Long-term monitoring of Ca2+ dynamics in C. elegans pharynx: an in vivo energy balance sensor.

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.

Melatonin ameliorates neurologic damage and neurophysiologic deficits in experimental models of stroke.

This review summarizes the numerous reports that have documented the neuroprotective actions of melatonin in experimental models of ischemia/reperfusion injury (stroke). In these investigations, which have used three species (rat, gerbil, and cat), melatonin was universally found to reduce brain damage that normally occurs as a consequence of the temporary interruption of blood flow followed by the reflow of oxygenated blood to the brain. The exogenous administration of melatonin in these experimental stroke models reduced infarct volume, lowered the frequency of apoptosis, increased the number of surviving neurons, reduced reactive gliosis, lowered the oxidation of neural lipids and oxidatively damaged DNA, induced bcl-2 gene expression (the activity of which improves cell survival), upregulated excision repair cross-complementing factor 6 (an essential gene for preferential DNA excision repair), restrained poly(ADP ribose) synthetase (which depletes cellular NAD resulting in the loss of ATP) activity, and improved neurophysiologic outcomes. Under no circumstances did melatonin exacerbate the damage associated with ischemia/reperfusion injury. As well as the beneficial pharmacologic actions of melatonin, several studies show that a relative deficiency of endogenous melatonin exaggerates neural damage due to stroke; this suggests that even physiologic concentrations of melatonin normally serve to protect the brain against damage. The primary action to explain melatonin's protective effects may relate to its ubiquitous direct and indirect antioxidative actions, although other beneficial functions of melatonin are not precluded.

Oxidative damage to catalase induced by peroxyl radicals: functional protection by melatonin and other antioxidants.

Thermal decomposition by the azo initiator 2,2' azobis(2-amidinopropane) dihydrochloride (AAPH) has been widely used as a water-soluble source of free radical initiators capable of inducing lipid peroxidation and protein damage. Here, in a lipid-free system, AAPH alone (40 mM) rapidly induced protein modification and inactivation of the enzyme catalase (EC 1.11.1.6). Using SDS-PAGE, it was shown that protein band intensity is dramatically reduced after 4 h of incubation with AAPH, leading to protein aggregation. Several antioxidants including melatonin, glutathione (GSH) and trolox prevented catalase modification when used at a 250 microM concentration whereas ascorbate was only effective at 1 mM concentration. All the antioxidants tested reduced carbonyl formation although melatonin was the most effective in this regard. Enzyme inactivation caused by AAPH was also significantly reduced by the antioxidants and again melatonin was more efficient than the other antioxidants used in this study. Results shown here demonstrate that alkyl peroxyl radicals inactivate catalase and reduce the effectiveness of cells to defend against free radical damage; the damage to catalase can be prevented by antioxidants, especially melatonin.

Mechanistic and comparative studies of melatonin and classic antioxidants in terms of their interactions with the ABTS cation radical.

Melatonin and classic antioxidants possess the capacity to scavenge ABTSb+ with IC50s of 4, 11, 15.5, 15.5, 17 and 21 microm for melatonin, glutathione, vitamin C, trolox, NADH and NADPH, respectively. In terms of scavenging ABTSb+, melatonin exhibits a different profile than that of the classic antioxidants. Classic antioxidants scavenge one or less ABTSb+, while each melatonin molecule can scavenge more than one ABTSb+, probably with a maximum of four. Classic antioxidants do not synergize when combined in terms of scavenging ABTSb+. However, a synergistic action is observed when melatonin is combined with any of the classic antioxidants. Cyclic voltammetry indicates that melatonin donates an electron at the potential of 715 mV. The scavenging mechanism of melatonin on ABTSb+ may involve multiple-electron donations via intermediates through a stepwise process. Intermediates including the melatoninyl cation radical, the melatoninyl neutral radical and cyclic 3-hydroxymelatonin (cyclic 3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) seem to participate in these reactions. More interestingly, the pH of the solution dramatically modifies the ABTSb+ scavenging capacity of melatonin while pH changes have no measurable influence on the scavenging activity of classic antioxidants. An acidic pH markedly reduces the ABTSb+ scavenging capacity of melatonin while an increased pH promotes the interaction of melatonin and ABTSb+. The major melatonin metabolites that develop when melatonin interacts with ABTSb+ are cyclic 3-OHM and AFMK. Cyclic 3-OHM is the intermediate between melatonin and AFMK, and cyclic 3-OHM also has the ability to scavenge ABTSb+. Melatonin and the metabolites which are generated via the interaction of melatonin with ABTSb+, i.e. the melatoninyl cation radical, melatoninyl neutral radical and cyclic 3-OHM, all scavenge ABTSb+. This unique cascade action of melatonin, in terms of scavenging, increases its efficiency to neutralized ABTSb+; this contrasts with the effects of the classic antioxidants.

Melatonin, xanthurenic acid, resveratrol, EGCG, vitamin C and alpha-lipoic acid differentially reduce oxidative DNA damage induced by Fenton reagents: a study of their individual and synergistic actions.

DNA damage generated by oxygen-derived free radicals is related to mutagenesis, carcinogenesis and aging. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. In the present in vitro study, we have examined the efficacy of three polyphenolic antioxidants, i.e. xanthurenic acid, resveratrol (3,4',5-trihydroxy-trans-stilbene) and (-)-epigallocatechin-3-gallate (EGCG) and two classical non-polyphenolic antioxidants, i.e. vitamin C (ascorbic acid) and alpha-lipoic acid (LA, 1,2-dithiolane-3-pentanoic acid) in inhibiting *OH-induced oxidative DNA damage. We compared the efficacy of these five antioxidants with the effectiveness of melatonin (N-acetyl-5-methoxytryptamine) and we also investigated the possible synergistic effects of melatonin with the other five molecules. Using high performance liquid chromatography (HPLC), the formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) (as CrCl3) plus hydrogen peroxide (H2O2) (Cr(III)/H2O2), was measured in the presence or absence of the antioxidants alone or in combination with melatonin. 8-OH-dG is considered a biomarker of oxidative DNA damage. Among the antioxidants tested, melatonin was the most effective of these with an IC50 = 3.6 +/- 0.1 micro m. For the other antioxidants the IC50 values were as follows: xanthurenic acid (IC50 = 7.9 +/- 0.3), resveratrol (IC50 = 10.9 +/- 0.3), EGCG (IC50 = 5.7 +/- 0.3), vitamin C (IC50 = 16.9 +/- 0.5) and LA (IC50 = 38.8 +/- 0.7). These values differ from that of melatonin with a P < 0.01. Melatonin (1 micro M) reversed the pro-oxidant effect of resveratrol (0.5 micro M) and vitamin C (0.5 micro M), had an antagonistic effect when used in combination with EGCG (1 micro M) and it exhibited synergism in combination with vitamin C (0.5 micro M) and with LA (5 micro M).

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation.

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.

Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin.

Melatonin, a derivative of an essential amino acid, tryptophan, was first identified in bovine pineal tissue and subsequently it has been portrayed exclusively as a hormone. Recently accumulated evidence has challenged this concept. Melatonin is present in the earliest life forms and is found in all organisms including bacteria, algae, fungi, plants, insects, and vertebrates including humans. Several characteristics of melatonin distinguish it from a classic hormone such as its direct, non-receptor-mediated free radical scavenging activity. As melatonin is also ingested in foodstuffs such as vegetables, fruits, rice, wheat and herbal medicines, from the nutritional point of view, melatonin can also be classified as a vitamin. It seems likely that melatonin initially evolved as an antioxidant, becoming a vitamin in the food chain, and in multicellular organisms, where it is produced, it has acquired autocoid, paracoid and hormonal properties.

Melatonin: detoxification of oxygen and nitrogen-based toxic reactants.

In the last decade, melatonin has been found to be highly protective against damage to macromolecules resulting from oxygen and nitrogen-based reactants. Considering this, numerous studies have examined the mechanisms whereby this indoleamine directly detoxifies these damaging agents. The evidence is compelling that melatonin scavenges several oxygen-derived reactive agents including the hydroxyl radical (OH), hydrogen peroxide (H2O2), singlet oxygen (1O2) and hypochlorous acid (HOCl). Additionally, melatonin reportedly reacts with nitric oxide (NO), the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH) to detoxify them. In some cases the products that are formed as a consequence of melatonin's scavenging actions have been identified. Whereas the ability of melatonin to neutralize these toxic agents likely accounts, in part, for the antioxidant activity of melatonin, it is not the only means by which melatonin serves to protect molecules from oxygen and nitrogen-based reactive metabolites.

Melatonin: reducing the toxicity and increasing the efficacy of drugs.

Melatonin (N-acetyl-5-methoxytryptamine) is a molecule with a very wide phylogenetic distribution from plants to man. In vertebrates, melatonin was initially thought to be exclusively of pineal origin recent studies have shown, however, that melatonin synthesis may occur in a variety of cells and organs. The concentration of melatonin within body fluids and subcellular compartments varies widely, with blood levels of the indole being lower than those at many other sites. Thus, when defining what constitutes a physiological level of melatonin, it must be defined relative to a specific compartment. Melatonin has been shown to have a variety of functions, and research in the last decade has proven the indole to be both a direct free radical scavenger and indirect antioxidant. Because of these actions, and possibly others that remain to be defined, melatonin has been shown to reduce the toxicity and increase the efficacy of a large number of drugs whose side effects are well documented. Herein, we summarize the beneficial effects of melatonin when combined with the following drugs: doxorubicin, cisplatin, epirubicin, cytarabine, bleomycin, gentamicin, ciclosporin, indometacin, acetylsalicylic acid, ranitidine, omeprazole, isoniazid, iron and erythropoietin, phenobarbital, carbamazepine, haloperidol, caposide-50, morphine, cyclophosphamide and L-cysteine. While the majority of these studies were conducted using animals, a number of the investigations also used man. Considering the low toxicity of melatonin and its ability to reduce the side effects and increase the efficacy of these drugs, its use as a combination therapy with these agents seems important and worthy of pursuit.

Validación del cuestionario aplicado al estudio "la Bioquímica en la Medicina: necesidad y relevancia" / Validation of the questionnaire applied to the project "Biochemistry in Medicine: need and relevance"

La docencia universitaria tiende cada vez con más fuerza hacia la integración y la coordinación, y dada la velocidad a la que avanza el conocimiento de los fenómenos bioquímicos implicados en la Medicina nos parece importante, desde un punto de vista docente, la identificación clara de los más relevantes y pertinentes para la comprensión de la propia Bioquímica y del resto de las asignaturas de la Licenciatura de Medicina. Con el objetivo de conocer las verdaderas necesidades de conceptos bioquímicos en el marco de una docencia integrada o al menos coordinada en las diferentes áreas de conocimiento de la Licenciatura de Medicina, hemos elaborado un cuestionario dirigido a los profesores de dicha facultad, dentro de un programa más amplio que denominamos "La Bioquímica en la Medicina: necesidad y relevancia", que abarca, además de la población de profesores, la de alumnos y la de médicos en ejercicio. El cuestionario ha sido validado según el método Delphi, con la colaboración de más de 30 profesores de la Universidad de Valladolid. De esta manera, hemos construido una herramienta válida para alcanzar su objetivo y queremos ponerla a disposición de otros investigadores interesados en el tema (AU)

Melatonin, longevity and health in the aged: an assessment.

This brief review considers the potential role of melatonin in the processes of aging, the prolongation of life span and health in the aged. Studies completed to date generally suggest that exogenously administered melatonin may serve to extend life span in invertebrates, but evidence supporting this conclusion in mammals is less compelling. Thus, any conclusion regarding a role for melatonin in extending normal longevity, particularly in mammals, would be premature. With regard to deferring the signs of chemically-induced neurodegenerative conditions in experimental animals, the data are remarkably strong and there is a modicum of evidence that in humans with debilitating diseases melatonin may have some beneficial actions. Indeed, this should be one focus of future research since as the number of elderly increases in the population, the frequency of costly age-related diseases will become increasingly burdensome to both the patient and to society as a whole.