Amiodarona causa vasodilatação dependente do endotélio em artérias coronárias caninas

OBJETIVO: Avaliar os efeitos vasodilatadores da amiodarona em artérias coronárias caninas empregando soluções de amiodarona dissolvida em polisorbato 80 ou em água. MÉTODOS: Anéis de artéria coronária, com e sem o endotélio íntegro, foram imersos em solução de krebs e conectadas a um transdutor para aferição de força isométrica promovida por contração vascular. As artérias foram expostas a concentrações crescentes de polisorbato 80, amiodarona dissolvida em água, amiodarona dissolvida em polisorbato 80 e uma apresentação comercial da amiodarona (Cordarone®). Os experimentos foram conduzidos na presença e na ausência dos seguintes bloqueadores enzimáticos: apenas indometacina, Nômega-nitro-L-arginina associada à indometacina e apenas Nômega-nitro-L-arginina. RESULTADOS: O polisorbato 80 causou pequeno relaxamento não dependente do endotélio. O Cordarone®, a amiodarona dissolvida em água e em polisorbato 80 promoveram relaxamento dependente do endotélio, que foi de maior magnitude para a amiodarona dissolvida em polisorbato e para o Cordarone®. Apenas a associação de indometacina com a Nômega-nitro-L-arginina foi capaz de abolir o relaxamento dependente do endotélio provocado pela amiodarona dissolvida em polisorbato 80. CONCLUSÃO: Os resultados obtidos indicam que a vasodilatação promovida pela amiodarona em artérias coronárias caninas é causada principalmente pela estimulação da liberação de óxido nítrico e fatores endoteliais relaxantes dependentes das ciclo-oxigenases.

Tryptophan metabolism in tsetse flies and the consequences of its derangement

Literature comparing salmon and wild type Glossina morsitans morsitans and that comparing tan and wild type Glossina palpalis palpalis is reviewed. New information is presented on behaviour and biochemistry of salmon and wild type G. m. morsitans. The eye color mutants result from two lesions in the tryptophan to xanthommatin pathway: lack of tryptophan oxygenase in G. m morsitans and failure to produce or retain xanthommatin in eyes (but not in testes) of G. p. palpalis. The salmon allele in G. m. morsitans is pleiotropic and profoundly affects many aspects of fly biology including longevity, reproductive capacity, vision, vectorial capacity and duration of flight, but not circadian rhythms. The tan allele in G. p. palpalis has little effect upon the biology of flies under laboratory conditions, except that tan flies appear less active than normal. Adult tsetse flies metabolize tryptophan to kynurenine which is excreted; fluctuations in activities of the enzymes producing kynurenine suggest this pathway is under metabolic control.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genes

Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Role of enhanced detoxication in a deltamethrin-resistant population of Triatoma infestans (Hemiptera, Reduviidae) from Argentina

Deltamethrin and other pyrethroids have been extensively used in Argentina since 1980, for the chemical control of Triatoma infestans Klug (Hemiptera: Reduviidae). Recently, resistance to deltamethrin was detected in field populations by the survival of bugs exposed by topical application to the diagnostic dose estimated on the CIPEIN susceptible strain. Results of the current study showed low resistant ratios (RRs) to deltamethrin for the resistant populations (RR ranged from 2.0 for San Luis colony to 7.9 for Salta colony). Biochemical studies were made on the most resistant colony (Salta) and the susceptible strain (CIPEIN), in order to establish the importance of degradative mechanisms as a cause of the detected resistance. Esterase activity was measured on 3 days old first instars through phenylthioacetate and a-naphtyl acetate activities. The results showed a significant difference in no cholinesterase esterase activity from susceptible (7.6 ± 0,7 µM S./i.min.) and Salta resistant colony (9.5 ± 0.8 µM S./i.min.). Cytochrome P450 mono-oxygenase (P450) activity was measured on individual insects through ethoxycoumarine deethylase (ECOD) activity using a fluorescence micro plate reader. The dependence of ECOD activity on age and body region of the nymphs, and pH and time of incubation were studied in order to optimize the measurement. As a result, comparative studies were performed on abdomens of 2 days old first instars at pH 7.2 and 4 h incubation time. ECOD activity of first nymphs was significantly lower in the susceptible colony (61.3 ± 9.08 pg ECOD/ insect) than in the resistant one (108.1± 5.7 pg ECOD/ insect). These results suggest that degradative esterases (no-cholinesterase) and mono-oxygenases cytochrome P450, play an important role in the resistance to deltamethrin in Salta colony from Argentina.

Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures.

Ochratoxin A (OTA), a mycotoxin and widespread food contaminant, is known for its patent nephrotoxicity and potential neurotoxicity. Previous observations in vitro showed that in the CNS, glial cells were particularly sensitive to OTA. In the search for the molecular mechanisms underlying OTA neurotoxicity, we investigated the relationship between OTA toxicity and glial reactivity, in serum-free aggregating brain cell cultures. Using quantitative reverse transcriptase-polymerase chain reaction to analyze changes in gene expression, we found that in astrocytes, non cytotoxic concentrations of OTA down-regulated glial fibrillary acidic protein, while it up-regulated vimentin and the peroxisome proliferator-activated receptor-gamma expression. OTA also up-regulated the inducible nitric oxide synthase and the heme oxygenase-1. These OTA-induced alterations in gene expression were more pronounced in cultures at an advanced stage of maturation. The natural peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-delta(12,14) prostaglandin J2, and the cyclic AMP analog, bromo cyclic AMP, significantly attenuated the strong induction of peroxisome proliferator-activated receptor-gamma and inducible nitric oxide synthase, while they partially reversed the inhibitory effect of OTA on glial fibrillary acidic protein. The present results show that OTA affects the cytoskeletal integrity of astrocytes as well as the expression of genes pertaining to the brain inflammatory response system, and suggest that a relationship exists between the inflammatory events and the cytoskeletal changes induced by OTA. Furthermore, these results suggest that, by inducing an atypical glial reactivity, OTA may severely affect the neuroprotective capacity of glial cells.

A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis

The intestinal anti-inflammatory effects of two probiotics isolated from breast milk, Lactobacillus reuteri and L. fermentum, were evaluated and compared in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis. Colitis was induced in rats by intracolonic administration of 10 mg TNBS dissolved in 50% ethanol (0.25 ml). Either L. reuteri or L. fermentum was daily administered orally (5 x 10(8) colony-forming units suspended in 0.5 ml skimmed milk) to each group of rats (n 10) for 3 weeks, starting 2 weeks before colitis induction. Colonic damage was evaluated histologically and biochemically, and the colonic luminal contents were used for bacterial studies and for SCFA production. Both probiotics showed intestinal anti-inflammatory effects in this model of experimental colitis, as evidenced histologically and by a significant reduction of colonic myeloperoxidase activity (P<0.05). L. fermentum significantly counteracted the colonic glutathione depletion induced by the inflammatory process. In addition, both probiotics lowered colonic TNFalpha levels (P<0.01) and inducible NO synthase expression when compared with non-treated rats; however, the decrease in colonic cyclo-oxygenase-2 expression was only achieved with L.fermentum administration. Finally, the two probiotics induced the growth of Lactobacilli species in comparison with control colitic rats, but the production of SCFA in colonic contents was only increased when L. fermentum was given. In conclusion, L. fermentum can exert beneficial immunomodulatory properties in inflammatory bowel disease, being more effective than L. reuteri, a probiotic with reputed efficacy in promoting beneficial effects on human health.

VEGF gene expression in adult human thymus fat: a correlative study with hypoxic induced factor and cyclooxygenase-2.

It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to adipogenesis and angiogenesis.

The systemic administration of oleoylethanolamide exerts neuroprotection of the nigrostriatal system in experimental Parkinsonism.

Oleoylethanolamide (OEA) is an agonist of the peroxisome proliferator-activated receptor α (PPARα) and has been described to exhibit neuroprotective properties when administered locally in animal models of several neurological disorder models, including stroke and Parkinson's disease. However, there is little information regarding the effectiveness of systemic administration of OEA on Parkinson's disease. In the present study, OEA-mediated neuroprotection has been tested on in vivo and in vitro models of 6-hydroxydopamine (6-OH-DA)-induced degeneration. The in vivo model was based on the intrastriatal infusion of the neurotoxin 6-OH-DA, which generates Parkinsonian symptoms. Rats were treated 2 h before and after the 6-OH-DA treatment with systemic OEA (0.5, 1, and 5 mg/kg). The Parkinsonian symptoms were evaluated at 1 and 4 wk after the development of lesions. The functional status of the nigrostriatal system was studied through tyrosine-hydroxylase (TH) and hemeoxygenase-1 (HO-1, oxidation marker) immunostaining as well as by monitoring the synaptophysin content. In vitro cell cultures were also treated with OEA and 6-OH-DA. As expected, our results revealed 6-OH-DA induced neurotoxicity and behavioural deficits; however, these alterations were less severe in the animals treated with the highest dose of OEA (5 mg/kg). 6-OH-DA administration significantly reduced the striatal TH-immunoreactivity (ir) density, synaptophysin expression, and the number of nigral TH-ir neurons. Moreover, 6-OH-DA enhanced striatal HO-1 content, which was blocked by OEA (5 mg/kg). In vitro, 0.5 and 1 μM of OEA exerted significant neuroprotection on cultured nigral neurons. These effects were abolished after blocking PPARα with the selective antagonist GW6471. In conclusion, systemic OEA protects the nigrostriatal circuit from 6-OH-DA-induced neurotoxicity through a PPARα-dependent mechanism.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

Cellular distribution of Hsp70 expression in rat skeletal muscles. Effects of moderate exercise training and chronic hypoxia.

Rat hindlimb muscles constitutively express the inducible heat shock protein 72 (Hsp70), apparently in proportion to the slow myosin content. Since it remains controversial whether chronic Hsp70 expression reflects the overimposed stress, we investigated Hsp70 cellular distribution in fast muscles of the posterior rat hindlimb after (1) mild exercise training (up to 30 m/min treadmill run for 1 h/day), which induces a remodeling in fast fiber composition, or (2) prolonged exposure to normobaric hypoxia (10%O(2)), which does not affect fiber-type composition. Both conditions increased significantly protein Hsp70 levels in the skeletal muscle. Immunohistochemistry showed the labeling for Hsp70 in subsets of both slow/type 1 and fast/type 2A myofibers of control, sedentary, and normoxic rats. Endurance training increased about threefold the percentage of Hsp70-positive myofibers (P < 0.001), and changed the distribution of Hsp70 immunoreactivity, which involved a larger subset of both type 2A and intermediate type 2A/2X myofibers (P < 0.001) and vascular smooth muscle cells. Hypoxia induced Hsp70 immunoreactivity in smooth muscle cells of veins and did not increase the percentage of Hsp70-positive myofibers; however, sustained exposure to hypoxia affected the distribution of Hsp70 immunoreactivity, which appeared detectable in a very small subset of type 2A fibers, whereas it concentrated in type 1 myofibers (P < 0.05) together with the labeling for heme-oxygenase isoform 1, a marker of oxidative stress. Therefore, the chronic induction of Hsp70 expression in rat skeletal muscles is not obligatory related to the slow fiber phenotype but reveals the occurrence of a stress response.

Low temperature impact on photosynthetic parameters of coffee genotypes

The objective of this work was to evaluate photoprotective mechanisms related to low positive temperatures in Coffea canephora (Conilon clones 02 and 153) and C. arabica ('Catucaí' IPR 102) genotypes, involved in cold temperature tolerance. To accomplish this, one-year-old plants were successively submitted to: temperature decrease of 0.5ºC day-1, from 25/20ºC to 13/8ºC; a three-day chilling cycle at 13/4ºC; and a recovery period of 14 days (25/20ºC). During the experiment, leaf gas exchange, chlorophyll a fluorescence and leaf photosynthetic pigment content were evaluated. Total activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and ribulose-5-phosphate kinase (Ru5PK) were quantified to measure the activity of photosynthesis key enzymes. All genotypes showed low temperature sensitivity, but displayed diverse cold impact and recovery capabilities regarding the photosynthetic-related parameters studied. Catucaí IPR 102 cultivar showed better ability to cope with cold stress than the Conilon clones, especially Conilon 02, and had full recovery of leaf gas exchange, fluorescence parameters, enzymatic activity, and higher contents of the photoprotective pigments zeaxanthin and lutein.