Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.     

Accessing the Two‐Electron Charge Storage Capacity of MnO2 in Mild Aqueous Electrolytes

Rechargeable batteries based on MnO₂ cathodes, able to operate in mild aqueous electrolytes, have attracted attention due to their appealing features for the design of low‐cost stationary energy storage devices. However, the charge/discharge mechanism of MnO₂ in such media is still a matter of debate. Here, an in‐depth quantitative spectroelectrochemical analysis of MnO₂ thin‐films provides a set of unrivaled mechanistic insights. A major finding is that charge storage occurs through the reversible two‐electron faradaic conversion of MnO₂ into Mn²⁺ in the presence of a wide range of weak Brønsted acids, including the [Zn(H₂O)₆]²⁺ or [Mn(H₂O)₆]²⁺ complexes present in aqueous Zn/MnO₂ batteries. Furthermore, it is shown that buffered electrolytes loaded with Mn²⁺ are ideal to achieve highly reversible conversion of MnO₂ with both high gravimetric capacity and remarkably stable charging/discharging potentials. In the most favorable case, a record gravimetric capacity of 450 mA·h·g^?1 is obtained at a high rate of 1.6 A·g^?1, with a Coulombic efficiency close to 100% and a MnO₂ utilization of 84%. Overall, the present results challenge the common view on MnO₂ the charge storage mechanism in mild aqueous electrolytes and underline the benefit of buffered electrolytes for high‐performance rechargeable aqueous batteries.     

Leptin Signaling in the Arcuate Nucleus Reduces Insulin’s Capacity to Suppress Hepatic Glucose Production in Obese Mice

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Environmental filtering determines community patterns in temporary wetlands: a multi-taxon approach

Climate characteristics appear to play a key role in filtering organisms based on their biological traits. If this trait filtering by climate indeed occurs, it should have effects on the composition, dynamics, taxonomic relatedness and co-occurrence patterns of local assemblages, regardless of the taxonomic group considered. This preliminary study aimed to assess the extent to which environmental variables might determine these patterns in local communities and to evaluate whether the ultimate cross-taxon congruence relationships are consistent across, or dependent on, the selected region. To this end, we studied the bryophyte, macrophyte, macroinvertebrate, and amphibian communities in two clusters of temporary wetlands on the NE Iberian Peninsula under mesothermal and semiarid climates. We observed effects of environmental filtering, with the communities differing between the climatic regions not only in their compositions but also in their dynamics and taxonomic relatedness patterns. Although the cross-taxon congruence in terms of species richness was high in the mesothermal climate, most of the congruent relationships were disrupted in the semiarid environment. Overall, because climate-dependent patterns appear to prevail over climate-consistent ones, we suggest that the use of surrogate taxa may be of limited value when aiming to assess wetland biodiversity across large areas.     

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.     

Prestress and Adhesion Site Dynamics Control Cell Sensitivity to Extracellular Stiffness

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.     

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68·7–97·5% similarity with other Polyporale laccases. The three laccases (59·5–62·9 kDa with 7–10% carbohydrate content) had high redox potentials (0·72–0·75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase‐1‐hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo‐ and pH stability, catalytic efficiency towards 2,2′‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale‐up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor‐made enzymes.