Juan Parrado

Protective effect of melatonin against the 1-methyl-4-phenylpyridinium-induced inhibition of Complex I of the mitochondrial respiratory chain

In the present study, a novel property of melatonin is shown: a protective effect of melatonin on the respiratory chain in isolated rat liver mitochondria and in striatal synaptosomes treated with 1‐methyl‐4‐phenylpyridinium ion (MPP+). The cellular damage caused by MPP+, a compound that produces a Parkinsonian‐like syndrome in humans, is the result of the mitochondrial respiration inhibition at the Complex I level and oxidative stress induction. Treatment of mitochondria with MPP+ inhibits the respiration rate. This effect was prevented by the inclusion of melatonin in the incubation mixture. This preventive effect, which is not related to the antioxidative properties of melatonin, seems to be due to the fact that melatonin prevents MPP+ interaction with Complex I. These results suggest that melatonin may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage.

Effects of aging on the various steps of protein synthesis: fragmentation of elongation factor 2

The possible mechanism responsible for the in vivo protein synthesis decline during aging was studied. In order to determine the effect of aging on the various steps of protein synthesis, we determined the ribosomal state of aggregation and the time of assembly and release of polypeptide chains in the process of protein synthesis in rat liver. The results suggest that elongation is the most sensitive step to aging. A molecular study of the Elongation Factor 2 (EF-2), the main protein involved in the elongation step, shows that this protein has a higher content of carbonyl groups and is less active in old rats. In addition, the molecular mass analysis of EF-2 shows that this protein becomes fragmented in old rats. A similar pattern of fragmentation is found in 3-month-old rats suffering oxidative stress, in that the decline in protein synthesis is similar to that found in old rats. These data suggest that: i) oxidative stress seems to be involved in the modifications of EF-2 observed during aging, and ii) the observed modifications (oxidation and fragmentation) of EF-2 could account for the decline in protein synthesis in old animals.

Age‐related increase in the immunoproteasome content in rat hippocampus: molecular and functional aspects

Alterations in the proteasome activity in the CNS have been described during aging. However, a detailed study of all proteasome subunits is actually lacking. We have analyzed, in vivo, the age‐related modifications in the molecular composition of hippocampal proteasomes. We found that the immunoproteasome/proteasome ratio was increased in aged hippocampus. The processing of the low‐molecular‐mass protein (LMP)7/β5i subunit, practically absent in young hippocampus, was increased in aged animals. Among the potential factors underlying these modifications we evaluated the neuroinflammation and the transcription factor Zif268. Lipopolysaccharide (LPS)‐induced neuroinflammation in young rats, up‐regulated the expression of immunoproteasome subunits and increased the processing of the LMP7/β5i protein. Moreover, the hydrophobicity of cellular peptides, analyzed by liquid chromatography, increased in both, young LPS‐injected animals and aged rats, suggesting that immunoproteasomes including the LMP7/β5i subunit could, at least in part, account for this modification. Also, the mRNA expression of the transcription factor Zif268, which down‐regulates the immunoproteasome subunit LMP7/β5i by binding to sequences within the promoter regions, was decreased in both, aged hippocampus and young LPS‐injected animals. Finally, we found that spatial memory training in young animals, a situation in which the expression of Zif268 is increased, modified the mRNA expression of the constitutive and catalytic subunits in an opposite manner. Based on present data, we propose that the age‐related increases in the content of hippocampal immunoproteasome is mostly because of neuroinflammatory processes associated to aging.

Isolation and characterization of carotenoproteins from crayfish (Procambarus clarkii)

Abstract Crayfish (Procambarus clarkii) were separated by solid filtration into a proteinaceous fraction (PF-1) and a chitinous fraction (CF). The PF-1 and CF were used as starting materials for carotenoprotein-1 and carotenoprotein-2 preparations, respectively. Crawfish carotenoprotein-1 was obtained by controlled enzymatic autodigestion of FP-1 and carotenoprotein-2 by in situ repeated batch lactic acid fermentation. Carotenoprotein-1 has a high content of essential amino acids, ω-3- and ω-6-fatty acids and carotene (mainly astaxanthin), and constitutes an excellent nutritional source for patients with malnutrition. Carotenoprotein-2, of lower nutritional quality but with a substantial carotene content, can be used as a good protein source for animal nutrition where coloration is required, such as for poultry or for salmonid fish bred by aquaculture.

Rice bran enzymatic extract restores endothelial function and vascular contractility in obese rats by reducing vascular inflammation and oxidative stress.

BACKGROUND Rice bran enzymatic extract (RBEE) used in this study has shown beneficial activities against dyslipidemia, hyperinsulinemia and hypertension. Our aim was to investigate the effects of a diet supplemented with RBEE in vascular impairment developed in obese Zucker rats and to evaluate the main mechanisms mediating this action. METHODS AND RESULTS Obese Zucker rats were fed a 1% and 5% RBEE-supplemented diet (O1% and O5%). Obese and their lean littermates fed a standard diet were used as controls (OC and LC, respectively). Vascular function was evaluated in aortic rings in organ baths. The role of nitric oxide (NO) was investigated by using NO synthase (NOS) inhibitors. Aortic expression of endothelial NOS (eNOS), inducible NOS (iNOS), tumor necrosis factor (TNF)-α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and superoxide production in arterial wall were determined. Endothelial dysfunction and vascular hyperreactivity to phenylephrine in obese rats were ameliorated by RBEE treatment, particularly with 1% RBEE. Up-regulation of eNOS protein expression in RBEE-treated aortas should contribute to this activity. RBEE attenuated vascular inflammation by reducing aortic iNOS and TNF-α expression. Aortas from RBEE-treated groups showed a significant decrease of superoxide production and down-regulation of NADPH oxidase subunits. CONCLUSION RBEE treatment restored endothelial function and vascular contractility in obese Zucker rats through a reduction of vascular inflammation and oxidative stress. These results show the nutraceutical potential of RBEE to prevent obesity-related vascular complications.

Anti-inflammatory activity of a honey flavonoid extract on lipopolysaccharide-activated N13 microglial cells.

Neuroinflammation is an important contributor to pathogenesis of age-related neurodegenerative disorders such as Alzheimers or Parkinsons disease. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for neurodegenerative processes. Flavonoids, widely distributed in the vegetable kingdom and in foods such as honey, have been suggested as novel therapeutic agents for the reduction of the deleterious effects of neuroinflammation. The present study investigated the potential protective effect of a honey flavonoid extract (HFE) on the production of pro-inflammatory mediators by lipopolysaccharide-stimulated N13 microglia. The results show that HFE significantly inhibited the release of pro-inflammatory cytokines such as TNF-α and IL-1β. The expressions of iNOS and the production of reactive oxygen intermediates (ROS) were also significantly inhibited. Accordingly, the present study demonstrates that HFE is a potent inhibitor of microglial activation and thus a potential preventive-therapeutic agent for neurodegenerative diseases involving neuroinflammation.

The endogenous amine 1-methyl-1,2,3,4-tetrahydroisoquinoline prevents the inhibition of complex I of the respiratory chain produced by MPP+

Abstract : The endogenous monoamine 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline has been shown to prevent the neurotoxic effect of MPP+ and other endogenous neurotoxins, which produce a parkinsonian‐like syndrome in humans. We have tested its potential protective effect in vivo by measuring the protection of 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline in the neurotoxicity elicited by MPP+ in rat striatum by tyrosine hydroxylase immunocytochemistry. Because we know that cellular damage caused by MPP+ is primarily the result of mitochondrial respiratory inhibition at the complex I level, we have extended the study further to understand this protective mechanism. We found that the inhibitory effect on the mitochondrial respiration rate induced by MPP+ in isolated rat liver mitochondria and striatal synaptosomes was prevented by addition of 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline. This compound has no antioxidant capacity ; therefore, this property is not involved in its protective effect. Thus, we postulate that the preventive effect that 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline has on mitochondrial inhibition for MPP+ could be due to a “shielding effect,” protecting the energetic machinery, thus preventing energetic failure. These results suggest that this endogenous amine may protect against the effect of several parkinsonism‐inducing compounds that are associated with progressive impairment of the mitochondrial function.

Betalain profile, phenolic content, and color characterization of different parts and varieties of Opuntia ficus-indica.

Three different varieties of Opuntia ficus-indica (R, red; Y, yellow; RY, red-yellow) have been considered in this study. Attention was focused on differential tristimulus colorimetry and on the analysis of individual betalains (HPLC-DAD-ESI-ToF-MS) and phenolic content, scarcely previously reported in these kinds of samples. The importance of this research stems from the elucidation of the parts and varieties of cactus pear more optimal for use as natural colorants and sources of phenolics and betalains. Thus, the RY pulp was appropriate to obtain colorants with high color intensity (C*(ab) = 66.5), whereas the whole Y fruit and R pulp reached powerful and stable yellow and red colors, respectively (C*(ab)/h(ab), 57.1/84.7 and 61.1°/81.8°). This choice was also based on the visually appreciable differences (ΔE*(ab) > 5) among samples, mainly quantitative (%Δ(2)L, %Δ(2)C). In addition, seeds of all Opuntia varieties showed significantly (p < 0.05) similar phenolic content (around 23.3 mg/g) and color characteristics.

Endothelium-dependent vasodilator and antioxidant properties of a novel enzymatic extract of grape pomace from wine industrial waste.

The aim of the present study was to evaluate the vascular effects of an enzymatic extract of grape pomace (GP-EE) on isolated arteries, focusing our attention on endothelium-derived relaxation and on its antioxidant properties. Grape pomace derived from wine making was extracted by an enzymatic process and its composition of polyphenols was evaluated by HPLC and ESI-MS/MS, detecting kaempferol, catechin, quercetin and procyanidins B1 and B2, trace levels of resveratrol and tracing out gallocatechin and anthocyanidins. GP-EE induced endothelium- and NO-dependent vasodilatation of both rat aorta and small mesenteric artery (SMA) segments and reduced Phe-induced response in aortic rings. Both ORAC and DPPH assays confirmed antioxidant scavenging properties of GP-EE, which also prevented O(2)(·-) production (assessed by DHE fluorescence) and contraction elicited by ET-1. These results provide evidence that GP-EE possesses interesting antioxidant and protective vascular properties and highlight the potential interest of this extract as a functional food.

Use of Rotofor preparative isoelectrofocusing cell in protein purification procedure

The Rotofor cell is a preparative isoelectric focusing (IEF) apparatus, in which IEF is performed entirely in free solution. Electrofocusing in Rotofor cell has been described as well-suited for use at any stage of a purification scheme. However, it has some important limitations in resolving complex mixtures of proteins. This paper describes the advantages and disadvantages of using the Rotofor cell in purification protocols.