Antioxidant properties of MitoTEMPOL and its hydroxylamine

Piperidine nitroxides such as TEMPOL have been widely used as antioxidants in vitro and in vivo. MitoTEMPOL is a mitochondria-targeted derivative of TEMPOL designed to protect mitochondria from the oxidative damage that they accumulate, but once there is rapidly reduced to its hydroxylamine, MitoTEMPOL-H. As little is known about the antioxidant efficacy of hydroxylamines, this study has assessed the antioxidant activity of both MitoTEMPOL and MitoTEMPOL-H. The hydroxylamine was more effective at preventing lipid-peroxidation than MitoTEMPOL and decreased oxidative damage to mitochondrial DNA caused by menadione. In contrast to MitoTEMPOL, MitoTEMPOL-H has no superoxide dismutase activity and its antioxidant actions are likely to be mediated by hydrogen atom donation. Therefore, even though MitoTEMPOL is rapidly reduced to MitoTEMPOL-H in cells, it remains an effective antioxidant. Furthermore, as TEMPOL is also reduced to a hydroxylamine in vivo, many of its antioxidant effects may also be mediated by its hydroxylamine.     

American Glass

Exploring the complicated issues of assessment in the arts, the authors discuss assessment of arts education and arts programs from a qualitative perspective: experiential, naturalistic, and ethnographic interpretation. With special attention to the practices of teaching, learning, and administration of education in the arts, quality is sought with emphasis on observations and judgment rather than instruments and measurement.     

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)₂D₃, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)₂D₃-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)₂D₃. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)₂D₃. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)₂D₃ effects. 1,25(OH)₂D₃ blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)₂D₃ down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

Mining the Giardia genome and proteome for conserved and unique basal body proteins

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.     

Whole-genome sequence of Streptococcus pseudopneumoniae isolate IS7493

Streptococcus pseudopneumoniae is a member of the viridans group streptococci (VGS) whose pathogenic significance is unclear. We announce the complete genome sequence of S. pseudopneumoniae IS7493. The genome sequence will assist in the characterization of this new organism and facilitate the development of accurate diagnostic assays to distinguish it from Streptococcus pneumoniae and Streptococcus mitis.     

Disentanglement of heterogeneous dynamics in mixed lipid systems

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of ³¹P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.     

Influence of organic residues and soil incorporation on temporal measures of microbial biomass and plant available nitrogen

Aims

Despite our current understanding of plant nitrogen (N) uptake and soil N dynamics in arable systems, the supply and demand of N are infrequently matched as a result of variable seasonal and soil conditions. Consequently, inefficiencies in N utilisation often lead to constrained production and can contribute to potential environmental impacts. The aim of this study was to examine the influence of plant residue quality (C/N ratio) and extent of residue incorporation into soil on temporal changes in soil mineral N and the associated plant N uptake by wheat in the semi-arid agricultural production zone of Western Australia.

Methods

Oat (Avena sativa); lupin (Lupinus angustifolius) and field pea (Pisum sativum) were incorporated into a Red-Brown Earth using varying degrees of mechanical disturbance (0 to 100% residue incorporated). Soil samples for inorganic N (NO ₃ ^? and NH ₄ ⁺ ) profiles (0?C50?cm), microbial biomass-C (0?C50?cm) and plant N uptake were taken throughout the growing season of the subsequent wheat (Triticum aestivum) crop. Grain yield and yield components were determined at harvest.

Results

Despite observed treatment effects for plant residue type and soil disturbance, fluctuations in inorganic N were more readily influenced by seasonal variability associated with wet-dry cycles. Treatment effects resulting from residue management and extent of soil disturbance were also more readily distinguished in the NO ₃ ^? pool. The release of N from crop residues significantly increased (p?=?0.05) with greater soil-residue contact which related to the method of incorporation; the greater the extent of soil disturbance, the greater the net supply of inorganic N. Differences in microbial biomass-C were primarily associated with the type of plant residue incorporated, with higher microbial biomass generally associated with legume crops. No effect of residue incorporation method was noted for microbial biomass suggesting little effect of soil disturbance on the microbial population in this soil.

Conclusions

Despite differences in the magnitude of N release, neither crop type nor incorporation method significantly altered the timing or pattern of N release. As such asynchrony of N supply was not improved through residue or soil management, or through increased microbial biomass in this semi-arid environment. N fluxes were primarily controlled by abiotic factors (e.g. climate), which in this study dominated over imposed agricultural management practices associated with residue management.     

Discovery of three novel coccidian parasites infecting California sea lions (Zalophus californianus), with evidence of sexual replication and interspecies pathogenicity

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites "A," "B," and "C." Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the "C" sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.     

Comparison of family 9 cellulases from mesophilic and thermophilic bacteria

Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.