P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Alteration of striatal dopaminergic neurotransmission in a mouse model of DYT11 myoclonus-dystonia

Background

DYT11 myoclonus-dystonia (M-D) syndrome is a neurological movement disorder characterized by myoclonic jerks and dystonic postures or movement that can be alleviated by alcohol. It is caused by mutations in SGCE encoding ε-sarcoglycan (ε-SG); the mouse homolog of this gene is Sgce. Paternally-inherited Sgce heterozygous knockout (Sgce KO) mice exhibit myoclonus, motor impairment and anxiety- and depression-like behaviors, modeling several clinical symptoms observed in DYT11 M-D patients. The behavioral deficits are accompanied by abnormally high levels of dopamine and its metabolites in the striatum of Sgce KO mice. Neuroimaging studies of DYT11 M-D patients show reduced dopamine D2 receptor (D2R) availability, although the possibility of increased endogenous dopamine, and consequently, competitive D2R occupancy cannot be ruled out.

Methodology/Principal Findings

The protein levels of striatal D2R, dopamine transporter (DAT), and dopamine D1 receptor (D1R) in Sgce KO mice were analyzed by Western blot. The striatal dopamine release after amphetamine injection in Sgce KO mice were analyzed by microdialysis in vivo. The striatal D2R was significantly decreased in Sgce KO mice without altering DAT and D1R. Sgce KO mice also exhibited a significant increase of dopamine release after amphetamine injection in comparison to wild-type (WT) littermates.

Conclusion/Significance

The results suggest ε-SG may have a role in the regulation of D2R expression. The loss of ε-SG results in decreased striatal D2R, and subsequently leads to increased discharge of dopamine which could contribute to the behavioral impairment observed in DYT11 dystonia patients and in Sgce KO mice. The results suggest that reduction of striatal D2R and enhanced striatal dopamine release may contribute to the pathophysiology of DYT11 M-D patients.     

Dual deficiency of angiotensin‐converting enzyme‐2 and Mas receptor enhances angiotensin II‐induced hypertension and hypertensive nephropathy

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1‐ERK1/2‐Smad3 and NF‐κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II‐induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1‐7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity

Background

The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding.

Methodology/Principal Findings

After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V0₂) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance.

Conclusions/Significance

We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Mycotoxin-Producing Ability and Chemotype Diversity of Aspergillus Section Flavi from Soils of Peanut-Growing Regions in Iran

Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9 %) were able to produce either CPA or AFB₁, while eight of the isolates (15.1 %) were non-toxigenic. The amounts of CPA and AFB₁ produced by the isolates were reported in the range of 18.2–403.8 μg/g and 53.3–7446.3 μg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4 % were producers of CPA, AFB₁ and sclerotia (group I), 13.2 % of CPA and AFB₁ (group II), 9.4 % of AFB₁ and sclerotia (group III), 13.2 % of AFB₁ (group IV), 5.7 % of CPA and sclerotia (group V) and 15.1 % were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB₁ and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.     

Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis

Background

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.

Principal Findings

Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K⁺-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca²⁺ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K⁺ (K_ATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.

Conclusions/Significance

We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K_ATP channel responsiveness and the subsequent activity of Ca²⁺ channels through pathways other than CDK5-mediated regulation.     

Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.     

An update analysis of two polymorphisms in encoding ribonuclease L gene and prostate cancer risk: involving 13,372 cases and 11,953 controls

Encoding ribonuclease L (RNASEL) is a ubiquitously expressed latent endoribonuclease involved in the mediation of antiviral and pro-apoptotic activities of the interferon-inducible 2-5A system. Although the relationship between RNASEL gene polymorphisms and prostate cancer (PCa) risk has been widely reported, results were somewhat controversial and underpowered. Now, we performed an update analysis of 14 publications evaluating the association between RNASEL R462Q and D541E polymorphisms and PCa risk. We conducted a literature search of PubMed database to identify all eligible articles that examined the association of RNASEL R462Q and D541E polymorphisms with PCa. Odds ratios (OR) with 95% confidence intervals (CI) were estimated to assess these association. R462Q showed a significantly elevated effect on Africans (QQ vs. RR: OR = 2.50, 95% CI = 1.28–4.87, P_{heterogeneity} = 0.231). In addition, PCa men who contain 462Q genotype had a higher Gleason score ≥ 7 (OR = 1.16, 95% CI = 1.05–1.28, P_{heterogeneity} = 0.906). On the other hand, D541E was associated with increased total PCa. In the stratified analysis by race, there was also significantly increased PCa in Africans and Caucasians, as well as in sporadic PCa studies (OR = 1.09, 95% CI = 1.04–1.15, P_{heterogeneity} = 0.078). Our update analysis showed evidence that RNASEL R462Q and D541E polymorphisms were associated with PCa risk. Still more well-designed studies should be performed to clarify the role of these two polymorphisms in the development of PCa.     

Germination responses to temperature and water potential in Jatropha curcas seeds: a hydrotime model explains the difference between dormancy expression and dormancy induction at different incubation temperatures

Background and Aims

Jatropha curcas is a drought-resistant tree whose seeds are a good source of oil that can be used for producing biodiesel. A successful crop establishment depends on a rapid and uniform germination of the seed. In this work we aimed to characterize the responses of J. curcas seeds to temperature and water availability, using thermal time and hydrotime analysis,

Methods

Thermal and hydrotime analysis was performed on germination data obtained from the incubation of seeds at different temperatures and at different water potentials.

Key Results

Base and optimum temperatures were 14·4 and 30 °C, respectively. Approximately 20 % of the seed population displayed absolute dormancy and part of it displayed relative dormancy which was progressively expressed in further fractions when incubation temperatures departed from 25 °C. The thermal time model, but not the hydrotime model, failed to describe adequately final germination percentages at temperatures other than 25 °C. The hydrotime constant, θ_H, was reduced when the incubation temperature was increased up to 30 °C, the base water potential for 50 % germination,Ψ_b(50), was less negative at 20 and 30 °C than at 25 °C, indicating either expression or induction of dormancy. At 20 °C this less negative Ψ_b(50) explained satisfactorily the germination curves obtained at all water potentials, while at 30 °C it had to be corrected towards even less negative values to match observed curves at water potentials below 0. Hence, Ψ_b(50) appeared to have been further displaced to less negative values as exposure to 30 °C was prolonged by osmoticum. These results suggest expression of dormancy at 20 °C and induction of secondary dormancy above 25 °C. This was confirmed by an experiment showing that inhibition of germination imposed by temperatures higher than 30 °C, but not that imposed at 20 °C, is a permanent effect.

Conclusions

This study revealed (a) the extremely narrow thermal range within which dormancy problems (either through expression or induction of dormancy) may not be encountered; and (b) the high sensitivity displayed by these seeds to water shortage. In addition, this work is the first one in which temperature effects on dormancy expression could be discriminated from those on dormancy induction using a hydrotime analysis.     

Connexin 43 and metabolic effect of fatty acids in stressed endothelial cells

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.     

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C

Background

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.

Methods and Findings

The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of N^G-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.

Conclusions

F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components.     

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells

AIM: To explore the approaches exerted by mesenchymal stem cells(MSCs) to improve Parkinson's disease(PD) pathophysiology.METHODS: MSCs were harvested from bone marrowof femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group(1) was control, Groups(2) and(3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group(2) was left untreated, while Group(3) was treated with single intravenous dose of bone marrow derived MSCs(BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1(TGF-β1), monocyte chemoattractant protein-1(MCP-1) and brain derived neurotrophic factor(BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase(TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1(489.7 ± 13.0 vs 691.2 ± 8.0, P 0.05) and MCP-1(89.6 ± 2.0 vs 112.1 ± 1.9, P 0.05) levels associated with significant increase in serum BDNF(3663 ± 17.8 vs 2905 ± 72.9, P 0.05) and brain DA(874 ± 15.0 vs 599 ± 9.8, P 0.05) levels as well as brain TH(1.18 ± 0.004 vs 0.54 ± 0.009, P 0.05) and nestin(1.29 ± 0.005 vs 0.67 ± 0.006, P 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin(293.2 ± 15.9 vs 271.5 ± 15.9, P 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.     

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway

Oxidized phospholipids have been shown to exhibit pleiotropic effects in numerous biological contexts. For example, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxidized phospholipid formed from alkyl phosphatidylcholines, is a peroxisome proliferator–activated receptor gamma (PPARγ) nuclear receptor agonist. Although it has been reported that PPARγ agonists including thiazolidinediones can induce plasma volume expansion by enhancing renal sodium and water retention, the role of azPC in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues and also investigated the effect of azPC on renal sodium handling in vivo. We showed using a microperfusion technique that azPC rapidly stimulated Na⁺/HCO₃⁻ cotransporter 1 (NBCe1) and luminal Na⁺/H⁺ exchanger (NHE) activities in a dose-dependent manner at submicromolar concentrations in isolated PTs from rats and humans. The rapid effects (within a few minutes) suggest that azPC activates NBCe1 and NHE via nongenomic signaling. The stimulatory effects were completely blocked by specific PPARγ antagonist GW9662, ERK kinase inhibitor PD98059, and CD36 inhibitor sulfosuccinimidyl oleate. Treatment with an siRNA against PPAR gamma completely blocked the stimulation of both NBCe1 and NHE by azPC. Moreover, azPC induced ERK phosphorylation in rat and human kidney cortex tissues, which were completely suppressed by GW9662 and PD98059 treatments. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via a CD36/PPARγ/mitogen-activated protein/ERK kinase/ERK pathway. We conclude that the stimulatory effects of azPC on PT transport may be partially involved in volume expansion.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Toll-like receptor 4 and high-mobility group box 1 are critical mediators of tissue injury and survival in a mouse model for heatstroke

The molecular mechanisms that initiate the inflammatory response in heatstroke and their relation with tissue injury and lethality are not fully elucidated. We examined whether endogenous ligands released by damaged/stressed cells such as high-mobility group box 1 (HMGB1) signaling through Toll-like receptor 4 (TLR4) may play a pathogenic role in heatstroke. Mutant TLR4-defective (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to heat stress in an environmental chamber pre-warmed at 43.5°C until their core temperature reached 42.7°C, which was taken as the onset of heatstroke. The animals were then allowed to recover passively at ambient temperature. A sham-heated group served as a control. Mutant mice displayed more histological liver damage and higher mortality compared with wild type mice (73% vs. 27%, respectively, P<0.001). Compared to wild type mice, mutant mice exhibited earlier plasma release of markers of systemic inflammation such as HMGB1 (206±105 vs. 63±21 ng/ml; P = 0.0018 and 209±100 vs. 46±32 ng/ml; P<0.0001), IL-6 (144±40 vs. 46±20 pg/ml; P<0.001 and 184±21 vs. 84±54 pg/ml; P = 0.04), and IL-1β (27±4 vs. 1.7±2.3 pg/ml; P<0.0001 at 1 hour). Both strains of mice displayed early release of HMGB1 into the circulation upstream of IL-1β and IL-6 responses which remained elevated up to 24 h. Specific inhibition of HMGB1 activity with DNA-binding A Box (600 µg/mouse) protected the mutant mice against the lethal effect of heat stress (60% A Box vs. 18% GST protein, P = 0.04). These findings suggest a protective role for the TLR4 in the host response to severe heat stress. They also suggest that HMGB1 is an early mediator of inflammation, tissue injury and lethality in heatstroke in the presence of defective TLR4 signaling.