Toxoplasma effectors targeting host signaling and transcription

Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by whichhas learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction

Application of botryosphaeran as a carbon black adherent on a glassy carbon electrode for the electrochemical determination of cyclobenzaprine

The present work describes the performance of a new voltammetric sensor based on the modification of glassy carbon electrodes (GCE) with carbon black (CB) and botryosphaeran (BOT) (CB-BOT/GCE) for the electroanalytical determination of cyclobenzaprine. BOT is a fungal exocellular (1→3)(1→6)-β-ᴅ-glucan, which was used to improve the adherence of CB onto the surface of GCE. The electrochemical characterisation was performed by electrochemical impedance spectroscopy which showed an improvement in the transfer of electrons on the surface of the sensor developed in relation to the unmodified (bare) GCE. The voltammetric behaviour of cyclobenzaprine was studied using bare GCE, BOT/GCE, CB/GCE, and CB-BOT/GCE. All electrodes presented an oxidation peak (+ 1.0 V) for cyclobenzaprine, while the cyclobenzaprine peak intensity on CB-BOT/GCE was found to be 480% higher than the bare GCE. Through employing square-wave voltammetry, the analytical curve was found to be linear over the concentration range of 2.0 to 20.6 μmol L−1 (in 0.1 mol L−1 NaCl solution) with a detection limit (based on 3-sigma) of 0.63 μmol L−1. The developed electrochemical sensor exhibited excellent sensitivity and selectivity and was successfully applied for the voltammetric determination of cyclobenzaprine in pharmaceutical, biological, and environmental samples for the first time using the CB-BOT/GCE electrochemical sensing platform

Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats

Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension, Allodynia and Metabolic Complications

BACKGROUND: Kinin B(1) receptor (B(1)R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B(1)R activation could perpetuate the oxidative stress which leads to diabetic complications. METHODS AND FINDINGS: Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8-12 weeks. A selective B(1)R antagonist (SSR240612) was administered acutely (3-30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B(1)R expression, aortic superoxide anion (O(2)(*-)) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3-30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O(2)(*-), NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B(1)R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O(2)(*-) in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10-100 microM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe(8)]des-Arg(9)-BK (20 microM; B(1)R agonist). Data show that the greater aortic O(2)(*-) production induced by the B(1)R agonist was blocked only by apocynin. CONCLUSIONS: Activation of kinin B(1)R increased O(2)(*-) through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B(1)R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B(1)R gene expression in this model

An interaction of renin-angiotensin and kallikrein-kinin systems contributes to vascular hypertrophy in angiotensin ii-induced hypertension: in vivo and in vitro studies

The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)–induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg9-Leu8-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184±5.9 vs 115±2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8±2.7 vs 6.0±1.8] and ERK1/2 phosphorylation (% of control: 218.3±29.4 vs 100±0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17±3.1) and ERK1/2 phosphorylation (137±20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings highlight an important cross-talk between the DABK and ANG II in the vascular system and contribute to a better understanding of the mechanisms involved in vascular remodeling in hypertension

Estrogen Protects the Female Heart from Ischemia/Reperfusion Injury through Manganese Superoxide Dismutase Phosphorylation by Mitochondrial p38β at Threonine 79 and Serine 106

A collective body of evidence indicates that estrogen protects the heart from myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism remains incompletely understood. We have previously delineated a novel mechanism of how 17β-estradiol (E2) protects cultured neonatal rat cardiomyocytes from hypoxia/reoxygenation (H/R) by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide dismutase (MnSOD) activity via p38β, leading to the suppression of reactive oxygen species (ROS) and apoptosis. Here we investigate these cytoprotective actions of E2 in vivo. Left coronary artery ligation and reperfusion was used to produce I/R injury in ovariectomized (OVX) female mice and in estrogen receptor (ER) null female mice. E2 treatment in OVX mice reduced the left ventricular infarct size accompanied by increased activity of mitochondrial p38β and MnSOD. I/R-induced infarct size in ERα knockout (ERKO), ERβ knockout (BERKO) and ERα and β double knockout (DERKO) female mice was larger than that in wild type (WT) mice, with little difference among ERKO, BERKO, and DERKO. Loss of both ERα and ERβ led to reduced activity of mitochondrial p38β and MnSOD at baseline and after I/R. The physical interaction between mitochondrial p38β and MnSOD in the heart was detected by co-immunoprecipitation (co-IP). Threonine 79 (T79) and serine 106 (S106) of MnSOD were identified to be phosphorylated by p38β in kinase assays. Overexpression of WT MnSOD in cardiomyocytes reduced ROS generation during H/R, while point mutation of T79 and S106 of MnSOD to alanine abolished its antioxidative function. We conclude that the protective effects of E2 and ER against cardiac I/R injury involve the regulation of MnSOD via posttranslational modification of the dismutase by p38β

Standardization and validation of a cytometric bead assay to assess antibodies to multiple <it>Plasmodium falciparum</it> recombinant antigens

<p>Abstract</p> <p>Background</p> <p>Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple <it>Plasmodium falciparum</it> antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to <it>P. falciparum</it> antigens, and to compare results of multiplex CBA to ELISA.</p> <p>Methods</p> <p>Antibodies to ten recombinant <it>P. falciparum</it> antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex <it>vs</it> multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared.</p> <p>Results</p> <p>Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (<it>r</it> = 0.88-0.99, <it>P</it> values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA.</p> <p>Conclusion</p> <p>With optimization, CBA may be the preferred method of testing for antibodies to <it>P. falciparum</it> antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.</p