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Background: Hemodiafiltration with on-line endogenous reinfusion (HFR) is an extracorporeal dialytic method that combines diffusion, convection and adsorption. HFR-Supra (HFR-S) is a second-generation system with increased convective permeability and adsorption capability. Previous studies suggested that HFR reduces oxidative stress compared to standard haemodialysis. The principal aim of the present study was to compare antioxidant vitamins behavior and oxidative status of hemodialysis patients treated with HFR and HFR-S., Methods: The study was designed as a multicenter, randomized, crossover trial. Forty-one patients were recruited from 19 dialysis centers and after a 4-month washout stabilization period in on-line hemodiafiltration (ol-HDF), each patient was randomized to a sequence of treatments (HFR-S followed by HFR or viceversa) with each treatment applied over 6 months. Plasma levels of Advanced Oxidation Protein Products, Total Antioxidant Status, vitamins C, A and E and their ligands (Retinol Binding Protein and total lipids) were measured at baseline and at the end of each treatment period., Results: Results show that the higher convective permeability of HFR-S with respect to HFR did not produce additional beneficial effects on the patients' oxidative status, a slight decrease of both Vitamin A and Retinol Binding Protein being the only difference registered in the long-term. However, as compared to ol-HDF, both the re-infusive techniques allowed to reduce the intradialytic loss of Vitamin C and, in the long-term, improve the patients' oxidative status and increase Retinol Binding Protein plasma values. No significant differences were found between the Vitamin C concentration of pre- and post cartridge UF neither in HFR-S nor in HFR showing that the sorbent resin does not adsorb Vitamin C., Conclusion: HFR-S and HFR are almost equivalent in term of impact on antioxidant vitamins and oxidative status of hemodialysis patients. Nonetheless, as compared to ol-HDF, both treatments produced a sensible sparing of Vitamin C and may represent a new approach for reducing oxidative stress and related complications in dialysis patients. Long-term effects of re-infusive treatments on patients' cardiovascular morbidity and mortality need to be evaluated., Trial Registration: ClinicalTrials.gov Identifier NCT01492491 , retrospectively registered in 10 December 2011.

Background: The objective of the study was to compare the convective treatment modes, on-line hemofiltration (HF) and on-line hemodiafiltration (HDF), regarding cardiovascular tolerance and effects on blood pressure, when applied under similar conditions in stable dialysis patients., Methods: 39 clinically stable dialysis patients were treated with HD for 6 months (run-in period), followed by HF and HDF in random order for 2x6 months. Similar biocompatibility (same membrane and fluid quality), similar treatment time and urea Kt/V were achieved using AK100/200 ULTRA machines, polyamide membranes in low-flux and high-flux versions and appropriate adjustment of blood flow rate (Qb) and dilution ratio (Qb/Qinf). Predilution was used for HDF (target dilution ratio = 2/1 ) as well as for HF (target dilution ratio = 1/1)., Results: 30 patients completed the study; 5 dropped out for non-study related reasons and 4 for non-compliance. Treatment with HF in comparison to HDF showed fewer hypotension episodes during the sessions per patient and month (HF: 0.5, HDF 1.1; p = 0.017), less plasma expander administration per patient and month (HF: 35.9 ml, HDF: 103.1 ml; p = 0.035), fewer episodes of intra-session headache (HF: 0.1, HDF: 0.4; p = 0.06), and higher pre-session MAP (HF: 98.4 mmHg, HDF: 93.8 mmHg; p = 0.037). No significant difference was found in inter-treatment weight gain, post-session MAP, or pre-session plasma sodium., Conclusions: HF and HDF provide good control of intra-session symptoms and blood pressure in stable patients. Treatment with HF resulted in a significant reduction in intra-session hypotension and a slight but significant increase in pre-session MAP, caused by an increase in systolic BP without any effect on the prevalence of hypertension or the dose of antihypertensive drugs, all compared to HDF.

Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of PhSeCl gave a series of different alpha-phenylselenyl aldehydes. Mukajiama reaction with silylketene acetals gave an intermediate product that was finally transformed into the desired oxiranyl peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-dependent reversible inhibitors of cysteine protease.

Conformational modifications and changes in the aggregation state of human alphaB-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH4SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH4SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of alphaB-crystallin in 2 M NH4SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated alphaB-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.

A pattern recognition method based on artificial neural networks (ANNs) to analyze and quantify the components of six alditol binary mixtures is presented. This method is suitable to classify the spectra of the 15 mixtures obtained from the six alditols and to produce quantitative estimates of the component concentrations. The system is user-friendly and is helpful in solving the problem of greatly overlapping signals, often encountered in NMR spectroscopy of carbohydrates. A "classification" ANN uses 200 intensity values of the 1H NMR spectrum in the range 3.5-4 ppm. When the correct mixture is identified, the quantification is solved by assigning a specific ANN to each mixture. These ANNs use the same 200 values of the spectrum and output the values of the two concentrations. The error in the ANN responses is studied, and a method is developed to estimate the accuracy in determining the concentrations. The networks' abilities to recognize previously unseen mixtures are tested. When the classification ANN (trained on the 15 binary mixtures) is exposed to complex (i.e., more than binary) mixtures of the six known alditols, it successfully identifies the components if their minimum concentration is 10%. Given the precision of the results and the small number of errors reported, we believe that the method can be used in all fields in which the recognition and quantification of components are necessary.

Different sets of geminal J coupling constants for the eight beta-CH2 protons in the iron-coordinating cysteines in Clostridium pasteurianum ferredoxin were detected by 2D TOCSY 1H NMR experiments on the oxidized protein. Four resonances were characterized by quite similar high values of J, two more resonances had a J value about half of the former ones, while the last two had extremely low J values. These findings suggest that the cysteines required for cubane symmetry around the iron atoms are constrained into different geometries. The simplified model used for fine tuning of tau m in these TOCSY experiments is also presented and discussed.

Incubation of native, reduced Clostridium pasteurianum ferredoxin with different metals gave a range of modifications in the electronic and EPR spectrum of the protein, or made the signals disappear. The reduced protein, isolated after incubation with different metals under identical conditions (50 microM protein, 1 mM metal, 1 h incubation) was found to contain amounts of foreign metals increasing with their thiophylicity, i.e. Cd2+ >> Zn2+ > Co2+. Little, if any, incorporation was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductant. The activity of substituted ferredoxins in a hydrogenase-coupled assay was proportional to the amount of residual iron, suggesting that the residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among individual molecules. The cadmium-substituted ferredoxin did not contain iron, but contained eight cadmium atoms and six labile sulfide atoms/mol. Folding of the isolated, substituted proteins was investigated by CD and 1H-NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent of the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to increase with the cadmium concentration. In the cadmium-substituted protein, cadmium was replaced by iron upon incubation with iron and mercaptoethanol in the absence of dithionite. In the presence of dithionite, cadmium was not replaced by iron upon incubation of the cadmium-substituted protein with excess iron and mercaptoethanol. In competition experiments, incubation of iron-containing ferredoxin with stoichiometric amounts of cadmium in the presence of dithionite and excess iron and mercaptoethanol resulted in quantifiable replacement of iron by cadmium. Therefore, substitution of iron by cadmium was only achieved under reducing conditions, and was only reversible in the absence of strong reductants.

The two zinc fingers of the DNA binding domain of the human retinoic acid receptor-beta were labelled with 113Cd. Two- and three-dimensional heteronuclear nuclear magnetic resonance (NMR) experiments show that the first eight conserved cysteine residues coordinate the two zinc ions tetrahedrally. The ninth conserved cysteine is not involved in metal coordination. In each finger one cysteine exhibits a heteronuclear 113Cd-1H coupling constant substantially smaller than those of the other metal binding cysteines.

Potentiometric, polarographic, and spectroscopic results obtained for Cu2+ and Ni(2+)-famotidine systems clearly indicated that this anti-ulcerogenic drug is a very potent chelating agent able to coordinate cupric ion that was at pH below 2. This drug exhibits excellent histamine H2 receptor blocking effects and its effective coordination to metal ions may have significant biological implications. Famotidine is found to be a very effective ligand for Ni2+ ions also.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.

The shortened analogue of insulin, des-(B26-B30)-pentapeptide insulin, has been characterized by two-dimensional 1H NMR. The 1H resonance assignments and the secondary structure in water solution are discussed The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1.