Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.     

Synthesis,semipreparative HPLC separation,biological evaluation,and 3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase B inhibitors

The present study reports on synthesis in high yields (70–99%), HPLC enantioseparation, inhibitory activity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large series of (4-aryl-thiazol-2-yl)hydrazones (1–45). Most of the synthesized compounds proved to be potent and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors. Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chromatographic enantioseparation of these compounds obtained by a stereoconservative pattern. The separated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further structural modification of these lead compounds.     

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

Metabolomics and lipid profile analysis of Coccomyxa melkonianii SCCA 048

Extremophiles - With an unsupervised GC–MS metabolomics approach, polar metabolite changes of the microalgae Coccomyxa melkonianii SCCA 048 grown under standard conditions for seven weeks...     

Aromatic and aliphatic mono- and bis-nitroxides: A study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV–Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2′-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.