is likely to occur in many tissues. The ability of proteins to bind this ethanol radical should be valuable in the diagnosis of alcohol abuse and may be relevant to some of the chronic effects of ethanol.

Animals↗
PubMed1999

Generation of acetate and production of ethyl-lysine in the reaction of acetaldehyde plus serum albumin.

We report that incubation of acetaldehyde with bovine serum albumin results in the generation of acetate in a reaction that is directly proportional to the levels of albumin and exponentially dependent on the concentration of acetaldehyde. Both reactants need to be present for acetate to be formed. The oxidation of acetaldehyde into acetate requires that a reduced product also be generated in the reaction. It was hypothesized that, at high concentrations, acetaldehyde itself may reduce the Schiff bases formed in the reaction of a second molecule of acetaldehyde with amino groups in the protein, resulting in the generation of ethyl-lysine moieties. Incubation of acetaldehyde (240 mM) with bovine serum albumin was found to generate ethyl-lysine moieties as determined by a specific monoclonal antibody. Immunization of rabbits with products of the reaction of bovine serum albumin with acetaldehyde led to the generation of antibodies that reacted to reduced adducts formed in the reaction of acetaldehyde and proteins in the presence of sodium cyanoborohydride. However, the generation of acetate from acetaldehyde plus albumin was 60-fold greater than could be explained by the reduction of Schiff bases, as determined by the maximal incorporation of [14C]-acetaldehyde into an acid-precipitable protein fraction. Thus, other mechanisms to generate acetate also occur. The present findings provide an explanation for earlier reports that acetaldehyde adducts formed under "nonreducing" conditions generate antibodies that recognize reduced acetaldehyde protein adducts. However, the mechanism by which the bulk of acetate is generated in the reaction of acetaldehyde and bovine serum albumin remains to be elucidated.

Acetaldehyde↗
PubMed1996

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics.

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.

Adult↗
PubMed1994

Effect of the non-NMDA receptor antagonist GYKI 52466 on the microdialysate and tissue concentrations of amino acids following transient forebrain ischaemia.

The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and gamma-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 microM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.

Amino Acids↗
PubMed1994

Ethanol-derived immunoreactive species formed by free radical mechanisms.

Recent studies have shown that the alpha-hydroxyethyl radical (CH3CHOH), a metabolite of ethanol, is produced in vitro and in vivo. We report studies that establish the immunogenicity of alpha-hydroxyethyl radical-derived protein adducts. Rat liver microsomes incubated in the presence of [14C]ethanol and NADPH (under aerobic conditions) incorporate 14C into acid-stable adducts. Incorporation was markedly inhibited by the free-radical scavenger alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. Rabbits immunized with rat liver microsomes that had been preincubated with ethanol and NADPH generated antibodies that recognized polylysine-acetaldehyde adducts and adducts formed by incubation of proteins with an alpha-hydroxyethyl radical-generating system (ethanol plus H2O2 plus Fe2+). Rabbits immunized with microsomes that had been preincubated with ethanol and NADPH plus alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone generated antibodies that recognized polylysine-acetaldehyde adducts. However, their reactivity against alpha-hydroxyethyl-derived protein epitopes was greatly reduced or was virtually abolished. Data indicate that microsomes metabolizing ethanol generate two types of adducts, acetaldehyde-derived adducts and alpha-hydroxyethyl radical-derived adducts, both of which are immunogenic. Immunization of rabbits with alpha-hydroxyethyl-bovine serum albumin adducts led to the production of antibodies that recognized alpha-hydroxyethyl-rabbit serum albumin adducts but did not recognize the native protein. Chronic alcohol feeding of rats led to the production of antibodies that recognized alpha-hydroxyethyl-rat serum albumin adducts but did not recognize rat serum albumin. The study (i) indicates that alpha-hydroxyethyl radical-derived protein adducts are immunogenic, (ii) supports earlier work that proposed that alpha-hydroxyethyl radicals generated in different systems bind covalently to proteins, and (iii) demonstrates the formation of antibodies to alpha-hydroxyethyl-derived protein adducts after chronic alcohol ingestion in vivo. The findings may have implications in the identification of chronic alcohol abuse and the pathogenesis of alcohol-induced organ damage.

Acetaldehyde↗
PubMed1993

Simple method for the preparation of antigen emulsions for immunization.

We have developed a rapid, safe, and reliable method to prepare emulsions of water-soluble antigens in an adjuvant oil phase for immunization purposes. The method, based on well established emulsification principles, employs a three-way 'T' connector to which three disposable syringes are attached. The system allows the stepwise addition of small volumes of the water phase, into the oil phase. We have compared the time required for emulsification, the rate of antigen release from the emulsion into a physiological phase, and the immunogenic properties of bovine serum albumin and transferrin contained in emulsions made by the new stepwise addition method, with those made by the widely used double-hubbed needle method. We report a significantly shorter (P < 0.001) and a more reproducible emulsification time for the stepwise addition method (6.1 +/- 2.1 min; mean +/- SD) than for the double-hubbed needle method (41.1 +/- 28.0 min). The stepwise addition method always yielded water-in-oil emulsions, while the double-hubbed needle method failed, about 20% of the time, to produce a water-in-oil emulsion after 120 min of mixing. Since the stepwise addition method employs a connector with a larger inner diameter (1.75 mm) than the one required for the double-hubbed needle method (0.84 mm); the pressure required for the former is markedly reduced compared with that required for the latter, thus making the new method safer and less labor-intensive. The rate of antigen release from the emulsions was significantly slower when the stepwise addition method was employed (P < 0.01). There were no differences in viscosity and stability in the emulsions prepared by the two methods. The ability of antigen-containing emulsions to elicit an immune response was found to be identical by the two methods; no significant differences were found in antibody titers as determined by enzyme-linked immunosorbent assays. These characteristics make the stepwise addition system the method of choice.

Adjuvants, Immunologic↗
PubMed1992

The non-NMDA antagonists, NBQX and GYKI 52466, protect against cortical and striatal cell loss following transient global ischaemia in the rat.

The cerebroprotective action of non-NMDA receptor blockade has been assessed in a model of transient global ischaemia using NBQX, 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, and GYKI 52466, 1-(amino-phenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine. HCl. In Wistar rats, prior cauterisation of the vertebral arteries was followed by occlusion of the common carotid arteries for 20 min, with a 7 day survival period before histological evaluation. NBQX, 40 mg/kg, or GYKI 52466, 40 mg/kg, was administered intravenously starting directly after the end of carotid occlusion and ending 3 h later. Both compounds produced significant protection against selective cell loss in the striatum and cortex. Less consistent changes were seen in the hippocampus; protection by NBQX was significant in CA3 but neither compound produced significant protection in CA1. This pattern of protection is interpreted in terms of a blockade of glutamate's action at non-NMDA receptors limited to the initial 3 h of reperfusion.

Animals↗
PubMed1992

GYKI 52466 blocks the increase in extracellular glutamate induced by ischaemia.

In vivo microdialysis has been used to study the effect of pre- or post-ischaemic administration of the non-NMDA antagonist 1-(4-amino-phenyl)-4-methyl-7, 8-methyl-endioxyl-5H-2,3- benzodiazepine hydrochloride (GYKI 52466), on the increases in extracellular glutamate levels induced by 20 minutes of four vessel occlusion in rats. In control rats, ischaemia resulted in transient increases in glutamate (4 fold), aspartate (6 fold) and gamma-aminobutyric acid (GABA) (15 fold) and decreases in glutamine (0.5 fold). Intravenous administration of GYKI 52466 (10 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion) beginning 20 minutes prior to the induction of ischaemia abolished ischaemia-induced glutamate release without affecting the increases in aspartate and GABA and the decrease in glutamine. Administration of GYKI 52466 immediately post-ischaemia resulted in a more rapid return of glutamate levels to basal values.

Animals↗
PubMed1992

Effect of NO synthase inhibition on NMDA- and ischaemia-induced hippocampal lesions.

We assessed the effect of NG-Nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase, on the hippocampal lesions induced either by the focal injection of N-methyl-D-aspartate (NMDA) or (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (s-AMPA) or by 10 min of severe forebrain ischaemia (4-vessel occlusion), in the rat. We find that L-NAME, 20 or 40 mg kg-1, selectively decreases NMDA-induced CA1 lesions while it has no effect on AMPA toxicity. L-NAME, 20 mg kg-1, does not decrease hippocampal damage induced by ischaemia. These results suggest that NO contributes to NMDA toxicity and support data indicating that NMDA receptor antagonists fail to protect against hippocampal CA1 lesions in the 4-vessel occlusion model.

Amino Acid Oxidoreductases↗
PubMed1991

Non-NMDA antagonists protect against kainate more than AMPA toxicity in the rat hippocampus.

Single focal injection of the excitatory amino acids (EAAs) kainic acid (KA, 1.1 nmol/microliters) and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S)-AMPA, 6 nmol/microliters) into rat dorsal hippocampus resulted in widespread neurodegeneration with 90-100% loss of hippocampal pyramidal cells in CA1, CA2, CA3 and CA4 subfields, and 50-70% loss of dentate granule (DG) cells. Focal injection of NMDA (30 nmol/microliters) under the same conditions resulted in 70-90% loss of CA1 cells with less damage in CA2, CA3, CA4 and DG cells (30-50%, 10-30%, and 30-50%, respectively). The non-NMDA antagonists NBQX (2,3-dihydro-6-nitro-7-sulphamoyl-benzo(f) quinoxaline) and GYKI 52466 (1-(amino)phenyl-4-methyl-7,8-methylendioxy-5H-2,3,benzodiazepine. HCl) co-injected (24 nmol/microliters) with EAAs or given as i.v. infusion (30 mg/kg/3h), protected against KA toxicity in CA1, CA2 and DG cells, with no protection in CA3 and CA4. NBQX i.v. protected against (S)-AMPA toxicity in the DG cells but no protection was observed against (S)-AMPA toxicity in hippocampal subfields (CA1, CA2 and CA4). Intravenous administration of NBQX and GYKI 52466 (30 mg/kg/3 h) also failed to protect against NMDA toxicity in the hippocampus. Systemic injections of D(-)-CPPene, (E)-4-(3-phos-phonoprop-2-enyl)-piperazine-2-carboxylic acid, (10 and 5 mg/kg, i.p., 20 min prior and 3 h post EAA injection) protected against NMDA and KA toxicity in the CA1, CA2 and DG subfield with no protective effect against (S)-AMPA toxicity.

Animals↗
PubMed1991

Memory retraining to support educational reintegration.

A memory retraining package specifically designed to facilitate reintegration of head injured patients into an educational environment is described. Two adolescent patients who had severe head injuries were administered the memory retraining package approximately three months postinjury. A single case study and multiple baseline design was used to evaluate the efficacy of the memory retraining program. The results suggested that this is a promising avenue for improving memory functioning and facilitating educational reintegration, but only where moderate rather than severe memory deficits are involved. Studies involving groups of patients and the collection of data on generalization are required to confirm usefulness.

Adolescent↗
PubMed1989

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs.

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.

Animals↗