Angiotensin II Induces Vascular Endocannabinoid Release, Which Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors

In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II.     

Comparison of Boolean analysis and standard phylogenetic methods using artificially evolved and natural mt-tRNA sequences from great apes

Boolean analysis (or BOOL-AN; Jakó et al., 2009. BOOL-AN: A method for comparative sequence analysis and phylogenetic reconstruction. Mol. Phylogenet. Evol. 52, 887-97.), a recently developed method for sequence comparison uses the Iterative Canonical Form of Boolean functions. It considers sequence information in a way entirely different from standard phylogenetic methods (i.e. Maximum Parsimony, Maximum-Likelihood, Neighbor-Joining, and Bayesian analysis). The performance and reliability of Boolean analysis were tested and compared with the standard phylogenetic methods, using artificially evolved - simulated - nucleotide sequences and the 22 mitochondrial tRNA genes of the great apes. At the outset, we assumed that the phylogeny of Hominidae is generally well established, and the guide tree of artificial sequence evolution can also be used as a benchmark. These offer a possibility to compare and test the performance of different phylogenetic methods. Trees were reconstructed by each method from 2500 simulated sequences and 22 mitochondrial tRNA sequences. We also introduced a special re-sampling method for Boolean analysis on permuted sequence sites, the P-BOOL-AN procedure. Considering the reliability values (branch support values of consensus trees and Robinson-Foulds distances) we used for simulated sequence trees produced by different phylogenetic methods, BOOL-AN appeared as the most reliable method. Although the mitochondrial tRNA sequences of great apes are relatively short (59-75 bases long) and the ratio of their constant characters is about 75%, BOOL-AN, P-BOOL-AN and the Bayesian approach produced the same tree-topology as the established phylogeny, while the outcomes of Maximum Parsimony, Maximum-Likelihood and Neighbor-Joining methods were equivocal. We conclude that Boolean analysis is a promising alternative to existing methods of sequence comparison for phylogenetic reconstruction and congruence analysis.     

Treatment with insulin inhibits poly(ADP-ribose)polymerase activation in a rat model of endotoxemia

In critically ill patients various conditions may lead to the activation of poly(ADP-ribose) polymerase (PARP). By promoting cellular energetic dysfunction, and by enhancing pro-inflammatory gene expression, PARP activation significantly contributes to the pathogenesis of shock. PARP activation is usually triggered by DNA strand breakage, which is typically the result of the overproduction of various reactive oxidant species. One of the pathophysiological conditions associated with PARP activation is hyperglycemia, where the reactive species are produced from the mitochondria and other cellular sources. In the present study we tested whether endotoxin-induced PARP activation and pro-inflammatory mediator production can be modified by insulin therapy. Rats subjected to bacterial lipopolysaccharide (LPS) with or without insulin co-treatment were studied. LPS-induced PARP activation in circulating lymphocytes was measured by flow cytometry, tumor necrosis factor alpha (TNF-alpha) production was measured by ELISA. The direct effect of insulin on the PARP activity of mononuclear leukocytes and human umbilical vein endothelial cells (HUVEC) in elevated glucose conditions was tested in vitro. LPS-induced significant hyperglycemic response activated PARP in circulating lymphocytes and induced TNF-alpha production. Insulin treatment prevented LPS-induced hyperglycemic response, blocked PARP activation and blunted LPS-induced TNF-alpha response. Insulin treatment caused a slight reduction in the PARP activity of mononuclear cells and HUVECs in vitro. We demonstrate that insulin treatment blocks LPS-induced PARP activation in vivo. We propose that this effect is mainly indirect, and occurs due to the prevention of stress induced hyperglycemia, with a direct cellular effect of insulin playing a potential minor supplemental role. The current findings may have significant implications in the context of the emerging concept of tight glycemic control and insulin treatment for critically ill patients.     

Body mass and body fat in Hungarian schoolboys: differences between 1980-2005

The prevalence of juvenile excess weight keeps growing in the more developed world (WHO, 1998). The aim of the study was to compare the prevalence of overweight and obesity in Hungarian schoolboys in 1980 and 2005.Two independent representative data collections were performed in volunteer boys aged between 6.51 and 18.50 years in the same 90 settlements of the country in 1980 (n=13,061) and 2005 (n=13,060). Height, body mass, and five skinfolds were measured by the same investigators in both instances. Overweight and obesity were estimated by using BMI (Cole et al., 2000), respectively skinfold thicknesses (Parízková, 1961).The pair-wise differences between height means were consistently significant in the 12 age groups studied. Body mass differences were not exactly proportionate with height. The boys of 2005 had significantly more relative body fat than those of 1980. The prevalence of overweight and obesity was remarkably higher in 2005.Taller height and a part of the heavier body mass in 2005 was attributed to a positive secular growth trend. The increases in BMI and fat content are negative consequences of a markedly changed lifestyle associated with inactivity and dietary habits. Because of its public health importance the trends of childhood obesity should be closely monitored.     

Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA)

Phospholipases A(2) (PLA(2)s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA(2) that we named AdPLA (adipose-specific phospholipase A(2)). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF(3), 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA(2). Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA(2)s, group XVI.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

The interactions between β-lactamase inhibitory proteins (BLIPs) and β-lactamases have been used as model systems to understand the principles of affinity and specificity in protein-protein interactions. The most extensively studied tight binding inhibitor, BLIP, has been characterized with respect to amino acid determinants of affinity and specificity for binding β-lactamases. BLIP-II, however, shares no sequence or structural homology to BLIP and is a femtomolar to picomolar potency inhibitor, and the amino acid determinants of binding affinity and specificity are unknown. In this study, alanine scanning mutagenesis was used in combination with determinations of on and off rates for each mutant to define the contribution of residues on the BLIP-II binding surface to both affinity and specificity toward four β-lactamases of diverse sequence. The residues making the largest contribution to binding energy are heavily biased toward aromatic amino acids near the center of the binding surface. In addition, substitutions that reduce binding energy do so by increasing off rates without impacting on rates. Also, residues with large contributions to binding energy generally exhibit low temperature factors in the structures of complexes. Finally, with the exception of D206A, BLIP-II alanine substitutions exhibit a similar trend of effect for all β-lactamases, i.e., a substitution that reduces affinity for one β-lactamase usually reduces affinity for all β-lactamases tested.     

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Abstract

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.