Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.     

Nitrous oxide and methane fluxes of a pristine slope mire in the German National Park Harz Mountains

Pristine peatlands covered by Histosols (bogs and fens) with high water table and a restricted oxygen (O₂) availability are known to have low emissions of nitrous oxide (N₂O) but may be a significant source for atmospheric methane (CH₄) which are both important greenhouse gases. For the first time N₂O and CH₄ fluxes of a pristine slope mire in the German Harz Mountains have been monitored. Previously reported peatlands are characterised by anaerobic conditions due to high water table levels. Slope mires monitored here receive O₂ through slope water inflow. Gas fluxes have been monitored deploying closed chamber method on a central non-forested area and a forested area at the periphery of the slope mire. By means of groundwater piezometers water table levels, ammonium and nitrate contents as well as hydro-chemical variables like oxygen content and redox potential of the mire pore water have been concurrently measured with trace gas fluxes at both monitoring sites of the slope mire. The slope mire took up small amounts of atmospheric methane at a rate of −0.02 ± 0.01 kg C ha⁻¹ year⁻¹ revealing no significant difference between the forested and non-forested site. Higher uptake rates were observed during low water table level. In contrast to pristine peatlands influx of oxygen containing pore water into slope mire does limit reduction processes and resultant CH₄ emission. N₂O fluxes of the forested and non-forested sites of the slope mire did not differ and amounted to 0.25 ± 0.44 kg N ha⁻¹ year⁻¹. Higher emissions were observed at low water table levels and during thawing periods. In spite of favourable conditions N₂O fluxes of the slope mire have been comparable to those of pristine peatlands.     

Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

Polymorphism and glycoprotein character of Ricinus communis lectins purified by affinity chromatography]

Two lectin fractions (S20W = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM celluloes. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.