Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii

A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38°C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38°C and for 4 months of storage at −18°C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior K _m value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.     

Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4-HNE (4-hydroxy-2-nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4-HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4-HNE analogue trans-cinnamate showed essentially the same characteristics as activation by 4-HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDP-sensitive proton conductance by palmitate and 4-HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation.     

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.     

Determining body fuels of wintering mallards

Measuring body lipids and proteins of wild animals such as mallards is essential to determine the impact of the environment on their body condition. A major difficulty, however, is that biochemical analysis of carcass is tedious and therefore cannot be applied at a large scale. The main objective of this study was therefore to find out if simple measurements can be used as indices of total body lipids and proteins. Four classes of lipid and protein indices, derived on a 'source' group, were defined according to their complexity and condition of application (field or laboratory). Accuracy of the indices was evaluated on an independent group, of which the body composition calculated from indices was compared to carcass analysis. In live birds, body mass was an accurate and convenient predictor for both lipid and protein masses. If carcasses are available, extensive analysis provides a higher accuracy for body lipids only. This can be simply obtained through multiple regressions using abdominal fat mass and/or dry body mass.     

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.     

Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: Report of a new case and review of the literature

Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA). The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient’s bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.     

CRIM1 Complexes with ?-catenin and Cadherins,Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.     

Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background

Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF.

Methods/Principal Findings

Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7^th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels.

Conclusions

Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.