Resveratrol Binding to Human Serum Albumin

Abstract

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure.

Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K_Res = 2.56× 10⁵ M^?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

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.     

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.     

In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells

Aim:  To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form.
Methods and Results:  Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD₅₀ showed that the fibroblasts were able to tolerate up to 80  μ g ml⁻¹ for 24 h, dropping thereafter to 62  μ g ml⁻¹ after 72 h of contact, compared to 160  μ g ml⁻¹ after 24 h, and 80  μ g ml⁻¹ after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25  μ g ml⁻¹) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition.
Conclusion:  These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans .
Significance and Impact of the Study:  This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.