Bile duct ligation and oxidative stress in the rat: effects in liver and kidney

In the liver, seven days of bile duct ligation (BDL) decreases the cytochrome P-450 content and the UDP-glucuronyl transferase activity. Also, a decrease in the water soluble antioxidant mechanism reflected in the activities of the enzymes superoxide dismutase (SOD), catalase and the glutathione peroxidase (GTPx) was found in the liver but not in the kidney. Despite an increase in the amount of the GSH in the liver, increased lipid peroxidation is produced in the BDL rats, as indicated by the levels of malondialdehyde (MDA). The kidney responded in a different way to cholestasis, decreasing only the UDP-glucuronyl transferase activity and increasing the levels of GSH and MDA. In the red blood cells the activity of the antioxidant enzymes SOD, GTPx and catalase and the content of GSH were not modulated by cholestasis. In conclusion, disturbance of the oxidant-antioxidant balance might be responsible for cholestatic liver injury and impaired renal function in BDL rats.     

Increased Stability and Catalytic Efficiency of Yeast Hexokinase Upon Interaction with Zwitterionic Micelles. Kinetics and Conformational Studies

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micellesof lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammoniumpropanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at35°C. The thermal inactivation kinetics followed one-exponentialdecay. The effect of ligands on protecting the enzyme against inactivationfollowed the order: glucose>glucose/Mg2+>ATP/Mg2+Mg2+bufferonly. Both LPC and HPS micelles increased the enzyme stability only whenthe incubation medium contained glucose or glucose/Mg2+,suggesting that the protein conformation is a key prerequisite for theenzyme-micelle interaction to take place. This enzyme-micelle interactionresulted in an increased catalytic efficiency (with a decrease in Km forATP and increase in Vmax as well as in changes on the tertiary (intrinsicfluorescence) structure of the yeast hexokinase.     

Signs of Ancient and Modern Exon-Shuffling Are Correlated to the Distribution of Ancient and Modern Domains Along Proteins

Exon-shuffling is an important mechanism accounting for the origin of many new proteins in eukaryotes. However, its role in the creation of proteins in the ancestor of prokaryotes and eukaryotes is still debatable. Excess of symmetric exons is thought to represent evidence for exon-shuffling since the exchange of exons flanked by introns of the same phase does not disrupt the reading frame of the host gene. In this report, we found that there is a significant correlation between symmetric units of shuffling and the age of protein domains. Ancient domains, present in both prokaryotes and eukaryotes, are more frequently bounded by phase 0 introns and their distribution is biased towards the central part of proteins. Modern domains are more frequently bounded by phase 1 introns and are present predominantly at the ends of proteins. We propose a model in which shuffling of ancient domains mainly flanked by phase 0 introns was important in the ancestor of eukaryotes and prokaryotes, during the creation of the central part of proteins. Shuffling of modern domains, predominantly flanked by phase 1 introns, accounted for the origin of the extremities of proteins during eukaryotic evolution.     

Flow mixing enhancement from balloon pulsations in an intravenous oxygenator

Computational investigations of flow mixing and oxygen transfer characteristics in an intravenous membrane oxygenator (IMO) are performed by direct numerical simulations of the conservation of mass, momentum, and species equations. Three-dimensional computational models are developed to investigate flow-mixing and oxygen-transfer characteristics for stationary and pulsating balloons, using the spectral element method. For a stationary balloon, the effect of the fiber placement within the fiber bundle and the number of fiber rings is investigated. In a pulsating balloon, the flow mixing characteristics are determined and the oxygen transfer rate is evaluated. For a stationary balloon, numerical simulations show two well-defined flow patterns that depend on the region of the IMO device. Successive increases of the Reynolds number raise the longitudinal velocity without creating secondary flow. This characteristic is not affected by staggered or non-staggered fiber placement within the fiber bundle. For a pulsating balloon, the flow mixing is enhanced by generating a three-dimensional time-dependent flow characterized by oscillatory radial, pulsatile longitudinal, and both oscillatory and random tangential velocities. This three-dimensional flow increases the flow mixing due to an active time-dependent secondary flow, particularly around the fibers. Analytical models show the fiber bundle placement effect on the pressure gradient and flow pattern. The oxygen transport from the fiber surface to the mean flow is due to a dominant radial diffusion mechanism, for the stationary balloon. The oxygen transfer rate reaches an asymptotic behavior at relatively low Reynolds numbers. For a pulsating balloon, the time-dependent oxygen-concentration field resembles the oscillatory and wavy nature of the time-dependent flow. Sherwood number evaluations demonstrate that balloon pulsations enhance the oxygen transfer rate, even for smaller flow rates.     

An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.     

The effects of selective and nonselective cyclooxygenase inhibitors on endothelin-1-induced fever in rats

It was previously shown that sustained fever can be induced in rats by central injection of endothelin-1 (ET-1). This peptide appears to participate in the mechanism(s) of LPS-induced fever, which is reduced by pretreatments with ET(B) receptor antagonists. In this study, we compared the effects of a nonselective cyclooxygenase (COX) inhibitor, indomethacin, with those of two selective COX-2 inhibitors, celecoxib and lumiracoxib, on ET-1-induced fever in rats. Fever induced in conscious animals by ET-1 (1 pmol icv) or LPS (5 mug/kg iv) was prevented by pretreatments with celecoxib (5 and 10 mg/kg) or lumiracoxib (5 mg/kg) given by oral gavage 1 h before stimuli. Lower doses of celecoxib had partial (2.5 mg/kg) or no effect (1 mg/kg). Indomethacin (2 mg/kg ip) partially inhibited fever induced by LPS but had no effect on ET-1-induced fever. The levels of PGE(2) and PGF(2alpha) in the cerebrospinal fluid (CSF) of pentobarbital sodium-anesthetized rats were significantly increased 3 h after the injection of LPS or ET-1. The latter increase was abolished by celecoxib at all tested doses and by indomethacin. In conclusion, selective COX-2 inhibitors were able to prevent ET-1-induced fever, indicating a role for COX-2 in this phenomenon. However, the fact that reduced CSF PG levels obtained with indomethacin and a low dose of celecoxib are not accompanied by changes in fever induced by ET-1, along with the lack of inhibitory effects of indomethacin on ET-1 fever, suggests that the latter might also involve COX-2-independent mechanisms.     

Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells

We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentrations tested. In contrast, long-term CO₂ production was reduced by FATP1 and FAT at all doses of palmitate and at the lower concentrations of oleate. Neither FATP1 nor FAT markedly altered the production of acid-soluble metabolic intermediates from palmitate or oleate. The intracellular localization of fusion constructs of FATP1 and FAT with enhanced green fluorescent protein (EGFP) was examined. Independently of fatty acid treatment, FATPGFP was observed throughout the cytosol in a reticular pattern and concentrated in the perinuclear region, partly overlapping with the Golgi marker GM-130. FATGFP was found in the extracellular membrane and in cytosolic vesicles not coincident with GM-130. Neither FATP1 nor FAT proteins colocalized with lipid droplets in oleate-treated cells. We conclude that whereas FAT is localized on the extracellular membrane, FATP1 is active in the cytosol and imports fatty acids into myotubes. Overall, both FATP1 and FAT stimulated transport and consumption of palmitate and oleate, which they channeled away from complete oxidation and toward TAG synthesis. palmitate; oleate; fatty acid binding proteins; skeletal muscle     

Neuroprotection and Protein Damage Prevention by Estradiol Replacement in Rat Hippocampal Slices Exposed to Oxygen-Glucose Deprivation

Here we investigated the effects of estradiol replacement in ovariectomized female rats using hippocampal slices exposed to oxygen-glucose deprivation (OGD). OGD induced lactate dehydrogenase (LDH) release to the incubation medium, what was assumed as a parameter of cellular death. In the estradiol-treated group the LDH release was markedly decreased by 23% as compared to the vehicle-treated group. In attempt to study a possible mechanism by which estradiol acts, we investigated some parameters of oxidative stress. In both vehicle-treated and estradiol-treated groups, OGD significantly increased the free radical production by 34% and 16%, respectively, although no significant differences on total antioxidant capacity were observed. Interestingly, estradiol replacement prevented the significant reduction in tryptophan and tyrosine contents caused by OGD observed in vehicle-treated animals. Our results show that estradiol replacement in ovariectomized female rats decreases cellular susceptibility to an ischemic-like injury and suggest a role for the hormone on protein damage prevention.     

Different roles of adenosine A1, A2A and A3 receptors in controlling kainate-induced toxicity in cortical cultured neurons

Adenosine is a neuromodulator that can control brain damage through activation of A(1), A(2A) and A(3) receptors, which are located in both neurons and other brain cells. We took advantage of cultured neurons to investigate the role of neuronal adenosine receptors in the control of neurotoxicity caused by kainate and cyclothiazide. Both A(1), A(2A) and A(3) receptors were immunocytochemically identified in cortical neurons. Activation of A(1) receptors with 100 nM CPA did not modify the extent of neuronal death whereas the A(1) receptor antagonist, DPCPX (50 nM), attenuated neurotoxicity by 28 +/- 5%, and effect similar to that resulting from the removal of endogenous adenosine with 2U/ml of adenosine deaminase (27 +/- 3% attenuation of neurotoxicity). In the presence of adenosine deaminase, DPCPX had no further effect and CPA now exacerbated neurotoxicity by 42 +/- 4%. Activation of A(2A) receptor with 30 nM CGS21680 attenuated neurotoxicity by 40 +/- 8%, an effect prevented by the A(2A) receptor antagonists, SCH58261 (50 nM) or ZM241385 (50 nM), which by themselves were devoid of effect. Finally, neither A(3) receptor activation with Cl-IB-MECA (100-500 nM) nor blockade with MRS1191 (5 microM) modified neurotoxicity. These results show that A(1) receptor activation enhances and A(2A) receptor activation attenuates neurotoxicity in cultured cortical neurons, indicating that these two neuronal adenosine receptors directly control neurodegeneration. Interestingly, the control by adenosine of neurotoxicity in cultured neurons is similar to that observed in vivo in newborn animals and is the opposite of what is observed in adult brain preparations where A(1) receptor activation and A(2A) receptor blockade are neuroprotective.     

Pretraining but not Preexposure to the Task Apparatus Prevents the Memory Impairment Induced by Blockade of Protein Synthesis,PKA or MAP Kinase in Rats

Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA footshock, 24 h training-test interval). Fifteen minutes before or 0, 1.5 or 3 hours after training, animals received a 0.8 l intrahippocampal infusion of the protein synthesis inhibitor anisomycin (80 g), the PKA inhibitor Rp-cAMP (0.05 g), the MAPK kinase inhibitor PD 098059 (50 M solution) or vehicle (phosphate buffer in saline, pH 7.4). Anisomycin, Rp-cAMP and PD 098059 impaired retention test performance in animals injected at different times, prior and after training. Pretraining with a low footshock intensity (0.2 mA) 24 h before training prevented the amnestic effect of all drugs studied. However, simple preexposure to the inhibitory avoidance apparatus did not alter the amnestic effects of all drugs. The results suggest that memory processing requires hippocampal mechanisms dependent on protein synthesis, PKA and MAPK kinase at different times after training. These findings suggest that weak training must be sufficient to produce some lasting cellular expression of the experience so that the enhancement of consolidation of a previously acquired memory is not dependent on protein synthesis, PKA or MAPK.