Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.     

Protective Effects of Selenium,Zinc, or Their Combination on Cadmium-Induced Oxidative Stress in Rat Kidney

The present study was conducted to investigate whether the combined treatment with Se and Zn offers more beneficial effects than that provided by either of them alone in reversing Cd-induced oxidative stress in the kidney of rat. For this purpose, 30 adult male Wistar albino rats, equally divided into control and four treated groups, received either 200 ppm Cd (as CdCl₂), 200 ppm Cd + 500 ppm Zn (as ZnCl₂), 200 ppm Cd + 0.1 ppm Se (as Na₂SeO₃), or 200 ppm Cd + 500 ppm Zn + 0.1 ppm Se in their drinking water for 35 days. The results showed that Cd treatment decreased significantly the catalase (CAT) and glutathione peroxidase (GSH-Px) activities, whereas the superoxide dismutase (SOD) activity and the renal levels of lipid peroxidation (as malondialdehyde, MDA) were increased compared to control rats. The treatment of Cd-exposed rats with Se alone had no significant effect on the Cd-induced increase in the MDA concentrations but increased significantly the CAT activities and reversed Cd-induced increase in SOD activity. It also partially prevented Cd-induced decrease in GSH-Px activity. The treatment of Cd-exposed animals with Zn alone increased significantly the CAT activity and partially protected against Cd-induced increase in the MDA concentrations, whereas it had no significant effect on the Cd-induced increase in SOD activity and decrease in GSH-Px activity. The combined treatment of Cd-exposed animals with Se and Zn was more effective than that with either of them alone in reversing Cd-induced decrease in CAT and GSH-Px activities and Cd-induced increase in MDA concentrations. Results demonstrated beneficial effects of combined Se and Zn treatment in Cd-induced oxidative stress in kidney and suggest that Se and Zn can have a synergistic role against Cd toxicity. I. Messaoudi and J. El Heni have equally contributed to this work.     

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C₁₂H₂₅-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₁₂-TAC) among many other detergents for extracting the yeast F₁F₀ ATP-synthase. H₁₂-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H₁₂-TAC or fluorinated surfactants such as C₂H₅-C₆F₁₂-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₂F₆-TAC) or C₆F₁₃-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F₆-TAC), two surfactants exhibiting a comparable polar head to H₁₂-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H₁₂-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H₁₂-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H₂F₆-TAC and F₆-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F₆-TAC than with H₂F₆-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H₂F₆-TAC or F₆-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.     

Chemical Composition and Biological Screening of the Essential Oils of Micromeria macrosiphon and M. arganietorum (Lamiaceae)

The chemical composition and in vitro biological activities of the essential oil (EO) of Micromeria macrosiphon Coss. and M. arganietorum (J. Emb.) R. Morales, two Lamiaceae endemic to south Morocco, were investigated. GC/MS analysis resulted in the identification of 36 metabolites from the EO of M. macrosiphon, 45 from M. arganietorum. Borneol was the major metabolite in both oils and together with related derivatives such as camphor, accounted for 2/3 of the EO of M. macrosiphon, 1/3 of those of M. arganietorum. Pinene and terpinene derivatives were also present in high proportions. From a chemotaxonomic point of view, the composition of the examined samples may be related to those of other species endemic to Macaronesia. Both EOs showed significant toxicity towards liver HepG2 and melanoma B16 4A5 tumor cell lines at 100 μg/mL; however, they were also cytotoxic towards S17 normal cell lines, with a selectivity index <1. No antibacterial activity was noticed against 52 strains at 100 μg/mL.     

Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure.     

Chemical Composition and Bioactivities of Quercus canariensis Flour Acorns Extracts Growing in Tunisia

The current study was conducted to investigate the chemical composition of Quercus canariensis flour acorns extracts as well as its biological activities in regards to the growing area using spectrophotometric and chromatographic techniques. The phenolic profile was composed of 19 compounds identified through HPLC-DAD analysis. Coumarin was the most abundant compound quantified in BniMtir, Nefza and ElGhorra and gallic (12.58–20.52 %), syringic (4.70–7.64 %) and trans-ferulic (2.28–2.94 %) acids were the abundant phenolic acids while kaempferol was the major flavonoid compounds quantified only in Quercus canariensis growing in BniMtir. On the other hand, Ain Snoussi acorn extract was characterized by its high content in luteolin-7-O-glucoside (58.46 %). The in-vitro antioxidant activities of the studied extracts were investigated and the results showed that Nefza ethanolic extract's has the highest activities. A bactericidal effect against Staphylococcus aureus was observed only by Elghorra population. On the other hand, Ain Snoussi acorn extract was efficient to inhibit growth of pathogenic bacteria, mentioned the highest activity against Escherichia coli. These results is the first study highlighted that zeen oak acorns are an excellent source of natural antioxidants and antibacterial compounds related to their lysozyme activity which could be exploited in the pharmaceutical and food sectors.     

A β-lactamase produced by a thermophilic Bacillus

Abstract A β-lactamase was purified from a thermophilic Bacillus strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the β-lactamase produced by Bacillus licheniformis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain.