Bioremoval of diethylketone by the synergistic combination of microorganisms and clays : uptake, removal and kinetic studies

The performance of two bacteria, Arthrobacter viscosus and Streptococcus equisimilis, and the effect of the interaction of these bacteria with four different clays on the retention of diethylketone were investigated in batch experiments. The uptake, the removal percentages and the kinetics of the processes were determined. S. equisimilis,by itself, had the best performance in terms of removal percentage, for all the initial diethylketone concentrations tested: 200, 350 and 700 mg/L. The uptake values are similar for both bacteria. A possible mechanism to explain the removal of diethylketone includes its degradation by bacteria, followed by the adsorption of the intermediates/sub-products by the functional groups present on the cells surfaces. The assays performed with bacteria and clays indicated that the uptake values are similar despite of the clay used, for the same microorganism and mass of clay, but in general higher values are reached when S. equisimilis is used, compared to A. viscosus. Kinetic data were described by pseudo-first and pseudo-second order models.The authors would like to gratefully acknowledge the financial support of this project by the Fundacao para a Ciencia e Tecnologia, Ministerio da Ciencia e Tecnologia, Portugal and co-funding by FSE (programme QREN-POPH). Cristina Quintelas thanks FCT for a post-doc grant. The authors would like also to thank Minas de Barqueiros, S. A., Prof. Rui Boaventura (FEUP-Portugal) and Prof. Isabel Correia Neves (Dep Quimica, UM, Portugal) who gently offered the clays

Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC

Beta-synemin expression in cardiotoxin-injected rat skeletal muscle

Background: β-synemin was originally identified in humans as an α-dystrobrevin-binding protein through a yeast two-hybrid screen using an amino acid sequence derived from exons 1 through 16 of α-dystrobrevin, a region common to both α-dystrobrevin-1 and -2. α-Dystrobrevin-1 and -2 are both expressed in muscle and co-localization experiments have determined which isoform preferentially functions with β-synemin in vivo. The aim of our study is to show whether each α-dystrobrevin isoform has the same affinity for β-synemin or whether one of the isoforms preferentially functions with β-synemin in muscle. Methods: The two α-dystrobrevin isoforms (-1 and -2) and β-synemin were localized in regenerating rat tibialis anterior muscle using immunoprecipitation, immunohistochemical and immunoblot analyses. Immunoprecipitation and co-localization studies for α-dystrobrevin and β-synemin were performed in regenerating muscle following cardiotoxin injection. Protein expression was then compared to that of developing rat muscle using immunoblot analysis.Results: With an anti-α-dystrobrevin antibody, β-synemin co-immunoprecipitated with α-dystrobrevin whereas with an anti-β-synemin antibody, α-dystrobrevin-1 (rather than the -2 isoform) preferentially co-immunoprecipitated with β-synemin. Immunohistochemical experiments show that β-synemin and α-dystrobrevin co-localize in rat skeletal muscle. In regenerating muscle, β-synemin is first expressed at the sarcolemma and in the cytoplasm at day 5 following cardiotoxin injection. Similarly, β-synemin and α-dystrobrevin-1 are detected by immunoblot analysis as weak bands by day 7. In contrast, immunoblot analysis shows that α-dystrobrevin-2 is expressed as early as 1 day post-injection in regenerating muscle. These results are similar to that of developing muscle. For example, in embryonic rats, immunoblot analysis shows that β-synemin and α-dystrobevin-1 are weakly expressed in developing lower limb muscle at 5 days post-birth, while α-dystrobrevin-2 is detectable before birth in 20-day post-fertilization embryos. Conclusion: Our results clearly show that β-synemin expression correlates with that of α-dystrobrevin-1, suggesting that β-synemin preferentially functions with α-dystrobrevin-1 in vivo and that these proteins are likely to function coordinately to play a vital role in developing and regenerating muscle

Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention

α-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts

α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression

A new simplified comorbidity score as a prognostic factor in non-small-cell lung cancer patients: description and comparison with the Charlson's index

Treatment of non-small-cell lung cancer (NSCLC) might take into account comorbidities as an important variable. The aim of this study was to generate a new simplified comorbidity score (SCS) and to determine whether or not it improves the possibility of predicting prognosis of NSCLC patients. A two-step methodology was used. Step 1: An SCS was developed and its prognostic value was compared with classical prognostic determinants in the outcome of 735 previously untreated NSCLC patients. Step 2: the SCS reliability as a prognostic determinant was tested in a different population of 136 prospectively accrued NSCLC patients with a formal comparison between SCS and the classical Charlson comorbidity index (CCI). Prognosis was analysed using both univariate and multivariate (Cox model) statistics. The SCS summarised the following variables: tobacco consumption, diabetes mellitus and renal insufficiency (respective weightings 7, 5 and 4), respiratory, neoplastic and cardiovascular comorbidities and alcoholism (weighting=1 for each item). In step 1, aside from classical variables such as age, stage of the disease and performance status, SCS was a statistically significant prognostic variable in univariate analyses. In the Cox model weight loss, stage grouping, performance status and SCS were independent determinants of a poor outcome. There was a trend towards statistical significance for age (P=0.08) and leucocytes count (P=0.06). In Step 2, both SCS and well-known prognostic variables were found as significant determinants in univariate analyses. There was a trend towards a negative prognostic effect for CCI. In multivariate analysis, stage grouping, performance status, histology, leucocytes, lymphocytes, lactate dehydrogenase, CYFRA 21-1 and SCS were independent determinants of a poor prognosis. CCI was removed from the Cox model. In conclusion, the SCS, constructed as an independent prognostic factor in a large NSCLC patient population, is validated in another prospective population and appears more informative than the CCI in predicting NSCLC patient outcome