Mitochondrial Aconitase Enzymatic Activity: A Potential Long-Term Survival Biomarker in the Blood of ALS Patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multisystemic, progressive, neurodegenerative disorder. Despite it being generally fatal within a period of 2-4 years, it is highly heterogeneous; as a result, survival periods may vary greatly among individual patients. Biomarkers can serve as tools for diagnosis, prognosis, indicators of therapeutic response, and future therapeutics. Free-radical-dependent mitochondrial damage is believed to play a crucial role in neurodegeneration in ALS. Mitochondrial aconitase, which is also known as aconitase 2 (Aco2), is a key Krebs cycle enzyme and is involved in the regulation of cellular metabolism and iron homeostasis. Aco2 is very sensitive to oxidative inactivation and can aggregate and accumulate in the mitochondrial matrix, causing mitochondrial dysfunction. Loss of Aco2 activity may therefore reflect increased levels of mitochondrial dysfunction due to oxidative damage and could be relevant to ALS pathogenesis. The aim of our study was to confirm changes in mitochondrial aconitase activity in peripheral blood and to determine whether such changes are dependent on, or independent of, the patient's condition and to propose the feasibility of using them as possible valid biomarkers to quantify the progression of the disease and as a predictor of individual prognosis in ALS. METHODS: We measured the Aco2 enzymatic activity in the platelets of blood samples taken from 22 controls and 26 ALS patients at different stages of disease development. We then correlated antioxidant activity with clinical and prognostic variables. RESULTS: Aco2 activity was significantly lower in the 26 ALS patients than in the 22 controls (p < 0.05). Patients with higher levels of Aco2 activity survived longer than those with lower levels (p < 0.05). Aco2 activity was also higher in patients with earlier onset (p < 0.05) and in those with predominantly upper motor neuron signs. CONCLUSIONS: Aco2 activity seems to be an independent factor that could be used in the long-term survival prognosis of ALS. Our findings suggest that blood Aco2 could be a leading candidate for use as a biomarker to improve prognosis. More studies are needed to confirm these results.

Effect of Castration Type and Diet on Growth Performance, Serum Sex Hormones and Metabolites, and Carcass Quality of Heavy Male Pigs.

A trial was carried out to study the effect of type of castration and diet on pigs destined for Teruel ham production, which is a Spanish protected designation of origin for dry-cured ham. A total of 144 Duroc × (Landrace × Large White) male pigs were used. Half of them were surgically castrated and the other half were immunocastrated with three doses at approximately 25, 58 and 79 kg of body weight. Furthermore, three diets (control vs. high energy vs. low crude protein-CP- and amino acids-AA) were tested from 80 to 137 kg of body weight. Growth performance, serum sex hormones and metabolites, and carcass quality were evaluated. Immunocastrated males grew faster and had better feed conversion ratio than surgically castrated males, but presented lower carcass fatness. Pigs fed the high-energy diet and the low-CP and -AA diet were more efficient at transforming feed into gain than those fed the control diet, but no effect was detected on carcass quality. In conclusion, surgically castrated males are preferable than immunocastrated males for Teruel dry-cured ham elaboration. Besides, a high-energy diet or a low-CP and -AA diet might improve productive performances, but does not provide any benefit in terms of carcass quality.

Influence of Immunocastration and Diet on Meat and Fat Quality of Heavy Female and Male Pigs.

Two experiments were carried out; one with female pigs and the other with male pigs destined for Teruel dry-cured ham production, to evaluate the effect of immunocastration (entire gilts-EG vs. immunocastrated gilts-IG and surgically castrated males vs. immunocastrated males-IM) and diet (control vs. high energy vs. low crude protein and amino acids) on meat quality and fat composition. Fifteen meat samples and eight fat samples of each treatment were analyzed in both experiments. In the case of males, six fat samples per treatment were analyzed to determine boar taint. Immunocastration is a good strategy in gilts intended for dry-cured ham production because improves meat composition; however, in males, immunocastration impairs the results of pork chemical composition compared with surgical castration. The IG presented a lower polyunsaturated/saturated fatty acids ratio than EG, improving fat technological quality. Diets had little effect on pork or fat quality in gilts, but a high-energy level using oilseeds and a low-crude-protein and -amino-acids diet from 80 to 137 kg of body weight could be interesting in IM to maintain or increase fat consistency, respectively. Moreover, in general, immunocastration is effective in avoiding boar taint in males.

Effect of Immunocastration and Diet on Growth Performance, Serum Metabolites and Sex Hormones, Reproductive Organ Development and Carcass Quality of Heavy Gilts.

It is desirable to increase fatness in gilts destined for Teruel dry-cured ham production. A total of 192 Duroc × (Landrace × Large White) gilts of 40.3 ± 4.80 kg body weight (BW) were used to assess the impact of immunocastration and feeding on growth performance, serum metabolites and sex hormones, reproductive organ development, and carcass quality. Six treatments were arranged factorially (2 × 3) with two types of gilt (entire gilts (EG) vs. immunocastrated gilts (IG)) and three experimental diets (control vs. high energy vs. low crude protein and amino acids) provided from 76 to 134 kg BW (n = 4 per treatment, being the replicate the pen with eight pigs). Immunocastration was carried out at 58 and 77 kg BW. The IG grew faster and showed lighter reproductive tracts and greater fatness than EG. The experimental feeds had limited effect on carcass quality, but the high-energy diet improved gain-to-feed ratio and the low-protein and -amino-acids diet did not impair growth performance. In conclusion, immunocastration was a better strategy than the tested diets to increase the fatness of gilts intended for Teruel dry-cured ham, although increasing energy or decreasing crude protein and amino acid levels in the diet could be beneficial strategies for pig farmers.

Immunocastration in Gilts: A Preliminary Study of the Effect of the Second Dose Administration Time on Growth, Reproductive Tract Development, and Carcass and Meat Quality.

Increasing fatness and avoiding puberty are desirable in gilts intended for high-quality dry-cured ham production. A total of 48 Duroc x (Landrace x Large White) females of 26.5 ± 3.70 kg body weight (BW) were used to evaluate the impact of immunocastration and to find the optimum application time of the second dose for immunocastration on growth; sex hormones; reproductive tract development; and carcass, meat, and fat quality. Gilts were allocated to four experimental treatments (n = 12): control (entire gilts, EG) and immunocastrated gilts (IG), providing the second dose at 12, 9, or 7 weeks before slaughter (with approximately 60, 75, or 90 kg BW, respectively). Mean slaughter BW was 125 kg. Immunocastrated gilts had lighter reproductive tracts and greater fat thickness than EG. Fat from IG was more saturated and less polyunsaturated than that from EG. Numerically, gilts immunocastrated 9 and 12 weeks before slaughter presented higher fatness than those immunocastrated 7 weeks before slaughter. In conclusion, immunocastration is a good strategy to improve the fatness of gilts destined to dry-cured ham elaboration, with the optimum time for the second dose application seemingly between 9 and 12 weeks before slaughter.

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis.

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SOD(G93A) mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SOD(G93A) animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SOD(G93A) mice.

Monitoring systemic oxidative stress in an animal model of amyotrophic lateral sclerosis.

A mutant form of the ubiquitous copper/zinc superoxide dismutase (SOD1) protein has been found in some patients with amyotrophic lateral sclerosis (ALS). We monitored oxidative stress in an animal model of ALS, the SOD(G93A) mouse, which develops a disease similar to ALS with an accelerated course. The aim of this work was to show that ALS damages several organs and tissues, from an oxidative stress point of view. We measured lipid and protein oxidative damage in different tissue homogenates of SOD(G93A) mice. The biomarkers that we analyzed were malondialdehyde + 4-hydroxyalkenal (MDA + 4-HDA) and carbonyls, respectively. The spinal cord and brain of SOD(G93A) mice showed increased lipid peroxidation after 100 or 130 days compared to age-matched littermate controls. The CNS was most affected, but lipid peroxidation was also detected in the skeletal muscle and liver on day 130. No changes were observed in protein carbonylation in the homogenates. Our results are consistent with a multisystem etiology of ALS and suggest that oxidative stress may play a primary role in ALS pathogenesis. Thus, oxidative stress represents a potential biomarker that might be useful in developing new therapeutic strategies for ALS.

Melatonin reduces lipid and protein oxidative damage in synaptosomes due to aluminium.

Prolonged exposure to excessive aluminium (Al) concentrations is involved in the ethiopathology of certain dementias and neurological disorders. Melatonin is a well-known antioxidant that efficiently reduces lipid peroxidation due to oxidative stress. Herein, we investigated in synaptosomal membranes the effect of melatonin in preventing Al promotion of lipid and protein oxidation when the metal was combined with FeCl(3) and ascorbic acid. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the membrane suspension and protein carbonyls were measured in the synaptosomes as an index of oxidative damage. Under our experimental conditions, the addition of Al (0.0001-1mmol/L) enhanced MDA+4-HDA formation in the synaptosomes. In addition, Al (1mmol/L) raised protein carbonyl contents. Melatonin reduced, in a concentration-dependent manner, lipid and protein oxidation due to Al, FeCl(3) and ascorbic acid in the synaptosomal membranes. These results show that melatonin confers protection against Al-induced oxidative damage in synaptosomes and suggest that this indoleamine may be considered as a neuroprotective agent in Al toxicity because of its antioxidant activity.

Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates.

Numerous data indicate that hyperhomocysteinemia is a risk factor for cardio- and cerebrovascular diseases. At least in part, homocysteine (HCY) impairs cerebrovascular function because it generates large numbers of free radicals. Since melatonin is a well-known antioxidant, which reduces oxidative stress and decreases HCY concentrations in plasma, the aim of this study was to investigate the effect of melatonin in preventing HCY-induced protein and lipid oxidation in rat brain homogenates. Brain homogenates were obtained from Sprague-Dawley rats and were incubated with or without HCY (0.01-5 mM) or melatonin (0.01-3 mM). Carbonyl content of proteins, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the brain homogenates were used as an index of protein and lipid oxidation, respectively. Under the experimental conditions used, the addition of HCY (0.01-5 mM) to the homogenates enhanced carbonyl protein and MDA+4-HDA formation. Melatonin reduced, in a concentration-dependent manner, protein and lipid oxidation due to HCY in the brain homogenates. These data suggest that preserving proteins from oxidative insults is an additional mechanism by which melatonin may act as an agent in potentially decreasing cardiovascular and cerebrovascular diseases related to hyperhomocysteinemia.

Fisiología del envejecimiento y del estrés oxidativo (B40) / No disponible

No disponible

Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in mature mouse motor nerve terminals.

In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). Because this traffic involves presynaptic activity, probably via the secretion of active molecules, we examined whether it is affected by brain-derived neurotrophic factor (BDNF). Quantitative confocal microscopy and a fluorimetric assay for beta-gal activity revealed that co-injecting BDNF and the fusion proteins significantly increased the kinetics and amount of the proteins' localization at the NMJ and their internalization by motor nerve terminals. The observed increases were independent of synaptic vesicle recycling because BDNF did not affect spontaneous quantal acetylcholine release. In addition, injecting anti-BDNF antibody shortly before injecting GFP-TTC, and before co-injecting GFP-TTC and BDNF, significantly reduced the fusion protein's localization at the NMJ. Co-injecting GFP-TTC with neurotrophin-4 (NT-4) or glial-derived neurotrophic factor (GDNF), but not with nerve growth factor, neurotrophin-3 or ciliary neurotrophic factor, also significantly increased the fusion protein's localization at the NMJ. Thus, TTC probes may use for their neuronal internalization endocytic pathways normally stimulated by BDNF, NT-4 and GDNF binding. Different tyrosine kinase receptors with similar signalling pathways are activated by BDNF/NT-4 and GDNF binding. Thus, activated components of these signalling pathways may be involved in the TTC probes' internalization, perhaps by facilitating localization of receptors of TTC in specific membrane microdomains or by recruiting various factors needed for internalization of TTC.

Melatonina en Psiquiatría: Una actualización / Melatonin in Psychiatry: An update

La melatonina es una molécula sintetizada en muchos órganos y tejidos del organismo incluida la glándula pineal. La regulación de la síntesis de melatonina por la pineal se conoce bien, y depende principalmente del fotoperíodo. Durante el día, la síntesis de melatonina está inhibida, mientras que la oscuridad activa el enzima limitante de su síntesis, aumentando su producción y secreción a la sangre y el líquido cefalorraquídeo. En consecuencia, el fotoperíodo induce un ritmo circadiano de melatonina plasmática con una acrofase alrededor de las 02:00 h en humanos. Este ritmo de melatonina sincroniza una serie de ritmos endocrinos y no endocrinos tales como la reproducción estacional en animales y el ciclo sueño-vigilia. Recientemente se ha demostrado que la melatonina ejerce otras funciones incluyendo la modulación del sistema inmune. En los últimos años han aparecido una gran cantidad de publicaciones demostrando tanto in vitro como in vivo, el efecto antioxidante y depurador de radicales libres de la melatonina. Probablemente, estas funciones antioxidantes de la melatonina reflejan una función protectora frente al estrés oxidativo en aquellos órganos y tejidos que la producen. El ritmo circadiano de secreción de melatonina en plasma puede alterarse en varias enfermedades psiquiátricas y su uso terapéutico se ha planteado en patologías que cursan con alteraciones del sueño, enfermedades neurodegenerativas y cáncer. El objetivo de este trabajo es revisar el papel de la melatonina en el tratamiento del trastorno depresivo mayor y en otras patologías psiquiátricas (AU)

Melatonin is a molecule synthesized in many organs and tissues, including the pineal gland. The regulation of the melatonin synthesis by the pineal is well known, and it mainly depends on the photoperiod. During daytime, melatonin synthesis is inhibited, whereas the darkness activates the limiting enzyme of the melatonin synthesis, increasing its production and release to blood and cerebrospinal fluid. Consequently, the photoperiod induces a circadian rhythm in melatonin levels with an acrophase at 02:00 h in humans. The circadian rhythm of melatonin synchronizes a number of endocrine and non-endocrine rhythms including the seasonal periods of reproduction in animals and the sleep-wake cycle. It was recently showed that melatonin exerts other functions including the modulation of the immune system. For the last years many publications have shown the antioxidant and free radical scavenger properties of the indoleamine, either in vitro or in vivo. Probably, the antioxidant functions of melatonin reflect a protective role against oxidative damage into the organs and tissues that produce this molecule. The circadian rhythm of plasma melatonin may be altered in several psychiatric disorders. Therapeutical use of melatonin has been implicated in sleep disorders, neurodegenerative diseases and cancer. The aim of this review is to uptake melatonin's role in the treatment of mayor depressive disorder and other psychiatric disorders (AU)

An in vitro study of hematopoietic progenitor cells from peripheral blood of rabbits.

The purpose of this study was to isolate and cultivate a subpopulation of pluripotent stem cells (PSCs) from the peripheral blood of rabbits, which are frequently used in veterinary research as an animal model. Pluripotent stem cells, as described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific from other hematopoietic stem cells. Commonly used human commercial media has been researched for culturing rabbit PSCs. These findings allow us to contemplate the direct application of this simple and standardized methodology in several areas of study, such as of the pharmacological effect of many drugs on hematopoietic cells, veterinary practice, and even the study of new strategies in cellular therapy for some human diseases.

Neuronal activity-dependent membrane traffic at the neuromuscular junction.

During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates beta-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ.