Effects of various iron supply on oxidative stress development and ferritin formation in the common ice plants

Mesembryanthemum crystallinum L. plants were grown from seeds in perlite. At the age of 4 weeks (juvenile plants) or 6 weeks (adult plants), they were transferred on nutrient media with different Fe³⁺ content brought in as Fe₂(SO₄)₃—EDTA complex (pH 6.0): control, iron deficit, and iron “excess”. Adult plants grown in media differing in iron content were subjected to salinity (300 mM NaCl) during the last 8 days of growth. Biochemical analyses were performed after plant fixation in liquid nitrogen; simultaneously, the samples for electron microscopy were taken. Different content of available Fe³⁺ in medium, especially under salinity conditions, changed sharply the content of chlorophyll and proline, the rate of lipid peroxidation, the level of H₂O₂, the activities of antioxidant enzymes in the leaves and roots, the number and sizes of plastoglobules, and ferritin formation in plastids. Joint action of salinity and iron deficit enhanced oxidative stress development, whereas iron excess hampered oxidative reaction development, reduced the rate of lipid peroxidation, and increased the chlorophyll content. At iron excess, plastoglobule lysis in plastids did not occur, their number and sizes increased, and ferritin deposits appeared, whereas the latter were absent at iron deficit.     

Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.     

Daily copper supplement effects on copper balance in trained subjects during prolonged restriction of muscular activity

The aim of this study was to assess the effect of a daily intake of copper supplements on negative copper balance during prolonged exposure to hypokinesia (decreased number of kilometers per day). During hypokinesia (HK), negative copper balance is shown by increased, not by decreased, serum copper concentration, as it happens in other situations. Studies were done during a 30-d prehypokinetic period and a 364-d hypokinetic period. Forty male trained volunteers aged 22–26 yr with a peak oxygen uptake of 66.4 mL/min/kg and with an average of 13.7 km/d running distance were chosen as subjects. They were equally divided into four groups: unsupplemented ambulatory control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented hypokinetic subjects (SHKS), and supplemented ambulatory control subjects (SACS). The SACS and SHKS groups took 0.09 mg copper carbonate/kg body weight daily. The SHKS and UHKS groups were maintained under an average running distance of 1.7 km/d, whereas the SACS and UACS groups did not experience any modifications in their normal training routines. During the 30-d prehypokinetic period and the 346-d hypokinetic period, urinary excretion of copper, calcium, and magnesium and serum concentrations of copper, calcium, and magnesium were measured. Copper loss in feces and copper balance was also determined. In both UHKS and SHKS groups, urinary excretion of copper, calcium, and magnesium and concentrations of copper, magnesium, and calcium in serum increased significantly when compared with the SACS and UACS groups. Loss of copper in feces was also increased significantly in the SHKS and UHKS groups when compared with the UACS and SACS groups. Throughout the study, the copper balance was negative in the SHKS and UHKS groups, whereas in the SACS and UACS groups, the copper balance was positive. It was concluded that a daily intake of copper supplements cannot be used to prevent copper deficiency shown by increased copper concentration. Copper supplements also failed to prevent negative copper balance and copper losses in feces and urine in endurancetrained subjects during prolonged exposure to HK.     

Analysis of the diversity of diazotrophic bacteria in peat soil by cloning of the <Emphasis Type="Italic">nifH</Emphasis> gene

The diversity of nitrogen-fixing microorganisms in the soil of an oligotrophic Sphagnum peat bog was studied by molecular cloning of fragments of the nifH gene encoding one of the main components of the nitrogenase complex. The fragments were amplified from the DNA isolated from the peat samples collected at the same site in January (library I) and November (library II), 2005. Analysis of the nifH sequence libraries revealed high diversity of diazotrophic bacteria in peat soil: the first library consisted of 237 clones and 55 unique sequence types, the second one included 171 clones and 52 sequence types. Comparison of the two clone libraries showed that the composition and population structure of the nitrogen-fixing community depended greatly on the sampling time; they shared only 11 phylotypes. The sequences of representatives of the class Alphaproteobacteria prevailed in both libraries (27% and 57% of clones in libraries I and II, respectively). Representatives of the classes Deltaproteobacteria and Chlorobea were minor components of library I (6% and 7% of clones, respectively), whereas they prevailed in library II (18% and 24% of clones, respectively). Members of the class Chloroflexi were present only in library I, while members of the classes Bacilli, Clostridia, and Methanomicrobia were present only in library II. Our studies demonstrated that, for complete evaluation of the diversity of natural nitrogen-fixing communities, nifH libraries should consist of at least 200–300 clones.     

Characterization of plant phenolic compounds by cyclic voltammetry

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (E_ap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The E_ap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (E_1/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one-and two-electron oxidation are proposed.     

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.     

At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking

The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 colocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.     

Deposition of iron and beta-amyloid plaques is associated with cortical cellular damage in rabbits fed with long-term cholesterol-enriched diets

Hypercholesterolemia is a potential trigger of Alzheimer's disease, and is thought to increase brain levels of beta-amyloid (Abeta) and iron. However, animal models to address the mechanisms by which Abeta and iron accumulation may cause neuronal damage are poorly defined. To address this question, we fed adult rabbits a 1% cholesterol-enriched diet for 7 months. This diet was associated with increased regional deposition of both iron and Abeta peptide in the brain. Iron preferentially accumulated around Abeta plaques in the adjacent cortex, but was not found in the hippocampus. Co-localization of iron and Abeta was accompanied by apoptosis, DNA damage, blood-brain barrier (BBB) disruption, as well as dysregulation in the level of the iron-regulatory proteins, ferritin and heme-oxygenase-1. We further demonstrate that the cholesterol diet-induced apoptosis is mediated by the activation of the endoplasmic reticulum stress pathway, involving the down-regulation of the endoplasmic reticulum chaperones, calreticulin, grp78 and grp94, and the activation of the growth and arrest DNA damage protein, gadd153. Our results suggest that BBB damage and disturbances in iron metabolism may render the cortex more vulnerable than the hippocampus to the cholesterol-induced cellular stress.