Elaboration and characterization of whey protein beads by an emulsification/cold gelation process: application for the protection of retinol

Whey protein beads were successfully produced using a new emulsification/cold gelation method. The principle of this method is based on an emulsifying step followed by a Ca(2+)-induced gelation of pre-denatured (80 degreesC/30 min) whey protein. Beads are formed by the dropwise addition of the suspension into a calcium chloride (CaCl(2)) solution. IR results show that bead formation has a pronounced effect on the secondary structure of whey protein, which leads to the formation of intermolecular hydrogen-bonded beta-sheet structures. Their preparation conditions (CaCl(2) concentrations of 10, 15, and 20% (w/w)) influence their sphericity and homogeneity: an increase in CaCl(2) favors regular-shaped beads. The physicochemical and mechanical characterizations of beads were also carried out. Their properties, such as swelling, elasticity, deformability, and resistance at fracture, change according to pH levels (1.9, 4.5, and 7.5) and preparation conditions. Indeed, protein chain networks exhibit different behavior patterns with respect to their charge. Finally, bead degradation by enzymatic hydrolysis reveals that beads are gastroresistant and form good matrixes to protect fat-soluble bioactive molecules such as retinol, that have in vivo intestinal absorption sites. The experiment demonstrated the potential of whey protein beads to protect molecules sensitive (i.e., vitamins) to oxidation.     

Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Curly bare (cub), a new mouse mutation on chromosome 11 causing skin and hair abnormalities,and a modifier gene (mcub) on chromosome 5

In the outcrossing of a new recessive mouse mutation causing hair loss, a new wavy-coated phenotype appeared. The two distinct phenotypes were shown to be alternative manifestations of the same gene mutation and attributable to a single modifier locus. The new mutation, curly bare (cub), was mapped to distal Chr 11 and the modifier (mcub) was mapped to Chr 5. When homozygous for the recessive mcub allele, cub/cub mice appear hairless. A single copy of the dominant Mcub allele confers a full, curly coat in cub/cub mice. Reciprocal transfer of full-thickness skin grafts between mutant and control animals showed that the skin phenotype was tissue autonomous. The hairless cub/cub mcub/mcub mice show normal contact sensitivity responses to oxazolone. The similarity of the wavy coat phenotype to those of Tgfa and Egfr mutations and the map positions of cub and mcub suggest candidate genes that interact in the EGF receptor signal transduction pathway.     

Conformational changes of pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication

Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating-cooling cycle of 20-80 degrees C. At pD 6, (i.e. pediocin's most active form), the FTIR results show that pediocin adopts an unordered structure with a small contribution of beta-turn. After a heating-cooling cycle, thermally-induced changes in pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I' band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of pediocin.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

Potential inhibitors of angiogenesis. Part I: 3-(imidazol-4(5)-ylmethylene)indolin-2-ones

The synthesis and pharmacological evaluation of new 3-(imidazol-4(5)-ylmethylene)indolin-2-ones, analogues of SU-5416, are reported. The final compounds 20-51 were obtained by Knoevenagel coupling between the substituted indolin-2-ones 1-15 and either the formylimidazole derivatives 16-18 or 2-formyl-3,5-dimethylpyrrole 19. Methylation at the nitrogen atom of the indolin-2-one and/or imidazole moities was carried out in the presence of the couple NaH/DMF. A Mannich reaction afforded the 1-dimethylaminomethyl derivatives 43 and 48. The antiangiogenic activity of these compounds was evaluated in a three dimensional in vitro rat aortic ring assay. In this test, compound 20 induced a decrease of angiogenesis comparable to that observed with SU-5416; the vascular density indexes at 1 microM were 30 +/- 18 and 22 +/- 4% of control, respectively. The compounds were also evaluated, in an independent manner, as inhibitors of the human EGF-receptor tyrosine kinase activity. As expected, only minor activities were observed with four compounds, out of thirty-one, exerting inhibitory effects in the range of 40-55% at 10 microM concentration.     

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.     

Nitric oxide and inducible nitric oxide synthase expression are downregulated in acute cholestasis in the rat accompanied by liver ischemia

Hepatic blood flow decreases under cholestasis and there is evidence that NO regulates liver microvascular perfusion. Thus, the aim of the present study was to evaluate NO synthesis in cholestasis. Cholestasis was induced by bile-duct ligation (BDL) in male Wistar rats. Bilirubins and enzyme activities were measured in serum. Lipid peroxidation, GSH, GSSG and glycogen were determined in liver. Histopathological analysis was performed. Serum NO2- + NO3- concentration was measured by the Gries reaction. iNOS immunoblot analysis was carried out using an iNOS polyclonal antibody. After 7 days of BDL lipid peroxidation increased while GSH/GSSG ratio decreased. Serum NO2- + NO3- and liver iNOS protein were reduced, accompanied by ischemia as revealed by the histopathological analysis. GSH upregulates NO synthesis by increasing iNOS mRNA levels and iNOS activity, thus the reduction of GSH/GSSG ratio may be responsible for the downregulation of iNOS protein and NO synthesis, which in turn may explain the observed ischemia and the decreased hepatic blood perfusion in cholestasis reported by others.