Comparison of two dimensional electrophoresis mouse colon proteomes before and after knocking out Aquaporin 8

Aquaporin (AQP) family plays a fundamental role in transmembrane water and small solutes movement. Within this family, aquaporin 8 (AQP8), showed to be widely distributed in the digestive system especially colon. To investigate the possible protein alterations involved in AQP8 regulation and trafficking, we extensively compared between wild type and AQP8 knockout mouse colon using semi-quantitative fluorescence- stained two dimensional gel electrophoresis (2-DE) coupled with nano LC-Ms/Ms. Our analysis revealed identification and regulation of 21 proteins, most notably, actin-related family which suggests its possible involvement in regulating AQP8 secretory vesicles migration to be integrated as a cell membrane protein.     

A biologically effective fullerene (C60) derivative with superoxide dismutase mimetic properties

Superoxide, a potentially toxic by-product of cellular metabolism, may contribute to tissue injury in many types of human disease. Here we show that a tris-malonic acid derivative of the fullerene C60 molecule (C3) is capable of removing the biologically important superoxide radical with a rate constant (k(C3)) of 2 x 10(6) mol(-1) s(-1), approximately 100-fold slower than the superoxide dismutases (SOD), a family of enzymes responsible for endogenous dismutation of superoxide. This rate constant is within the range of values reported for several manganese-containing SOD mimetic compounds. The reaction between C3 and superoxide was not via stoichiometric "scavenging," as expected, but through catalytic dismutation of superoxide, indicated by lack of structural modifications to C3, regeneration of oxygen, production of hydrogen peroxide, and absence of EPR-active (paramagnetic) products, all consistent with a catalytic mechanism. A model is proposed in which electron-deficient regions on the C60 sphere work in concert with malonyl groups attached to C3 to electrostatically guide and stabilize superoxide, promoting dismutation. We also found that C3 treatment of Sod2(-/-) mice, which lack expression of mitochondrial manganese superoxide dismutase (MnSOD), increased their life span by 300%. These data, coupled with evidence that C3 localizes to mitochondria, suggest that C3 functionally replaces MnSOD, acting as a biologically effective SOD mimetic.     

A unique mutation underlying carbonic anhydrase II deficiency syndrome in patients of Arab descent

We have investigated, in the genomic DNA of ten Tunisian patients, the presence of a splice junction mutation at the 5 end of intron 2 in the carbonic anhydrase II gene (CAII) previously described in six CAII-deficient patients presumed to be of Arab origin. All our patients were homozygous for this mutation and were mentally retarded, a characteristic feature of the phenotype of patients with an Arabic background. This mutation is found exclusively in patients with an Arabic background and thus may be confined to this ethnic group.     

Synthesis and preliminary biological evaluation of truncated zoanthenol analogues

Zoanthamines are a family of marine alkaloids that have complex heptacyclic structures and are reported to be interleukin-6 modulators. While the structure of zoanthamines, especially the ABC-ring portion, is similar to that of steroids, the CDEFG-ring portion, composed of aminoacetal and lactone core, is a unique structural element. In this report, we designed and synthesized ABC-ring 6 and CDEFG-ring 7, which are truncated analogues of the northern and southern hemispheres of zoanthenol 5, respectively, and which incorporate all of the functionality of each hemisphere. A preliminary SAR study suggested that the hydrochloride of the CEFG-ring portion is an active pharmacophore for suppressing the growth of interleukin-6-dependent MH60 cells.     

Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure.     

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

AIMS: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. METHODS AND RESULTS: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0.2 and 6.7 g l(-1)) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. CONCLUSIONS: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.     

Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO₂ hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.     

Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2–16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88−M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88−M2e2-16 phages in the presence of incomplete Freund’s adjuvant, induced robust M2e-specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.     

Chemokine-Like Receptor 1 Deficiency Does Not Affect the Development of Insulin Resistance and Nonalcoholic Fatty Liver Disease in Mice

The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1), are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD), which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1^-/-) mice and wild type (WT) mice into low-density lipoprotein receptor knock-out (Ldlr^-/-) mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS). Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1^-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr^-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.