Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.     

Chronic hyperhomocysteinemia impairs vascular function in ovariectomized rat carotid arteries

Homocysteine is an independent risk factor for coronary heart disease, as well as for cerebrovascular and peripheral vascular diseases. The purpose of this study was to investigate the effects of hyperhomocysteinemia (HHcy) on vascular reactivity within carotid artery segments isolated from ovariectomized female rats. Treatment with dl-Hcy thiolactone (1 g/kg body weight per day) reduced the phenylephrine-induced contraction of denuded rings. However, the treatment did not alter KCl-induced contractions, or relaxations induced by sodium nitroprusside or acetylcholine. We report elevated expressions of iNOS, eNOS, and nitrotyrosine in homocysteine-treated rat artery sections. Moreover, the inhibition of NOS by l-NAME, 1,400 W, or l-NNA restored phenylephrine-induced vasoconstriction in carotid artery segments from Hcy-treated rats. In conclusion, our findings show that severe HHCy can promote an acute decrease in the endothelium-independent contractile responses of carotid arteries to adrenergic agonists. This effect was restored by nitric oxide synthase inhibitors, which further supports the involvement of nitric oxide in HHcy-derived vascular dysfunction.     

High levels of active quiescin Q6 sulfhydryl oxidase (QSOX) are selectively present in fetal serum

The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.     

Mild mitochondrial uncoupling and calorie restriction increase fasting eNOS, akt and mitochondrial biogenesis

Enhanced mitochondrial biogenesis promoted by eNOS activation is believed to play a central role in the beneficial effects of calorie restriction (CR). Since treatment of mice with dinitrophenol (DNP) promotes health and lifespan benefits similar to those observed in CR, we hypothesized that it could also impact biogenesis. We found that DNP and CR increase citrate synthase activity, PGC-1α, cytochrome c oxidase and mitofusin-2 expression, as well as fasting plasma levels of NO^• products. In addition, eNOS and Akt phosphorylation in skeletal muscle and visceral adipose tissue was activated in fasting CR and DNP animals. Overall, our results indicate that systemic mild uncoupling activates eNOS and Akt-dependent pathways leading to mitochondrial biogenesis.     

A new species of Pelomus Reiss, 1989 (Diptera: Chironomidae: Chironominae) from the Neotropical Region,with emendation of the generic diagnosis

A new species of Pelomus Reiss, 1989 Reiss, F. 1989. ‘Pelomus gen. nov., ein weiterer potamobionter Vertreter des Harnischia-Komplexes aus dem Amazonasbecken (Diptera, Chironomidae)’. Acta Biologica Debrecina Oecologia Hungarica, 2: 305–314.  [Google Scholar] (Diptera: Chironomidae: Chironominae), P. sophiae sp. n., is described and figured as male, pupa and larva. Diagnoses for male and pupa of the genus are emended. The larvae, reared in the laboratory to obtain all life stages, were collected on bottom sand of reservoir and ponds, in southeast Brazil.     

Spatial Distribution of the Risk of Dengue and the Entomological Indicators in Sumaré, State of S?o Paulo,Brazil

Dengue fever is a major public health problem worldwide, caused by any of four virus (DENV-1, DENV-2, DENV-3 and DENV-4; Flaviviridae: Flavivirus), transmitted by Aedes aegypti mosquito. Reducing the levels of infestation by A. aegypti is one of the few current strategies to control dengue fever. Entomological indicators are used by dengue national control program to measure the infestation of A. aegypti, but little is known about predictive power of these indicators to measure dengue risk. In this spatial case-control study, we analyzed the spatial distribution of the risk of dengue and the influence of entomological indicators of A. aegypti in its egg, larva-pupa and adult stages occurring in a mid-size city in the state of São Paulo. The dengue cases were those confirmed by the city''s epidemiological surveillance system and the controls were obtained through random selection of points within the perimeter of the inhabited area. The values of the entomological indicators were extrapolated for the entire study area through the geostatistical ordinary kriging technique. For each case and control, the respective indicator values were obtained, according with its geographical coordinates and analyzed by using a generalized additive model. Dengue incidence demonstrated a seasonal behavior, as well as the entomological indicators of all mosquito''s evolutionary stages. The infestation did not present a significant variation in intensity and was not a limiting or determining factor of the occurrence of cases in the municipality. The risk maps of the disease from crude and adjusted generalized additive models did not present differences, suggesting that areas with the highest values of entomological indicators were not associated with the incidence of dengue. The inclusion of other variables in the generalized additive models may reveal the modulatory effect for the risk of the disease, which is not found in this study.     

Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation

Tartrate-resistant acid phosphatase (TRAP) is a well-known marker of osteoclasts and bone resorption. Here we have investigated whether osteoblast-like cells (hFOB 1.19) present TRAP activity and how would be its pattern of expression during osteoblastic differentiation. We also observed how the osteoblastic differentiation affected the reduced glutathione levels. TRAP activity was measured using the p-nitrophenylphosphate substrate. The osteogenic potential of hFOB 1.19 cells was studied by measuring alkaline phosphatase activity and mineralized nodule formation. Oxidative stress was determined by HPLC and DNTB assays. TRAP activity and the reduced glutathione-dependent microenvironment were modulated during osteoblastic differentiation. During this phase, TRAP activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day, decreasing thereafter. We demonstrate that TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell, since it seemed to depend on suitable levels of reduced glutathione.     

B-cell activation stimulated by monoclonal anti-Lyb2.1 antibody is mediated through a receptor distinct from the BSF-1 receptor

The experiments in this paper demonstrate that monoclonal anti-Lyb2.1 antibody enhances the proliferative response of anti-immunoglobulin (anti-Ig)-stimulated but not of dextran sulfate-stimulated B cells. The magnitude of this enhanced B-cell proliferation is comparable to that induced by BSF-1 on anti-Ig-stimulated cells. The ability of this antibody to enhance B-cell proliferation does not result from its ability to neutralize the suppressive effects on B-cell activation that is mediated by the Fc fragment of anti-Ig antibody as it is equally as effective in enhancing B-cell proliferative responses stimulated by F(ab')2 fragments of anti-Ig. BSF-1 and Anti-Lyb2.1 appear to stimulate nonoverlapping pathways leading to B-cell activation since the enhanced responses induced by the combination of BSF-1 and anti-Lyb2.1 on anti-Ig-stimulated cells are additive even when maximum quantities of these activators are employed. There is also a marked difference in their activity on T cells; while BSF-1 can enhance T-cell proliferation in synergy with phorbol ester, anti-Lyb2.1 is ineffective in this regard. These data, while consistent with the suggestion that the Lyb2 surface determinant on B cells may be involved in B-cell activation, indicate that it is distinct from the receptors for BSF-1 or BCGF-II.     

Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present student further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n=16). PAF infusion (1–9nmol/min) produced triphasic changes in CBF (n=7): an initial brief phase of coronary dilation (14 ± 2%) above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B₂ levels, while 6-keto-PGF_1α and leukotriene C₄-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A₂ mediated mechanism.