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Urea Concentrations (urea + concentration)
Selected AbstractsA comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgarisFEBS JOURNAL, Issue 1 2002Brian Ó Nuallain The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s,1 in 3 m urea in 25 mm sodium phosphate at 25 °C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein. [source] Prediction of hemodialysis sorbent cartridge urea nitrogen capacity and sodium release from in vitro testsHEMODIALYSIS INTERNATIONAL, Issue 2 2008Benjamin P. ROSENBAUM Abstract In sorbent-based hemodialysis, factors limiting a treatment session are urea conversion capacity and sodium release from the cartridge. In vitro experiments were performed to model typical treatment scenarios using various dialyzers and 4 types of SORBÔ sorbent cartridges. The experiments were continued to the point of column saturation with ammonium. The urea nitrogen removed and amount of sodium released in each trial were analyzed in a multi-variable regression against several variables: amount of zirconium phosphate (ZrP), dialysate flow rate (DFR), simulated blood flow rate (BFR), simulated patient whole-body fluid volume (V), initial simulated patient urea concentration (BUNi), dialyzer area permeability (KoA) product, initial dialysate sodium and bicarbonate (HCO3i) concentrations, initial simulated patient sodium (Nai), pH of ZrP, creatinine, breakthrough time, and average urea nitrogen concentration in dialysate. The urea nitrogen capacity (UNC) of various new SORBÔ columns is positively related to ZrP, BFR, V, BUNi, and ZrP pH and negatively to DFR with an R2adjusted=0.990. Two models are described for sodium release. The first model is related positively to DFR and V and negatively to ZrP, KoA product, and dialysate HCO3i with an R2adjusted=0.584. The second model incorporates knowledge of initial simulated patient sodium (negative relationship) and urea levels (negative relationship) in addition to the parameters in the first model with an R2adjusted=0.786. These mathematical models should allow for prediction of patient sodium profiles and the time of column urea saturation based on simple inputs relating to patient chemistries and the dialysis treatment. [source] New enzymatic assay for serum urea nitrogen using urea amidolyaseJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 2 2003Shigeki Kimura Abstract We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N,, N,-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80,3.76% (n = 10) at 2.8,19.0 mmol/L, respectively. The day-to-day CVs were 2.23,4.59%. Analytical recovery was 92,115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x , 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of ,0.18 ± 0.86 mmol/L [(values by reference method , that of present method) ± SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52,56, 2003. © 2003 Wiley-Liss, Inc. [source] Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A 1H-NMR and electronic absorbance studyPROTEIN SCIENCE, Issue 9 2002Daniela Delli Castelli Abstract The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N,X,D, where X represents a disordered globin structure (occurring at ,4 M urea) that still binds the heme. [source] Urea interactions with protein groups: A volumetric study,BIOPOLYMERS, Issue 10 2010Soyoung Lee Abstract We determined the partial molar volumes and adiabatic compressibilities of N -acetyl amino acid amides, N -acetyl amino acid methylamides, N -acetyl amino acids, and short oligoglycines as a function of urea concentration. We analyze these data within the framework of a statistical thermodynamic formalism to determine the association constants for the reaction in which urea binds to the glycyl unit and each of the naturally occurring amino acid side chains replacing two waters of hydration. Our determined association constants, k, range from 0.04 to 0.39M. We derive a general equation that links k with changes in free energy, ,Gtr, accompanying the transfer of functional groups from water to urea. In this equation, ,Gtr is the sum of a change in the free energy of cavity formation, ,,GC, and the differential free energy of solute,solvent interactions, ,,GI, in urea and water. The observed range of affinity coefficients, k, corresponds to the values of ,,GI ranging from highly favorable to slightly unfavorable. Taken together, our data support a direct interaction model in which urea denatures a protein by concerted action via favorable interactions with a wide range of protein groups. Our derived equation linking k to ,Gtr suggests that ,,GI and, hence, the net transfer free energy, ,Gtr, are both strongly influenced by the concentration of a solute used in the experiment. We emphasize the need to exercise caution when two solutes differing in solubility are compared to determine the ,Gtr contribution of a particular functional group. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 866,879, 2010. [source] High Recovery Refolding of rhG-CSF from Escherichia coli, Using Urea Gradient Size Exclusion ChromatographyBIOTECHNOLOGY PROGRESS, Issue 1 2008Chaozhan Wang Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli ( E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol·L -1 urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding&‐;in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 ,L of solubilized and denatured rhG-CSF in 8.0 mol·L -1 urea solution with a total protein concentration of 2.3 mg·mL -1 was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 × 108 IU·mg -1 was obtained, and the mass recovery was 46.1%. [source] Increasing renal mass improves survival in anephric rats following metanephros transplantationEXPERIMENTAL PHYSIOLOGY, Issue 1 2007Damian Marshall Renal failure and end-stage renal disease are prevalent diseases associated with high levels of morbidity and mortality, the preferred treatment for which is kidney transplantation. However, the gulf between supply and demand for kidneys remains high and is growing every year. A potential alternative to the transplantation of mature adult kidneys is the transplantation of the developing renal primordium, the metanephros. It has been shown previously, in rodent models, that transplantation of a metanephros can provide renal function capable of prolonging survival in anephric animals. The aim of the present study was to determine whether increasing the mass of transplanted tissue can prolong survival further. Embryonic day 15 rat metanephroi were transplanted into the peritoneum of anaesthetized adult rat recipients. Twenty-one days later, the transplanted metanephroi were anastomosed to the recipient's urinary system, and 35 days following anastomosis the animal's native renal mass was removed. Survival times and composition of the excreted fluid were determined. Rats with single metanephros transplants survived 29 h longer than anephric controls (P < 0.001); animals with two metanephroi survived 44 h longer (P < 0.001). A dilute urine was formed, with low concentrations of sodium, potassium and urea; potassium and urea concentrations were elevated in terminal serum samples, but sodium concentration and osmolality were comparable to control values. These data show that survival time is proportional to the mass of functional renal tissue. While transplanted metanephroi cannot currently provide life-sustaining renal function, this approach may have therapeutic benefit in the future. [source] The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier proteinFEBS JOURNAL, Issue 11 2004Characterization of cold denaturation, protein interactions Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 ± 0.3 kcal·mol,1·K,1, at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 ± 4 kcal·mol,1, the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m,Hi), entropy (m,Si) and heat capacity (m,Cpi), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m,Hi and the m,Si are negative at low temperatures, but as the temperature is increased, m,Hi makes a less favourable contribution than m,Si to the change in free energy upon urea binding. The m,Cpi is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small. [source] Effect of frequency of application of inorganic nitrogen fertilizer within a rotational paddock-grazing system on the performance of dairy cows and inputs of labourGRASS & FORAGE SCIENCE, Issue 2 2008C. P. Ferris Abstract As herd sizes and labour costs increase, and the availability of skilled labour decreases, efficient use of available labour becomes more important in dairy cow systems. Two experiments were conducted to examine the effect of reducing the frequency of application of inorganic nitrogen (N) fertilizer on inputs of labour and performance of dairy cows. Experiments 1 (duration of 169 d) and 2 (duration of 179 d) involved fifty-eight and forty multiparous Holstein,Friesian dairy cows, respectively, in mid-lactation. In each experiment, in the ,infrequent' treatment fertilizer was applied to all paddocks on a single occasion at the start of each grazing cycle, while in treatment ,frequent', fertilizer was applied on three occasions each week, within 2 or 3 d of each paddock having been grazed. The experimental treatments were started from 30 March and 29 March in Experiments 1 and 2 respectively. Total N application rates were approximately 360 and 250 kg N ha,1 in Experiments 1 and 2 respectively. Concentrate feed (4·0 kg per cow) was offered daily in both experiments. With the ,infrequent' treatment, highest concentrations of crude protein and nitrate in herbage were observed in swards grazed approximately 10 d after N fertilizer was applied. Treatment had no significant effect on milk yield, milk fat and protein concentrations, and final live weight and body condition score of cows in either experiment. Milk urea and plasma urea concentrations were not significantly affected by treatment. Calculated application times of fertilizer for a herd of 100 dairy cows were 107 and 83 min week,1 for the ,frequent' and ,infrequent' treatments respectively. [source] Effect of a high-protein diet on food intake and liver metabolism during pregnancy, lactation and after weaning in micePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2010Björn Kuhla Abstract Major hepatic metabolic pathways are involved in the control of food intake but how dietary proteins affect global metabolism to adjust food intake is incompletely understood, particularly under physiological challenging conditions such as lactation. In order to identify these molecular events, mice were fed a high-protein (HP) diet from pregnancy, during lactation until after weaning and compared with control fed counterparts. Liver specimens were analyzed for regulated proteins using 2-DE and MALDI-TOF-MS and plasma samples for metabolites. Based on the 26 differentially expressed proteins associated with depleted liver glycogen content, elevated urea and citrulline plasma concentrations, we conclude that HP feeding during lactation leads to an activated amino acid, carbohydrate and fatty acid catabolism while it activates gluconeogenesis. From pregnancy to lactation, plasma arginine, tryptophan, serine, glutamine and cysteine decreased, whereas urea concentrations increased in both groups. Concomitantly, hepatic glycogen content decreased while total fat content remained unaltered in both groups. Consideration of 59 proteins differentially expressed between pregnancy and lactation highlights different strategies of HP and control fed mice to meet energy requirements for lactation by adjusting amino acid degradation, carbohydrate and fat metabolism, citrate cycle, but also ATP-turnover, protein folding, secretion of proteins and (de)activation of transcription factors. [source] Differential expression of heat shock protein 27 and 70 in renal papillary collecting duct and interstitial cells , implications for urea resistanceTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Wolfgang Neuhofer The adaptation of renal medullary cells to their hyperosmotic environment involves the accumulation of compatible organic osmolytes and the enhanced synthesis of heat shock proteins (HSP) 27 and 70. While the mechanisms leading to osmolyte accumulation are similar in papillary collecting duct (PCD) and papillary interstitial (PI) cells, the present data demonstrate that HSP27 and HSP70 are expressed differentially in these cells both in vivo and in vitro. HSP70 is abundant in PCD, but not expressed in PI cells in the papilla in situ, while HSP27 is expressed in both PCD and PI cells. These observations could be reproduced by non-permeant solutes in cultured cells. Osmotic stress strongly induced HSP70 in MDCK cells (as a model for PCD cells), but not in PI cells, while HSP27 was constitutively expressed in MDCK cells and was up-regulated in PI cells. Since prior hypertonic stress (NaCl addition) protects MDCK against subsequent exposure to high urea concentrations, this effect was also assessed in PI cells. In both cell lines, hypertonic pretreatment prior to urea exposure (400 mm) strongly attenuated caspase-3 activation. Inhibition of HSP27 expression by antisense transfection diminished the protective effect of hypertonic preconditioning in PI cells, while attenuation of HSP70 expression in MDCK cells diminished the protective effect of hypertonic preconditioning in these cells. These observations indicate that PCD and PI cells employ cell-specific mechanisms for protection against high urea concentrations as present in the renal papilla during antidiuresis. [source] Lycopene, a Carotenoid, Attenuates Cyclosporine-Induced Renal Dysfunction and Oxidative Stress in RatsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2007Ahmet Ate, ahin Adult male Sprague-Dawley rats were randomly divided into four groups. The control group received physiological saline; animals in the lycopene group received only lycopene (10 mg/kg); animals in the cyclosporine A group received only cyclosporine A (15 mg/kg) and animals in cyclosporine plus lycopene group received cyclosporine and lycopene for 21 days. The effects of lycopene on cyclosporine A-induced nephrotoxicity were evaluated by plasma creatinine, urea, sodium and calcium concentrations; kidney tissue thiobarbituric acid reactive species, reduced glutathione (GSH), glutathione peroxidase (GSH-Px) and catalase activities and histopatological examinations. Administration of cyclosporine A to rats induced a marked renal failure, characterized with a significant increase in plasma creatinine and urea concentrations. Cyclosporine A also induced oxidative stress as indicated by increased kidney tissue concentrations of thiobarbituric acid reactive species and GSH, and reduced activities of GSH-Px and catalase. Moreover, the kidneys of cyclosporine A-treated rats showed tubular necrosis, degeneration, dilatation, thickened basement membranes, luminal cast formation and inter-tubular fibrosis. Lycopene markedly reduced elevated plasma creatinine, urea levels and counteracted the deleterious effects of cyclosporine A on oxidative stress markers. In addition, lycopene ameliorated cyclosporine A-induced pathological changes including tubular necrosis, degeneration, thickened basement membranes and inter-tubular fibrosis when compared to the alone cyclosporine A group. These data indicate that the natural antioxidant lycopene might have protective effect against cyclosporine-induced nephrotoxicity and oxidative stress in rat. [source] Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using "Phase Diagram" Method of FluorescenceCHINESE JOURNAL OF CHEMISTRY, Issue 12 2007Liu-Jiao BIAN Abstract The refolding of reduced and non-reducing egg white lysozymes in a urea solution was studied by a "phase diagram" method of fluorescence. The result showed that in the refolding of the reduced egg white lysozyme, an intermediate state of an egg white lysozyme exists at the urea concentrations in a final renaturation solution being about 4.5 mol/L, their refolding follows a three-state model; while in the refolding of the non-reducing egg white lysozyme, two intermediate states exist at the urea concentrations being separately 4.0 and 2.5 mol/L, and their refolding follows a four-state model. Through the comparison between the unfolding and refolding of an egg white lysozyme in the urea solution, it was found that both of the refolding of reduced and non-reducing egg white lysozyme molecules was irreversible to their unfolding in the urea solution. Finally, a suggested refolding was separately presented for the reduced and non-reducing egg white lysozymes in the urea solution. [source] Comparison of cysteinyl leukotriene concentrations between exhaled breath condensate and bronchoalveolar lavage fluidCLINICAL & EXPERIMENTAL ALLERGY, Issue 12 2008E. Ono Summary Background Collection of exhaled breath condensate (EBC) is a simple, non-invasive method of obtaining samples from the airways and it can be repeated in short intervals without side effects; therefore, it provides an opportunity to monitor the changes in concentration of inflammatory mediators in the airways. However, EBC analysis still has several unresolved issues. Objective To better understand the characteristics of EBC, we compared cysteinyl leukotriene (CysLT) concentrations between bronchoalveolar lavage fluid (BALF) and EBC. We also attempted to correct CysLT concentrations in BALF and EBC diluted with saline and water vapour using biological markers. Methods EBC was collected from 14 patients with idiopathic pulmonary fibrosis before bronchoscopy. We measured CysLT concentrations and also quantified tyrosine, urea and total protein as possible biomarkers for correcting dilution. Results (1) We have validated the quantification of CysLTs in EBC. (2) Although a significant correlation was observed among tyrosine and urea concentrations in BALF, urea and total protein concentrations were below the detection limit in EBC. (3) CysLT concentrations were higher in BALF than in EBC (median, 15.96 pg/mL vs. 5.5 pg/mL; P=0.001) and there was no correlation of CysLT concentrations in BALF with those in EBC. A significant correlation of the ratio of total CysLT concentration to tyrosine concentration (CysLT/Y) in EBC with that in BALF was observed (r=0.547, P=0.043). (4) CysLT/Y in EBC correlated with serum KL-6 concentration and total cell count in BALF, and CysLT/Y in BALF also correlated with exhaled NO concentration and %VC. Conclusions CysLT/Y in EBC significantly correlated with that in BALF and some clinical parameters correlated with CysLT/Y. Tyrosine concentration may be used to correct the dilution error for CysLT concentrations, and CysLT/Y in EBC can be a surrogate marker for CysLT concentrations in BALF. [source] | ||||||||||||||||||||||||||||