An optimization study of solid-state fermentation: xanthophylls extraction from marigold flowers

Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.     

The -308 polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene and ex vivo lipopolysaccharide-induced TNF-alpha expression in patients with aggressive periodontitis and/or type 1 diabetes mellitus

Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.     

Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli

DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.     

Ciprofibrate therapy in patients with hypertriglyceridemia and low high density lipoprotein (HDL)-cholesterol: greater reduction of non-HDL cholesterol in subjects with excess body weight (The CIPROAMLAT study)

Background

Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk.

Methods

The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion.

Results

In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246).

Conclusion

The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.     

Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly

The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers.     

Evidence of substantial carbon isotope fractionation among substrate, inorganic carbon, and biomass during aerobic mineralization of 1, 2-dichloroethane by Xanthobacter autotrophicus

Carbon isotope fractionation during aerobic mineralization of 1, 2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of (13)C in residual 1,2-DCA was observed, with a mean fractionation factor alpha +/- standard deviation of 0.968 +/- 0.0013 to 0.973 +/- 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor alpha to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of (13)C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S(N)2 nucleophilic substitution. S(N)2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.     

Medium Selection and Effect of Higher Oxygen Concentration Pulses on <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var<Emphasis Type="Italic">. lepidiotum</Emphasis> Conidial Production and Quality

Rice and oat flours were analyzed as media for the production of conidia by M. anisopliae var. lepidiotum. The presence of peptone increased conidia yield regardless of the substrate used; however, the highest yield was achieved on oat flour media. The effect of oxygen on conidia production using oat-peptone medium was also studied at two levels: Normal atmosphere (21% O₂) and Oxygen-rich pulses (26% O₂). Maximum conidia production (4.25 × 10⁷ conidia cm⁻²) was achieved using 26% O₂ pulses after 156 h of culture, which was higher than 100% relative to conidial levels under normal atmosphere. Conidia yield per gram of biomass was 2.6 times higher with 26% O₂ (1.12 × 10⁷ conidia mg⁻¹). Conidia quality parameters, such as germination and hydrophobicity, did not show significant differences (P < 0.05) between those treatments. Bioassays parameters, using Tenebrio molitor adults, were analyzed for conidia obtained in both atmospheres and data were fitted to an exponential model. The specific mortality rates were 2.22 and 1.26 days⁻¹, whereas lethal times for 50% mortality were 3.90 and 4.31 days, for 26% O₂ pulses and 21% O₂ atmosphere, respectively. These results are relevant for production processes since an oxygen increase allowed superior levels of conidia by M. anisopliae without altering quality parameters and virulence toward Tenebrio molitor adults.     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.