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.     

Mimicry Enhances Observational Learning in 16-Month-Old Infants

We examined the effect of mimicry on how 16-month-old infants learn by observation a novel tool use action, which consisted of using a rake to retrieve a toy. Across four conditions, we manipulated whether during an initial play phase, an adult mimicked the infant''s play or not (testing the effect of mimicry), the infant played with the adult or played alone (controlling the effect of interacting with a contingent partner) and whether the infant saw a demonstration of the tool''s use or not (evaluating baseline performance). We found that infants who had been mimicked learned best from a demonstration of the rake''s use and performed better than infants who only played with the experimenter without mimicry or played by themselves before the demonstration. As expected, infants did not learn from a demonstration of the rake''s use when they played by themselves and thus had no previous interaction with an experimenter. The mechanisms driving this powerful learning effect of mimicry are discussed.     

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.     

Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

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.     

New Insights into the Methodology of L-Arginine-Induced Acute Pancreatitis

Animal models are ideal to study the pathomechanism and therapy of acute pancreatitis (AP). The use of L-arginine-induced AP model is nowadays becoming increasingly popular in mice. However, carefully looking through the literature, marked differences in disease severity could be observed. In fact, while setting up the L-arginine (2×4 g/kg i.p.)-induced AP model in BALB/c mice, we found a relatively low rate (around 15%) of pancreatic necrosis, whereas others have detected much higher rates (up to 55%). We suspected that this may be due to differences between mouse strains. We administered various concentrations (5–30%, pH = 7.4) and doses (2×4, 3×3, or 4×2.5 g/kg) of L-arginine-HCl in BALB/c, FVB/n and C57BL/6 mice. The potential gender-specific effect of L-arginine was investigated in C57BL/6 mice. The fate of mice in response to the i.p. injections of L arginine followed one of three courses. Some mice (1) developed severe AP or (2) remained AP-free by 72 h, whereas others (3) had to be euthanized (to avoid their death, which was caused by the high dose of L-arginine and not AP) within 12 h., In FVB/n and C57BL/6 mice, the pancreatic necrosis rate (about 50%) was significantly higher than that observed in BALB/c mice using 2×4 g/kg 10% L–arginine, but euthanasia was necessary in a large proportion of animals, The i.p. injection of lower L-arginine concentrations (e.g. 5–8%) in case of the 2×4 g/kg dose, or other L-arginine doses (3×3 or 4×2.5 g/kg, 10%) were better for inducing AP. We could not detect any significant differences between the AP severity of male and female mice. Taken together, when setting up the L-arginine-induced AP model, there are several important factors that are worth consideration such as the dose and concentration of the administered L arginine-HCl solution and also the strain of mice.     

Tracing the genetic origin of Europe's first farmers reveals insights into their social organization

Farming was established in Central Europe by the Linearbandkeramik culture (LBK), a well-investigated archaeological horizon, which emerged in the Carpathian Basin, in today''s Hungary. However, the genetic background of the LBK genesis is yet unclear. Here we present 9 Y chromosomal and 84 mitochondrial DNA profiles from Mesolithic, Neolithic Starčevo and LBK sites (seventh/sixth millennia BC) from the Carpathian Basin and southeastern Europe. We detect genetic continuity of both maternal and paternal elements during the initial spread of agriculture, and confirm the substantial genetic impact of early southeastern European and Carpathian Basin farming cultures on Central European populations of the sixth–fourth millennia BC. Comprehensive Y chromosomal and mitochondrial DNA population genetic analyses demonstrate a clear affinity of the early farmers to the modern Near East and Caucasus, tracing the expansion from that region through southeastern Europe and the Carpathian Basin into Central Europe. However, our results also reveal contrasting patterns for male and female genetic diversity in the European Neolithic, suggesting a system of patrilineal descent and patrilocal residential rules among the early farmers.     

Structure-Function Analysis of Heterodimer Formation,Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.     

Effect of S-methylmethionine on the photosynthesis in maize at different chilling temperatures

The effect of the natural compound S-methylmethionine (SMM) on the functioning of the photosynthetic apparatus, the efficiency of photosynthesis and the synthesis of stress-induced phenoloids and anthocyanins involved in defence was investigated in young maize plants exposed to moderate and severe chilling stress. Damage to PSII was observed as a reduction in the value of variable fluorescence (Fv/Fm) which could be detected even after few hours of mild chilling stress. At temperatures below 10°C, the reduction in Fv/Fm was more pronounced. Changes in the value of net photosynthesis exhibited a similar tendency. SMM has a moderating effect on this reduction and its protective effect was more pronounced under long-lasting chilling stress and at the lowest temperatures. Monitoring of fluorescence intensities and ratios correlated with the levels of stress defence compounds. The fluorescence intensities were found to increase over the course of chilling stress in response to SMM, with the highest values being recorded in plants exposed to the longest period of stress. A similar tendency was observed for the quantity of anthocyanins. The results confirm the complex role of SMM, which is manifested both in preserving the ability of the photosynthetic apparatus to function and in stimulating the synthesis of metabolites involved in stress defence.