Inhibition of Xanthine Oxidase by Allopurinol Prevents Skeletal Muscle Atrophy: Role of p38 MAPKinase and E3 Ubiquitin Ligases

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.     

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus

The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3′ element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer-promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus.     

Neuroendocrine disruption without direct endocrine mode of action: Polychloro-biphenyls (PCBs) and bisphenol A (BPA) as case studies

Endocrine disruption is commonly thought to be restricted to a direct endocrine mode of action i.e. the perturbation of the activation of a given type of hormonal receptor by its natural ligand. Consistent with the WHO definition of an endocrine disrupter, a key issue is the “altered function(s) of the endocrine system”. Such altered functions can result from different chemical interactions, beyond agonistic or antagonistic effect at a given receptor. Based on neuroendocrine disruption by polychlorinated biphenyls and bisphenol A, this paper proposes different mechanistic paradigms that can result in adverse health effects. They are a consequence of altered endocrine function(s) secondary to chemical interaction with different steps in the physiological regulatory processes, thus accounting for a possibly indirect endocrine mode of action.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

Impact of the carbon chain length of novel platinum complexes derived from N-alkyl-propanediamines on their cytotoxic activity and cellular uptake

This work describes the synthesis and characterization of four new ligands derived from 1,3-propanediamine in addition to the preparation and characterization of their respective platinum(II) complexes by reaction with K₂PtCl₄. These ligands were obtained by the reaction of the corresponding alkyl mesylate with 1,3-propanediamine. We have prepared compounds having different carbon chains lengths in an attempt to correlate this factor, which influences the lipophilicity of the compounds, with cytotoxic activity. Octanol/water partition coefficients, the effect of the four complexes on the growth of two tumoral cell lines, and their cellular uptake were investigated. Increasing lipophilicity enhances the rate of cellular uptake and, consequently, the cytotoxic activity.     

Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process.     

A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure

The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated. Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein. Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template. The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport. In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA. These differences can be related to those of the transported substrates. Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein. These may be important for size selection and proper positioning of drugs for transport. The relevance of the modeled conformation to P-gp function is discussed.     

Spatial distribution patterns of interphase centromeres during retinoic acid-induced differentiation of promyelocytic leukemia cells

BACKGROUND: The pericentromeric heterochromatin is an important element for the regulation of gene silencing. Its spatial distribution during interphase appears to be cell-type specific. This study analyzes three-dimensional (3D) centromere distribution patterns during cellular differentiation along the neutrophil pathway. METHODS: Differentiation of the promyelocytic leukemia cell line NB4 was induced by retinoic acid. Centromeres in interphase nuclei were visualized by immunofluorescence staining of centromere-associated proteins with CREST serum. 3D images of nuclei were obtained by confocal microscopy. Automated methods for the segmentation of point-like objects in 3D images were implemented to detect the position of centromeres. Features of centromere localization patterns were determined by constructing the minimal spanning tree of the centromere distribution. RESULTS: In differentiated NB4 cells, the number of centromere conglomerates (chromocenters) was decreased and the distance between chromocenters was increased as compared with untreated controls. The nuclear volume did not differ between the two groups. CONCLUSIONS: The measured rearrangement of centromeres indicates a progressive clustering of heterochromatin and a global remodeling of interphase chromosome territories during differentiation of NB4 cells. The developed methods for the analysis of 3D centromere distribution patterns provide the opportunity for a fast and objective analysis of heterochromatin remodeling.     

H2-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni–Fe] and [Fe–Fe] hydrogenases

In this paper, the hydrogen (H₂)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni–Fe] hydrogenase (HyaB) and periplasmic [Fe–Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H₂ to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H₂-dependent conditions with concentration of 1.4 and 2.4 μmol g protein^-1, respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.