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.     

Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells

Human colorectal carcinoma (Caco-2) cells undergo in culture spontaneous enterocytic differentiation, characterized by polarization and appearance of the functional apical brush border membrane. To provide insights into the biology of differentiation, we have performed a comparative proteomic analysis of the plasma membranes from proliferating cells (PCs) and the apical membranes from differentiated cells (DCs). Proteins were resolved by SDS-PAGE, in-gel digested and analyzed by RP-LC and MS/MS. Alternatively, proteins were digested in solution, and tryptic peptides were labeled with isotopic tags and analyzed by 2-D LC followed by MS/MS. Among the 1125 proteins identified in both proteomes, 76 were found to be significantly increased in the membranes of DCs and 61 were increased in PCs. Majority of the proteins increased in the apical membranes were metabolic enzymes, proteins involved in the maintenance of cellular structure, transmembrane transporters, and proteins regulating vesicular transport. In contrast, majority of the proteins increased in the membranes of PCs were involved in gene expression, protein synthesis, and folding. Both groups contained many novel proteins with yet to be identified functions, which could provide potential new markers of the intestinal cells or of colorectal cancer.     

Tau-mediated process outgrowth is differentially altered by the expression of MAP2b and MAP2c in Sf9 cells

It is well documented that the MAPs, MAP2 and tau, play pivotal roles in neurite outgrowth. Several isoforms of MAP2 and tau are coexpressed in neurons, suggesting that the pattern of neurite outgrowth results from a functional equilibrium among these isoforms. In the present study, by coexpressing two of these MAPs at the same time in Sf9 cells, we demonstrated that tau-mediated process outgrowth is affected differently by MAP2b and MAP2c. MAP2b impairs tau ability to induce process outgrowth. Tau affects MAP2c capacity to induce the formation of multiple processes. There is evidence that actin microfilaments (F-actin) are involved in the elaboration of tau-mediated process outgrowth in Sf9 cells. We compared the effects of MAP2b and MAP2c with the effects of tau on F-actin distribution and stability in Sf9 cells. In MAP2b- and MAP2c-expressing cells with processes, F-actin was redistributed. However, in MAP2b-expressing cells without processes, the distribution of F-actin appears to be similar to the one in wild-type infected cells. Collectively, these results indicate that MAP2b could impair the ability of MAP2c and tau to redistribute F-actin in Sf9 cells, thereby decreasing their capacity to induce process formation. Furthermore, MAP2b and MAP2c patterns of process outgrowth were differentially modified by depolymerization of F-actin by cytochalasin D (CD). As previously reported for tau-expressing cells, the MAP2b-expressing cells developed a higher number of processes per cell and a higher number of cells presented processes in the presence of CD. However, the number of cells with multiple processes was lower in MAP2b-expressing cells than in tau-expressing cells treated with CD at 24 h postinfection. This suggests that MAP2b exerts an effect on F-actin stability at an earlier stage of infection than tau. MAP2c had also some stabilizing effects on F-actin at an early stage of infection, since the percentage of cells presenting one process was similar to the nontreated cells. Therefore, MAP2b seems to have less capacity than MAP2c to redistribute F-actin but, nonetheless, both of these MAP2 isoforms exert a stabilizing effect on F-actin at an early stage of infection. Finally, by modifying phosphorylation we showed that MAP2c capacity to induce multiple processes is related to protein phosphorylation in Sf9 cells. Therefore, the differential effect of MAP2c and MAP2b on process outgrowth seems also to depend on protein phosphorylation.     

(2-amino-phenyl)-amides of omega-substituted alkanoic acids as new histone deacetylase inhibitors

A variety of omega-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds were shown to inhibit recombinant human histone deacetylases (HDACs) with IC(50) values in the low micromolar range and induce hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.     

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

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.