Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l⁻¹ in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest.     

The Expression and Diversity of Catalases in Isolates of Genus Comamonas in Response to the Oxidative Stress of a Polluted Environment

We have evaluated the role of monofunctional heme-containing catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Our results indicate that this constitutively expressed catalase represents the major source for the defence of Comamonas terrigena cells against toxic peroxides but the cells can express also a second form of catalase that is bigger and its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Comamonas terrigena but in those strains that were not exposed to oxidative stress, no such sequence motif could be detected. The results obtained underline the importance of catalase expression in the defence mechanism against oxidative stress in bacterial cells.     

Analysis of flow and vascular resistance in a model of the circle of Willis

A very simple model of the flow in the circle of Willis is described in this paper. Disregarding pulsatility and vessel wall elasticity, fluxes in all segments of the circle of Willis and its afferent and efferent vessels are calculated by applying the Poiseuille-Hagen formula. Comparison with the fluxes calculated numerically from a more sophisticated mathematical model, including pulsatility, vessel wall elasticity and nonlinear effects, revealed only very slight differences. In short, fluxes in the afferent vessels and the segments of the circle of Willis are influenced by any change of resistance within the network, whereas the fluxes in the efferent segments are dominated by the efferent resistance distribution. However, a great advantage of the present simple model is that it offers the possibility of an analytical approach which yields both an easy sensitivity analysis of parameters and an insight into the mechanisms that govern the flow in a network like the circle of Willis. It can be concluded that these mechanisms are similar to the principles of the Wheatstone bridge, known from electrical circuit theory.     

BMP-7/TGF-β1 signalling in myoblasts: components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25 ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1 ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.     

Cleavage of von Willebrand factor by granzyme M destroys its factor VIII binding capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.     

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background  

Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.     

In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.