Antioxidant activity of new ruthenium compounds

Many biological properties have been attributed to ruthenium complexes including anti-tumor activity and the attenuation of reperfusion damage and infarct size. In this work, we characterize the antioxidant activity of trans-[RuCl2(nic)4] where nic is 3-pyridinecarboxylic acid and trans-[RuCl2(i-nic)4] where i-nic is 4-pyridinecarboxylic acid by (i) evaluation of total antioxidant potential (TRAP); (ii) prevention of DNA damage induced by hydrogen peroxide using the alkaline comet assay; and (iii) the prevention of lipid peroxidation and cell death induced by iron in liver slices. Our results suggest that nic has stronger antioxidant potential when compared to the i-nic. Higher doses (above 200 microM) of these compounds gave genotoxic effects, but the antioxidant potential could be obtained with the use lower doses (0.1-10 microM).     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

The etiological relation between serum iron level and infection incidence in hemodialysis uremic patients

Through the treatment of anaemia in dialysis patients part of the iron ions remain free in the serum which is at the bacterias disposal for growth and the strengthening of their virulence. The linear relation of the increased serum iron level and tissue iron stores in the body and the infection incidence in dialysed patients has become more emphasised. The need of a clearly defined upper threshold of the serum iron concentration limit has been mentioned in scientific journals intensely, and consequently the demand for more precise professional instructions for anaemia treatment. For the purpose of participating in these professional and scientific discussions, we have observed the relation between the iron overload of the organism and complication incidence in 120 of our haemodialysis uremic patients, with special emphasis on infections. It has been established that the sepses incidence is much higher in patients with a serum ferritin concentration above 500 microg/L, than in those patients with a ferritin level lower than the mentioned value ( 2 = 7.857, p = 0.005). The incidence of vascular access infection is significantly higher in those patients with a serum ferritin level above 500 microg/L than in those patients with a ferritin level lower than the mentioned value (Chi2 = 23.186, p = 0.001). Furthermore, it has been determined that the incidence of total infection in patients is 3.8 episodes per 100 patients months, which is in accordance to the referral values of other authors. CONCLUSION--In the analysis of the achieved results, it has been determined that the infection incidence is significantly higher in dialysed patients with a serum iron level higher than 500 g/L, than in those patients with lower values.     

A field test of the dilution effect hypothesis in four avian multi-host pathogens

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens–three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)–and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.     

LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF_1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF_1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.     

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K_d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R⁴⁹ that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.     

Reducing the impact of pesticides on biological control in Australian vineyards: pesticide mortality and fecundity effects on an indicator species, the predatory mite Euseius victoriensis (Acari: Phytoseiidae)

Laboratory bioassays on detached soybean, Glycine max (L.) Merr., leaves were used to test 23 fungicides, five insecticides, two acaricides, one herbicide, and two adjuvants on a key Australian predatory mite species Euseius victoriensis (Womersley) in "worst-case scenario" direct overspray assays. Zero- to 48-h-old juveniles, their initial food, and water supply were sprayed to runoff with a Potter tower; spinosad and wettable sulfur residues also were tested. Tests were standardized to deliver a pesticide dose comparable with commercial application of highest label rates at 1,000 liter/ha. Cumulative mortality was assessed 48 h, 4 d, and 7 d after spraying. Fecundity was assessed for 7 d from start of oviposition. No significant mortality or fecundity effects were detected for the following compounds at single-use application at 1,000 liter/ha: azoxystrobin, Bacillus thuringiensis (Bt) subsp. kurstaki, captan, chlorothalonil, copper hydroxide, fenarimol, glyphosate, hexaconazole, indoxacarb, metalaxyl/copper hydroxide, myclobutanil, nonyl phenol ethylene oxide, phosphorous acid, potassium bicarbonate, pyraclostrobin, quinoxyfen, spiroxamine, synthetic latex, tebufenozide, triadimenol, and trifloxystrobin. Iprodione and penconazole had some detrimental effect on fecundity. Canola oil as acaricide (2 liter/100 liter) and wettable sulfur (200 g/100 liter) had some detrimental effect on survival and fecundity and cyprodinil/fludioxonil on survivor. The following compounds were highly toxic (high 48-h mortality): benomyl, carbendazim, emamectin benzoate, mancozeb, spinosad (direct overspray and residue), wettable sulfur (> or = 400 g/100 liter), and pyrimethanil; pyrimethanil had no significant effect on fecundity of surviving females. Indoxacarb safety to E. victoriensis contrasts with its toxicity to key parasitoids and chrysopid predators. Potential impact of findings is discussed.     

Factors affecting the adsorption of bacteriocins ST194BZ and ST23LD to Lactobacillus sakei and Enterococcus sp

Bacteriocins ST194BZ and ST23LD, produced by Lactobacillus plantarum, inhibit Gram-positive and Gram-negative bacteria. Images obtained by atomic force microscopy showed clear signs of membrane damage of Lactobacillus sakei, accompanied by the leakage of DNA and beta-galactosidase. Adsorption of the bacteriocins to cells was increased when cells were treated with buffers at pH values above neutral. An increase in bacteriocin ST194BZ adsorption to cells of Enterococcus sp. and L. sakei was observed with an increase in incubation temperatures, but at different rates for the two species. Treatment of the two species with various inorganic salts and solvents gave different results regarding the adsorption of the two bacteriocins. In general, pre-treatment of the two sensitive cells with Triton X-100, Triton X-114 and chloroform increased the adsorption of the two bacteriocins. Increased adsorption of bacteriocin ST23LD to L. sakei was recorded when the cells were pre-treated with Tris and NH4-citrate. Treatment of Enterococcus sp. and L. sakei with Na-EDTA and SDS decreased the adsorption of the two bacteriocins. Variable results were recorded with inorganic salts.     

Twintrons are not unique to the Euglena chloroplast genome: structure and evolution of a plastome cpn60 gene from a cryptomonad

Introns within introns (twintrons) are known only from the Euglena chloroplast genome. Twintrons are group II or III introns, into which another group II or III intron has been transposed. In this paper we describe a non-Euglena twintron structure within a plastid-encoded chaperone gene (cpn60) of the cryptomonad alga Pyrenomonas salina. In addition, the evolutionary relationships between members of the Cpn60 protein family are determined. Our findings permit the inclusion of cryptomonad plastomes in phylogenetic studies of intron evolution and present further evidence for the origin of modern plastids from a cyanobacterial ancestor.This paper is dedicated to Prof. Dr. Peter Sitte on the occasion of his 65th birthday