An enhanced partition model is proposed for the distribution of 4-nitrophenol between polar quasi-immiscible solvents (water and 1-octanol). Monitoring both phases over an extend wavelengths range ...(200–450 nm), the presence of 4-nitrophenol and 4-nitrophenolate species in the aqueous and organic layers was emphasized. A genetic algorithm has been used to minimize the sum of squared residuals model-experiment using the non-linear equations system model resulted from mass and charge balances. Thus, improved values for the thermodynamic constants associated to partition, dissociation and dimerization equilibria occurring in both phases were found: 1-octanol dissociation constant in the aqueous phase, KOw = 3.2 × 10−13 mol∙L−1, 4-nitrophenol dissociation constant in aqueous phase, KΦw = 1.45 × 10−8 mol∙L−1, water dissociation constant in the organic phase, Kwo = 3.5 × 10−16 mol∙L−1, 1-octanol dissociation constant in the organic phase, KOo = 1.1 × 10−16 mol∙L−1, 4-nitrophenol dissociation constant in organic phase, KΦo = 2.9 × 10−10mol∙L−1, 1-octanol dimerization constant in the organic phase, KDo = 3 × 10−9 L∙mol−1 and 4-nitrophenol dimerization constant in organic phase, KDΦ = 1.4 × 10−8 L∙mol−1. Although the logarithms of calculated partition coefficients are rather similar to the values reported in the literature, in the 1.86–2.07 range, the collected experimental evidence demonstrates that the process had been oversimplified and the polar characteristics of the organic solvent had been neglected in the former studies. The enhanced partition model emphasized the nonlinear dependency of the partition coefficient upon the analyte concentrations.
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•Enhanced repartition model to account for mass balance closure and spectral behaviour.•4-Nitrophenol repartition accompanied by association-dissociation equilibria in both phases.•Non-linear behaviour of partition coefficient for polar third-party distribution in polar immiscible solvents.•Partition coefficients computed from non-dissociated third-party species concentrations only, prone to significant errors.•Evaluation of equilibrium constants of competitive reactions.
•Microalgae Nannochloris sp. were grown in mixotrophic conditions, under stress.•Two-step technologies for FAME obtaining from algae oil was performed.•A new heterogeneous catalyst with excellent ...activity and stability was prepared.•FAME technology was investigated in batch and as continuous flow process.•Under the optimal conditions, FAME yield reached 98.7%.
Intermediates for synthetic paraffinic kerosene with structures of fatty acid methyl ester (FAME), were synthesized from algal oil extracted from Nannochloris sp. biomass. Microalgae were grown under mixotrophic conditions to improve biomass production, using glycerin as additional source of organic carbon, by-product in FAME obtaining process. Microalgae were supplementary stressed to increase the lipid productivity, by reducing the inorganic nitrogen and partially replacing it with organic nitrogen (hydrolyzed proteins from the same microalgae). Since algal oil has high free fatty acids (FFA) content, a two-step process for FAME synthesis was necessary. In the first step, FFA was esterified with methanol over SO42−/TiO2–La2O3 solid acid catalyst. The second step, i.e. methanolysis of pre-treated oil, was performed over Na2SiO3/Mg–Al-hydrotalcite (Na2SiO3/Mg–Al-HTC) heterogeneous base catalyst. The Na2SiO3/Mg–Al-HTC catalyst was synthesized and then characterized by X-ray diffraction (XRD), differential thermal analysis (DTA), textural analysis (BET method), scanning electron microscopy (SEM), energy dispersive X-ray fluorescence analysis (EDXRF) and Fourier transform infrared (FTIR) spectroscopy. The influences of catalyst loading and stability, methanol-to-oil molar ratio and the reaction time on the FAME yield were carefully studied. FAME producing technology was investigated first in batch and then in a continuous flow process.
: We identified and quantified by LC-MS/MS 11 (quercetin, galangin, pinocembrin, kaempferol, vanillin, chrysin, gallic acid, p-coumaric acid, trans-ferulic acid, caffeic acid, and caffeic acid ...phenethyl ester) out of the 21 polyphenolic compounds we looked for in ethanolic (25% and 50%) and aqueous propolis extracts by comparison with standards and literature data.
We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage ...display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.
Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray ...technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies.
Abstract
Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to ...establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.
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