M. Ángeles Peinado

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Endothelial NOS-derived nitric oxide prevents injury resulting from reoxygenation in the hypoxic lung

Abstract To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.

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. (Mol Cell Biochem 268: 33–43, 2005)

Immunolocalization of the HNK-1 epitope in the autonomic innervation to the liver and upper digestive tract of the developing rat embryo.

The immunohistochemical analysis of the HNK-1 epitope presence in the liver and upper digestive tract nerves was carried out in 12- to 18-day-old rat embryos embedded in acrylamide–agarose and observed with laser scanning confocal microscopy. The vagus and sympathetic trunk were intensely immunostained at all ages; branches of both structures were also HNK-1 positive, and ramified ventrocaudally following the course of the thoracic and abdominal aorta, caval vein, portal vein and ductus venosus. As early as day 12, some immunostained cells were seen in the mesentery that formed the enteric nervous system. Clearly immunostained HNK-1-immunoreactive fibres were detected innervating the digestive wall after day 14, forming both myenteric and submucosal plexuses. After day 16, the Glisson sheath showed streams of HNK-1-positive fibres coming from dorsal areas, lining the peritoneal surface of the diaphragm, invading the capsule, and ramifying superficially around the lobes of the liver. We saw no immunoreactive structures pervading the hepatic lobes at all ages studied, with the exception of occasional HNK-1-positive cells in the superficial parenchyma, which were visualized after 16 days of gestation. Our findings can help to understand the development of the gastrointestinal and liver innervation in the rat.

Nitric oxide averts hypoxia‐induced damage during reoxygenation in rat heart

Nitric oxide (NO), synthesized by the hemoproteins NO synthases (NOS), is known to play important roles in physiological and pathological conditions in the heart, including hypoxia/reoxygenation (H/R). This work investigates the role that endogenous NO plays in the cardiac H/R‐induced injury. A follow‐up study was conducted in Wistar rats subjected to 30 min of hypoxia, with or without prior treatment using the nonselective NOS inhibitor L‐NAME (1.5 mM). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analysing parameters of cell, and tissue damage (lipid peroxidation, apoptosis, and protein nitration), as well as in situ NOS activity and NO production (NOx). The results showed that after L‐NAME administration, in situ NOS activity was almost completely eliminated in all the experimental groups, and consequently, NOx levels fell. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. These results reveal that NOS inhibition exacerbates the peroxidative and apoptotic damage observed before the treatment with L‐NAME in the hypoxic heart, pointing to a cardioprotective role of NOS‐derived NO against H/R‐induced injury. These findings could open the possibility of future studies to design new therapies for H/R‐dysfunctions based on NO‐pharmacology. Microsc. Res. Tech., 2011.

Endogenous Nitric Oxide Can Act as Beneficial or Deleterious in the Hypoxic Lung Depending on the Reoxygenation Time

Nitric oxide (NO) has been implicated in many pathophysiological situations in the lung, including hypoxia/reoxygenation. This work seeks to clarify the current controversy concerning the double protective/toxic role of endogenous NO under hypoxia/reoxygenation situations in the lung by using a nitric oxide synthase (NOS) inhibitor, in a novel approach to address the problems raised from assaults under such circumstances. A follow‐up study was conducted in Wistar rats submitted to hypoxia/reoxygenation (hypoxia for 30 min; reoxygenation of 0 h, 48 h, and 5 days), with or without prior treatment using the nonselective NOS inhibitor L‐NAME (1.5 mM, in drinking water). Lipid peroxidation, apoptosis level, protein nitration, in situ NOS activity and NO production (NOx) were analyzed. This is the first work to focus on the time‐course effects of L‐NAME in the adult rat lung submitted to hypoxia/reoxygenation. The results showed that after L‐NAME administration, in situ NOS activity was almost completely eliminated and consequently, NOx levels fell. Lipid peroxidation and the percentage of apoptotic cells rose at the earliest reoxygenation time (0 h), but decreased in the later period (48 h and 5 days). Also nitrated protein expression decreased at 48 h and 5 days posthypoxia. These results suggest that NOS‐derived NO exerts two different effects on lung hypoxia/reoxygenation injury depending on the reoxygenation time: NO has a beneficial role just after the hypoxic stimulus and a deleterious effect in the later reoxygenation times. Moreover, we propose that this dual role of NO depends directly on the producer NOS isoform. Anat Rec, 2010.

Polyphenol Oxidase and Oleuropein in Olives and their Changes During Olive Ripening

Publisher Summary Phenols are secondary metabolites of plants widely distributed throughout all plant organs and have important functions in the metabolism and physiology of plants. This complex group of substances has structures that vary from single phenolic molecules such as hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] to highly polymerized compounds such as lignins. These compounds have diverse and important functions such as: the maintenance of plant integrity (lignins), floral pigmentation (flavonoids), antibiotics (phytoalexins), and plant defense against pathogens or symbionts. The occurrence of these substances in food is broadly variable and reaches high levels in the olive fruit and oil. Currently, there is keen interest in dietary polyphenols due to their antioxidant capacity and consequent benefits to human health. Oleuropein, the main phenol of the olive, is a heterosidic ester of β-glucosylated elenolic acid and hydroxytyrosol. Many of the nutritional and organoleptic properties of olive oil depend on the content of phenols in general and of oleuropein and hydroxytyrosol in particular. Oleuropein and hydroxytyrosol have a high antioxidant capacity with high free-radical scavenging activity. The amount of phenols in olive oil is considered as an index of the quality of this product.