TEMPONE reduces renal dysfunction and injury mediated by oxidative stress of the rat kidney

Here we investigate the effects of the stable, water-soluble nitroxyl radical, TEMPONE, on renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the rat kidney in vivo. TEMPONE significantly improved both glomerular and tubular function (serum urea, creatinine, creatinine clearance, and fractional excretion of Na(+)) in a dose-dependent manner and significantly attenuated the reperfusion-injury associated with I/R (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase, assessment of renal histology). TEMPONE also markedly reduced the immunohistochemical evidence of the formation of nitrotyrosine and poly(ADP-ribose), indicating reduction of nitrosative and oxidative stress, respectively. The latter was reflected in vitro, where TEMPONE significantly reduced cellular injury of primary cultures of rat renal proximal tubular (PT) cells caused by hydrogen peroxide in a dose-dependent manner. Importantly, in contrast to its in vivo metabolite TEMPOL (which also provided protective effects against renal I/R and oxidative stress of PT cells), TEMPONE reduced renal dysfunction and injury without causing a significant reduction in blood pressure upon administration. These results suggest, for the first time, that TEMPONE can reduce the renal dysfunction and injury caused by I/R and the injury caused to PT cells by oxidative stress without producing the adverse cardiovascular effects observed when using other nitroxyl radicals.     

Aurora Mitochondrialis drives fission during mitosis

Mitochondria proliferate by growth and partition during every cell-division cycle. Recently, Kashatus et?al. (2011) reported that Aurora A kinase regulates the small GTPase RalA to mediate mitochondrial fission. This work illuminates the molecular mechanism behind mitochondrial inheritance in mammals and extends the functional repertoire of a key mitotic regulator.     

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization.The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C₂OHMIM] > [BIM] and [OMIM] ≈ [E₂-MPy] ? [E₃-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF₄ ? [C₂OHMIM]BF₄ ? [BIM]BF₄. Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E₃-MPy] and [E₂-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF₆⁻, BF₄⁻ and Tf₂N⁻) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL.The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.     

Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.     

Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.     

Speciation and structure of ferriprotoporphyrin IX in aqueous solution: spectroscopic and diffusion measurements demonstrate dimerization,but not μ-oxo dimer formation

Changes in epsilon (393) (the Soret band) of aqueous ferriprotoporphyrin IX [Fe(III)PPIX] with concentration indicate that it dimerizes, but does not form higher aggregates. Diffusion measurements support this observation. The diffusion coefficient of aqueous Fe(III)PPIX is half that of the hydrated monomeric dicyano complex. Much of the apparent instability of aqueous Fe(III)PPIX solutions could be attributed to adsorption onto glass and plastic surfaces. However, epsilon (347) was found to be independent of the aggregation state of the porphyrin and was used to correct for the effects of adsorption. The UV-vis spectrum of the aqueous dimer is not consistent with that expected for a mu-oxo dimer and the (1)H NMR spectrum is characteristic of five-coordinate, high-spin Fe(III)PPIX. Magnetic susceptibility measurements using the Evans method showed that there is no antiferromagnetic coupling in the dimer. By contrast, when the mu-oxo dimer is induced in 10% aqueous pyridine, characteristic UV-vis and (1)H NMR spectra of this species are observed and the magnetic moment is consistent with strong antiferromagnetic coupling. We propose a model in which the spontaneously formed aqueous Fe(III)PPIX dimer involves noncovalent interaction of the unligated faces of two five-coordinate H(2)O/HO-Fe(III)PPIX molecules, with the axial H(2)O/OH(-) ligands directed outwards. This arrangement is consistent with the crystal structures of related five-coordinate iron(III) porphyrins and accounts for the observed pH dependence of the dimerization constant and the spectra of the monomer and dimer. Structures for the aqueous dimer are proposed on the basis of molecular dynamics/simulated annealing calculations using a force field previously developed for modeling metalloporphyrins.