Influence of coffee roasting degree on inflammatory and oxidative stress markers in high-fructose and saturated fat-fed rats.

The objective of this study was to evaluate the effect of roasting coffee degree on inflammatory (NF-kß F-6 and TNF-α) and stress oxidative markers (malondialdehyde (MDA), nitric oxide (NO) end product concentrations, catalase (CAT), and superoxide dismutase (SOD) in high-fructose and saturated fat (HFSFD)-fed rats. Roasting was performed using hot air circulation (200 °C) for 45 and 60 min, obtaining dark and very dark coffee, respectively. Male Wistar rats were randomly assigned to receive a) unroasted coffee, b) dark coffee, c) very dark coffee, or distilled water for the control group (n = 8). Coffee brews (7.4 mL/per day equivalent to 75 mL/day in humans) were given by gavage for sixteen weeks. All treated groups significantly decreased NF-kß F-6 (∼30 % for unroasted, ∼50 % for dark, and âˆ¼ 75 % for very dark group) and TNF-α in the liver compared with the control group. Additionally, TNF-α showed a significant reduction in all treatment groups (∼26 % for unroasted and dark groups, and âˆ¼ 39 % for very dark group) in adipose tissue (AT) compared with the negative control. Regarding oxidative stress makers, all coffee brews exerted antioxidant effects in serum, AT, liver, kidney, and heart. Our results revealed that the anti-inflammatory and antioxidant effects of coffee vary according to the roasting degree in HFSFD-fed rats.

Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis.

Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5±0.2mgl(-1) and the replicative system produced 20.3±0.8mgl(-1) of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.

Stabilization of dimeric ß-glucosidase from Aspergillus niger via glutaraldehyde immobilization under different conditions.

The dimeric enzyme ß-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The stability of the free enzyme depended on enzyme concentration. Immobilization via ion exchange improved enzyme stability/activity, depending on the immobilization pH. However, the enzyme was desorbed in 75 mM NaCl at pH 7 and some stability/enzyme concentration dependence still existed. TREATMENT: of these biocatalysts with glutaraldehyde increased enzyme stability (e.g. at pH 5, after incubation under conditions where the enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in enzyme activity, but the stabilization was lower. While when measuring the enzyme activity at pH 4 there were no changes after immobilization, all immobilized enzymes were more active than the free enzyme at pH 6 and 7 (2-3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid enzyme subunit desorption, being the immobilization pH a key point in their design.

Expression and characterization of a recombinant psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica E. Desv. [Poaceae].

We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.