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Juan B Barroso

Publications and source records attributed to Juan B Barroso.

12 recordsLinked to original sources
PubMed2006

Downregulation in the expression of the serine dehydratase in the rat liver during chronic metabolic acidosis.

Blood pH controls the activity of important regulatory enzymes in the metabolism. Serine dehydratase (SerDH) transforms l-serine into pyruvate and ammonium and is involved in the regulation of gluconeogenesis from serine in the rat liver. In this work, we investigate the effect of chronic metabolic acidosis on the kinetics, specific protein level, tissue location, and mRNA levels of rat liver SerDH. Experimental acidosis was induced in rats by ingestion of 0.28 M ammonium chloride solution for 10 days. Acidosis significantly (P<0.05) decreased SerDH activity at all substrate concentrations assayed. Moreover, the Vmax value was 38.50+/-3.51 mU/mg (n=7) of mitochondrial protein in the acidotic rats and 92.49+/-6.79 mU/mg (n=7) in the control rats. Western blot analysis revealed a significant reduction (14%) in the level of SerDH protein content in the rat liver during acidosis. Immunohistochemical analysis showed that SerDH location did not change in response to chronic metabolic acidosis and confirmed previous results on SerDH protein levels. Moreover, the SerDH mRNA level, estimated by RT-PCR, was also significantly 33.8% lower than in control. These results suggest that during experimental acidosis a specific repression of rat-liver SerDH gene transcription could result, lowering the amount and activity of this enzyme. The changes found in SerDH expression are part of an overall metabolic response of liver to maintain acid-base homeostasis during acidosis.

Acid-Base Equilibrium↗
PubMed2006

The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves.

Superoxide dismutase (SOD) is a key antioxidant enzyme present in prokaryotic and eukaryotic cells as a first line of defense against the accumulation of superoxide radicals. In olive leaves, the SOD enzymatic system was characterized and was found to be comprised of three isozymes, an Mn-SOD, an Fe-SOD and a CuZn-SOD. Transcript expression analysis of whole leaves showed that the three isozymes represented 82, 17 and 0.8% of the total SOD expressed, respectively. Using the combination of laser capture microdissection (LCM) and real-time quantitative reverse transcription-PCR (RT-PCR), the expression of these SOD isozymes was studied in different cell types of olive leaves, including spongy mesophyll, palisade mesophyll, xylem and phloem. In spongy mesophyll cells, the isozyme proportion was similar to that in whole leaves, but in the other cells the proportion of expressed SOD isozymes was different. In palisade mesophyll cells, Fe-SOD was the most abundant, followed by Mn-SOD and CuZn-SOD, but in phloem cells Mn-SOD was the most prominent isozyme, and Fe-SOD was present in trace amounts. In xylem cells, only the Mn-SOD was detected. On the other hand, the highest accumulation of superoxide radicals was localized in vascular tissue which was the tissue with the lowest level of SOD transcripts. These data show that in olive leaves, each SOD isozyme has a different gene expression depending on the cell type of the leaf.

DNA, Plant↗
PubMed2006

Localization of S-nitrosoglutathione and expression of S-nitrosoglutathione reductase in pea plants under cadmium stress.

S-nitrosoglutathione (GSNO) is considered a natural nitric oxide (NO.) reservoir and a reactive nitrogen intermediate in animal cells, but little is known about this molecule and its metabolism in plant systems. In this work, using pea plants as a model system, the presence of GSNO in collenchyma cells was demonstrated by an immunohistochemical method. When pea plants were grown with a toxic Cd concentration (50 microM) the content of GSNO in collenchyma cells was drastically reduced. Determination of the nitric oxide (NO.) and gluthathione contents in leaves by confocal laser scanning microscopy and HPLC, respectively, showed a marked decrease of both compounds in plants treated with cadmium. The analysis of the S-nitrosoglutathione reductase (GSNOR) activity and its transcript expression in leaves showed a reduction of 31% by cadmium. These results indicate that GSNO is associated with a specific plant cell type, and this metabolite and its related catabolic activity, GSNOR, are both down-regulated under Cd stress.

Cadmium↗
PubMed2006

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development.

Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from L-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.

Arginine↗
PubMed2006

The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants.

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.

Antioxidants↗
PubMed2005

Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional analysis under environmental stress conditions.

In plant cells, ascorbate is a major antioxidant that is involved in the ascorbate-glutathione cycle. Monodehydroascorbate reductase (MDAR) is the enzymatic component of this cycle involved in the regeneration of reduced ascorbate. The identification of the intron-exon organization and the promoter region of the pea (Pisum sativum) MDAR 1 gene was achieved in pea leaves using the method of walking polymerase chain reaction on genomic DNA. The nuclear gene of MDAR 1 comprises nine exons and eight introns, giving a total length of 3,770 bp. The sequence of 544 bp upstream of the initiation codon, which contains the promoter and 5' untranslated region, and 190 bp downstream of the stop codon were also determined. The presence of different regulatory motifs in the promoter region of the gene might indicate distinct responses to various conditions. The expression analysis in different plant organs by northern blots showed that fruits had the highest level of MDAR. Confocal laser scanning microscopy analysis of pea leaves transformed with Agrobacterium tumefaciens having the binary vectors pGD, which contain the autofluorescent proteins enhanced green fluorescent protein and enhanced yellow fluorescent protein with the full-length cDNA for MDAR 1 and catalase, indicated that the MDAR 1 encoded the peroxisomal isoform. The functional analysis of MDAR by activity and protein expression was studied in pea plants grown under eight stress conditions, including continuous light, high light intensity, continuous dark, mechanical wounding, low and high temperature, cadmium, and the herbicide 2,4-dichlorophenoxyacetic acid. This functional analysis is representative of all the MDAR isoforms present in the different cell compartments. Results obtained showed a significant induction by high light intensity and cadmium. On the other hand, expression studies, performed by semiquantitative reverse transcription-polymerase chain reaction demonstrated differential expression patterns of peroxisomal MDAR 1 transcripts in pea plants grown under the mentioned stress conditions. These findings show that the peroxisomal MDAR 1 has a differential regulation that could be indicative of its specific function in peroxisomes. All these biochemical and molecular data represent a significant step to understand the specific physiological role of each MDAR isoenzyme and its participation in the antioxidant mechanisms of plant cells.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2005

Serine dehydratase expression decreases in rat livers injured by chronic thioacetamide ingestion.

Serine dehydratase (SerDH) is a gluconeogenic enzyme involved in the catabolism of serine, which is regulated by the composition of their diet and their hormonal status in rats. This study examines how chronic injury caused to the liver of rats by the ingestion of thioacetamide (TAA) affects SerDH protein, mRNA levels, enzyme kinetics and its tissue location. After 97 days' oral intake of TAA, the activity of SerDH at all substrate concentrations assayed was about 60% lower than in controls. No significant differences in Km values were found between the treated group and controls. Immunoblotting and immunohistochemistry revealed a significant reduction in the level of SerDH protein in the livers of the treated rats. SerDH was detected specifically in the periportal zone of the hepatic acinus and this location did not change in response to TAA treatment. The level of SerDH mRNA, quantified by reverse transcription and polymerase chain reaction, was significantly lower in treated rats than in the controls. The present findings suggest that the SerDH expression is rendered to be down regulatory during chronic liver injury induced by TAA. These results enhance our understanding about the biochemical mechanisms implied in the control and integration of serine catabolism during liver injury in rat.

Animals↗
PubMed2004

Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants.

The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.

Animals↗
PubMed2004

Nitric oxide and nitric oxide synthase activity in plants.

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.

Nitric Oxide↗
PubMed2003

Plant peroxisomes, reactive oxygen metabolism and nitric oxide.

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.

Nitric Oxide↗
PubMed2002

Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes.

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O2*(-)) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals O2*(-). Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP(+) can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO*), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).

Antioxidants↗