Heterodimer formation between superoxide dismutase and its copper chaperone

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.     

Hypolipidemic and antiatherosclerotic potential of Pleurotus ostreatus, cultived by submerged fermentation in the high-fat diet fed rats

The ability of Pleurotus ostreatus biomass, cultived by submerged fermentation, to produce beneficial effect on lipid profile and macrophages activity during a high-fat diet (HFD) for a long-term intake was investigated. Blood samples were collected through cardiac puncture to measure the plasma cholesterol, triglycerides, low-density protein (LDL), high-density protein (HDL), aspartate aminotransferase (AST) activity, urea-blood urea nitrogen (BUN)/creatinine ratio of rats fed on an HFD for 4 months. Dosage of lipid hydroperoxides was carried out on methanolic extract of liver tissue. Peritoneal macrophages activity was evaluated in relation to the superoxide anion, hydrogen peroxide and nitric oxide production, phagocytosis and lysosomal volume. The administration of P. ostreatus significantly altered the lipid profile and oxidative stress as related to the LDL and triglycerides decrease and inhibitory effects on superoxide anion and hydrogen peroxide production. All findings of this study lead us to suggest that the P. ostreatus maybe a beneficial agent in the hyperlipidemia and atherosclerosis treatments.     

Complexity of Semiarid Gypsophilous Shrub Communities Mediates the AMF Biodiversity at the Plant Species Level

The community composition of arbuscular mycorrhizal fungi (AMF) was analyzed in roots of Gypsophila struthium growing in gypsum soils under semiarid conditions. In order to investigate the effect of plant community degradation on the AMF biodiversity at the single species level, on the basis of the plant community complexity level, we selected four areas affected by degradation and shrub species spatial heterogeneity. The AM fungal community colonizing G. struthium was investigated from the morphological and molecular points of view. All plants were well colonized and showed a high level of infective AM propagules. Roots were analyzed by polymerase chain reaction, restriction fragment length polymorphism screening, and sequence analyses of the ribosomal DNA small subunit region. Four AM fungal types were identified and clustered into the AM fungal family: Glomeraceae, Glomus being the only taxon present. One fungal type was present in all the selected areas. Two fungal types are distinct from any previously published sequences and could be specific to gypsum soils. The chemical–physical properties of the soil were not correlated with the AMF diversity in roots. Our data show vegetation cover complexity-dependent differences in the AM fungal community composition.     

The effects of 1-year treatment with a haemodiafiltration with on-line regeneration of ultrafiltrate (HFR) dialysis on biomarkers of oxidative stress in patients with chronic renal failure

In the last few years haemodiafiltration with on-line regeneration of ultrafiltrate (HFR) has been shown to have a positive impact on inflammation and oxidative stress biomarkers, but its effect on antioxidant levels and on oxidative damage to biomolecules in the long-term is still unknown. This is a randomised clinical study over 12 months involving 40 patients on haemodialysis, comparing the effect of HFR (n = 25) dialysis with haemodialysis with polysulfone (HD-PS, n = 15) on oxidative stress. Total antioxidant capacity, enzymatic antioxidant [superoxide dismutase (SOD), catalase and glutathione peroxidase], non-enzymatic (GSH) and biomarkers of oxidative stress (TBARs, carbonyl groups and 8-OH-dG) were evaluated. The antioxidant activity decreased in the lymphocytes of patients dialysed with HFR, with a significant decrease in the enzyme SOD. In the oxidative stress biomarkers, an increase was seen in the levels of 8-OH-dG in patients on HD-PS dialysis but not in those treated with HFR. Throughout the year the changes in antioxidant levels and biomarkers of oxidative damage in patients dialysed with HFR were generally more modest and fluctuated less than those dialysed with HD-PS. Our study indicates that, in general, long-term dialysis with HFR does not modified antioxidant parameters or increases the oxidative damage to biomolecules. The HFR showed to be a biocompatible technique for long-term dialysis.     

Antioxidant activities of Ptychopetalum olacoides (“muirapuama”) in mice brain

Ptychopetalum olacoides (PO) roots are used by Amazonian peoples to prepare traditional remedies for treating various central nervous system conditions in which free radicals are likely to be implicated. Following the identification of PO ethanol extract (POEE) free-radical scavenging properties in vitro, the aim of this study was to verify the in vivo antioxidant effect of POEE. Aging mice (14 months) were treated (i.p.) with saline, DMSO (20%) or POEE (100mg/kg body wt.), and the hippocampi, cerebral cortex, striata, hypothalamus and cerebellum dissected out 60 min later to measure antioxidant enzyme activities, free-radical production and damage to macromolecules. POEE administration reduced free-radical production in the hypothalamus, lead to significant decrease in lipid peroxidation in the cerebral cortex, striatum and hypothalamus, as well as in the carbonyl content in cerebellum and striatum. In terms of antioxidant enzymes, catalase activity was increased in the cortex, striatum, cerebellum and hippocampus, while glutathione peroxidase activity was increased in the hippocampus. This study suggests that POEE contains compounds able to improve the cellular antioxidant network efficacy in the brain, ultimately reducing the damage caused by oxidative stress.     

Validation of QTLs for Orobanche crenata resistance in faba bean (Vicia faba L.) across environments and generations

Broomrape (Orobanche crenata Forsk.) is a major root–parasite of faba bean (Vicia faba L.), that seriously limits crop cultivation in the whole Mediterranean area. This parasitic weed is difficult to control, difficult to evaluate and the resistance identified so far is of polygenic nature. This study was conducted to identify genetic regions associated with broomrape resistance in recombinant inbred lines (RILs) and to validate their previous location in the original F₂ population derived from the cross between lines Vf6 and Vf136. A progeny consisting of 165 F₆ RILs was evaluated in three environments across two locations in 2003 and 2004. Two hundred seventy seven molecular markers were assigned to 21 linkage groups (9 of them assigned to specific chromosomes) that covered 2,856.7 cM of the V. faba genome. The composite interval mapping on the F₆ map detected more quantitative trait loci (QTL) than in the F₂ analysis. In this sense, four QTLs controlling O. crenata resistance (Oc2–Oc5) were identified in the RI segregant population in three different environments. Only Oc1, previously reported in the F₂ population, was not significant in the advanced lines. Oc2 and Oc3 were found to be associated with O. crenata resistance in at least two of the three environments, while the remaining two, Oc4 and Oc5, were only detected in Córdoba-04 and Mengíbar-04 and seemed to be environment dependent.     

Identification and characterization of NBS-LRR class resistance gene analogs in faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.).

A PCR approach with degenerate primers designed from conserved NBS-LRR (nucleotide binding site-leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from 5 faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologies to various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the sequences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identity of 70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). The number of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolated from faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-class identity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based on the deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6 known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1-4 from potato, N from tobacco, L6 from flax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1-4, L6, N, RGA05 to RGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS-LRR sequences. The development of suitable polymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment of their potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first to report on faba bean RGAs.