Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

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.     

Diazoxide preserves hypercapnia-induced arteriolar vasodilation after global cerebral ischemia in piglets

Diazoxide (Diaz), an activator of mitochondrial ATP-sensitive K+ (mitoKATP) channels, is neuroprotective, but the mechanism of action is unclear. We tested whether Diaz preserves endothelium-dependent (hypercapnia) or -independent [iloprost (Ilo)] cerebrovascular dilator responses after ischemia-reperfusion (I/R) in newborn pigs and whether the effect of Diaz is sensitive to 5-hydroxydecanoate (5-HD), an inhibitor of mitoKATP channels. Anesthetized, ventilated piglets (n = 48) were equipped with closed cranial windows. Changes in diameter of pial arterioles were determined with intravital microscopy in response to graded hypercapnia (5-10% CO2 - 21% O2-balance N2, n = 25) or Ilo (0.1-1 microg/ml, n = 18) before and 1 h after 10 min of global I/R. Experimental groups were pretreated with vehicle, NS-398 (a selective cyclooxygenase-2 inhibitor, 1 mg/kg), Diaz (3 mg/kg), or 5-HD (20 mg/kg) + Diaz. Potential direct effects of Diaz and 5-HD on hypercapnic vasodilation were also tested in the absence of I/R (n = 5). To confirm the direct effect of Diaz on mitochondria, mitochondrial membrane potential in cultured piglet cerebrovascular endothelial cells was monitored using Mito Tracker Red. Hypercapnia resulted in dose-dependent pial arteriolar vasodilation, which was attenuated by approximately 70% after I/R in vehicle- and NS-398-treated animals. Diaz and 5-HD did not affect the CO2 response. Diaz significantly preserved the postischemic vasodilation response to hypercapnia, but not to Ilo. Diaz depolarized mitochondria in cultured piglet cerebrovascular endothelial cells, and 5-HD completely abolished the protective effect of Diaz, both findings indicate a role for mitoKATP channels. In summary, preservation of arteriolar dilator responsiveness by Diaz may contribute to neuroprotection.     

Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.     

13C-31P couplings in the study of conformational properties of some diastereomeric nucleoside-3'-thiophosphate derivatives

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3'-phosphorothioate esters and salts are reported. Vicinal carbon phosphorus couplings reflect different predominance of the epsilon conformation in the isomeric (Rp and Sp) esters, while for the salts the epsilont conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.     

Two functional active conformations of the integrin {alpha}2{beta}1, depending on activation condition and cell type

For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin alpha2beta1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of alpha2beta1 because it only binds to the activated state of alpha2beta1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of alpha2beta1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of alpha2beta1 are dependent on the cell type where alpha2beta1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and alpha2beta1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of alpha2beta1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.