Increase In Urea Concentration Research Articles

Effect of foliar spray of urea and zinc on growth and flowering attributes of guava (Psidium guajava) cv. BHAVNAGAR RED

Study was conducted in experimental farm of Junagadh Agricultural University, Gujarat India to find out the effect of foliar application of 1.0 per cent, 1.5 per cent or 2.0 per cent urea and zinc sulfate with concentration of 0.2, 04 and 0.6 per cent on guava crop, respectively, just before blooming significantly improved shoot growth, leaf number, area and weight. The best results were obtained with treatments of 1.0 and 1.5 per cent foliar spray of urea. In case of flowering, fruit set and fruit retention was better in treated plants with fertilizer. The size of fruits and yield per tree increased with increasing urea concentration and the significant variation in number of flowers per shoot was observed while non significant effect was observed under interaction effect of urea and zinc. The highest fruit weight and maximum fruit girth was registered with 0.6 per cent zinc. Thus, it is proved that 1.5 per cent urea solution and 0.6 per cent zinc sulphate were effective for the augmentation of growth and flowering attributes of guava.

Concentration of injetion solution and its effects on soil and on yield of fertirrigated banana cv. Terra Maranhão

The Fertigation is the combined application of water and nutrients to a crop. It can be adapted to all types of agricultural crops. The objective of this study was to evaluate the effect of urea concentration in irrigation water on electrical conductivity of the soil solution and saturation extract along the first cycle of banana cv. Terra Maranhão. The experiment followed a completely randomized design with six treatments and ten replications. Treatments regarded for using three urea concentrations (1.0; 2.5 and 4.0 g L-1) in irrigation water applied by two micro irrigation systems (microsprinkler and drip). Results showed that there was a linear elevation of electrical conductivity of saturation extract and soil solution with the increase on concentration of urea in the injection solution. Urea should be used under concentrations up to 2.5 g L-1 in irrigation water without causing increase on electric conductivity of soil solution and saturation extract, considering 1.1 dS m-1 as the tolerated value for the crop. Nitrate in the soil solution increased significantly with the increase of urea concentration in the injection solution. The maximum concentration of nitrate in the soil occurred for 4,0 g L-1 concentration of the injection solution.

Study the effect of foliar spraying with different levels of urea in some anatomical side for eggplant Solanum melongena L.

The field experiment had been conducted in the Lath-house in the college of Agriculture , Anbar University by planting Solanum melongena L. Black beauty during agricultural season of 2010. Seedlings were distributed randomly according to complete randomized design (CRD) . The four concentrations of urea dissolved in distill water . The levels of urea were control (N0) ; 0.5 g/L(N1) ;1 g/L(N2);8 g/L(N3) . After 40 days of transplanting Eggplants were sprayed four times by urea concentrations . Anatomic study of cross-sections of root showed clear reduction in thickness of Epidermis, cortex and xylem tissues of plants treated with the concentration 8 gram urea/L where thickness of these tissues was 26.07 µm, 178.52 µm, 37.60 µm respectively. The phloem showed 224.32 um thickness. It was the highest among other treatments especially the treatment 1g urea/L which showed higher rate of xylem thickness of 373.68 µm. The cross-section of stem also showed clear difference in the epidermis thickness for all treated plants. In addition, 1gram urea/L showed higher cortex thickness of the stem,( 432.93 µm). Increasing urea concentration to 8 gram/L lead to reduce xylem thickness (104.72 µm) and decreasing pith thickness in the same treatment rated 426.67 µm. Plants treated with 1 gram urea/L, show increasing in the phloem and the pith thickness of 98.62 µm, 577.47µm rate respectively.The study showed that stoma distribution in the surface of leaves is of Anomocytic type with clear reduction of the outer epidermis thickness of 1gram urea/L treatment rated 16.05µm. This treatment showed highest rate in palasid tissues 98.40. The N2 treatment shows higher rate in stoma frequency on the upper and lower surfaces, 10 stoma/ml². The N3 treatment shows less number of stoma on the upper and lower surfaces, 6.04 stoma/ml². As for cell length in the two epidermis, the N2 treatment shows clear increase rated 59.08 in comparison with control treatment and othertreatments.

In-gel detection of esterase-like albumin activity: Characterization of esterase-free sera albumin and its putative role as non-invasive biomarker of hepatic fibrosis

Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA) (10mg/kg body weight) within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE) (7.5%). In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58×10−3mM; pH 7.5) as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10%) and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.

Macrophage depletion abates Porphyromonas gingivalis-induced alveolar bone resorption in mice.

The role of the macrophage in the immunopathology of periodontitis has not been well defined. In this study, we show that intraoral inoculation of mice with Porphyromonas gingivalis resulted in infection, alveolar bone resorption, and a significant increase in F4/80(+) macrophages in gingival and submandibular lymph node tissues. Macrophage depletion using clodronate-liposomes resulted in a significant reduction in F4/80(+) macrophage infiltration of gingival and submandibular lymph node tissues and significantly (p < 0.01) less P. gingivalis-induced bone resorption compared with controls in BALB/c and C57BL/6 mice. In both mouse strains, the P. gingivalis-specific IgG Ab subclass and serum cytokine [IL-4, IL-10, IFN-γ, and IL-12 (p70)] responses were significantly (p < 0.01) lower in the macrophage-depleted groups. Macrophage depletion resulted in a significant reduction in the level of P. gingivalis infection, and the level of P. gingivalis infection was significantly correlated with the level of alveolar bone resorption. M1 macrophages (CD86(+)), rather than M2 macrophages (CD206(+)), were the dominant macrophage phenotype of the gingival infiltrate in response to P. gingivalis infection. P. gingivalis induced a significant (p < 0.01) increase in NO production and a small increase in urea concentration, as well as a significant increase in the secretion of IL-1β, IL-6, IL-10, IL-12 (p70), eotaxin, G-CSF, GM-CSF, macrophage chemoattractant protein-1, macrophage inflammatory protein-α and -β, and TNF-α in isolated murine macrophages. In conclusion, P. gingivalis infection induced infiltration of functional/inflammatory M1 macrophages into gingival tissue and alveolar bone resorption. Macrophage depletion reduced P. gingivalis infection and alveolar bone resorption by modulating the host immune response.

Urea-Induced Dissociation of Nerve Growth Factor from Venom of Chinese Cobra by SEC

The urea-induced dissociation of nerve growth factor from venom of Chinese cobra (cNGF) was studied by intrinsic fluorescence emission spectra, SEC, urea-gradient polyacrylamide gel electrophoresis, assays of biological activity and thermodynamic parameters. The results showed that when urea concentration was lower than or equal to 4.0 mol L−1 or higher than or equal to 8.0 mol L−1, cNGF existed only in native homodimer form or monomer form, respectively; whereas when urea concentration was higher than 4.0 mol L−1 and lower than 8.0 mol L−1, they existed simultaneously in the native homodimer and monomer forms and the former decreased, while the latter increased with the increase in urea concentration. Based on the association–dissociation equilibrium between cNGF and urea molecules, an equation, which includes two characteristic dissociation parameters K and ∆m, was presented to describe the urea-induced dissociation process of cNGF. As the reaction temperature increased from 15 to 35 °C, positive enthalpy and entropy changes were observed, and the parameter K increased from 2.72 × 10−13 to 5.18 × 10−12 (L mol−1), while the parameters ∆m and ∆G, respectively, decreased from 10.18 to 8.42 and from −10.27 to −18.67 (kJ mol−1), which means that the urea-induced dissociation of cNGF was spontaneous and entropy-driven and the higher temperature was favorable for the dissociation process. Using the procedures and equations mentioned in the paper, the urea-induced dissociation of cNGF is first comprehensively described. Furthermore, this work presents a useful method for people to study the dissociation of dimer or multimer proteins induced by denaturants, inducers, pH, etc.

The Effect of Short-Term Interval Training during the Competitive Season on Physical Fitness and Signs of Fatigue: A Crossover Trial in High-Level Youth Football Players

To analyze performance and fatigue effects of small-sided games (SSG) vs high-intensity interval training (HIIT) performed during a 4-wk in-season period in high-level youth football. Nineteen players from 4 youth teams (16.5 [SD 0.8] y, 1.79 [0.06] m, 70.7 [5.6] kg) of the 2 highest German divisions completed the study. Teams were randomly assigned to 1 of 2 training sequences (2 endurance sessions per wk): One training group started with SSG, whereas the other group conducted HIIT during the first half of the competitive season. After the winter break, training programs were changed between groups. Before and after the training periods the following tests were completed: the Recovery-Stress Questionnaire for Athletes, creatine kinase and urea concentrations, vertical-jump height (countermovement jump [CMJ], drop jump), straight sprint, agility, and an incremental field test to determine individual anaerobic threshold (IAT). Significant time effects were observed for IAT (+1.3%, η(p)(2) = .31), peak heart rate (-1.8%, η(p)(2) = .45), and CMJ (-2.3%, η(p)(2) = .27), with no significant interaction between groups (P > .30). Players with low baseline IAT values (+4.3%) showed greater improvements than those with high initial values (± 0.0%). A significant decrease was found for total recovery (-5.0%, η(p)(2) = .29), and an increase was found for urea concentration (+9.2%, η(p)(2) = .44). Four weeks of in-season endurance training can lead to relevant improvements in endurance capacity. The decreases in CMJ height and total-recovery score together with the increase in urea concentration might be interpreted as early signs of fatigue. Thus, the danger of overtaxing players should be considered.

Avidity of anticardiolipin antibodies—A factor that could be important for their detection by ELISA methods

Avidity is an important feature of antibodies associated with their pathogenic effects. Anticardiolipin antibodies (aCLs) are the most commonly examined antiphospholipid antibodies, however, their avidity has been investigated only marginally. The aim of the study was to compare the avidity of the antibodies specifically bound to the cardiolipin-coated microtitrate wells with those bound to the wells bearing no cardiolipin in various conditions. We analysed 22 serum samples with high, medium and low levels of aCL IgG. The avidity of aCL IgG was determined in serially diluted sera by the ELISA method in the presence of increasing urea concentrations (from 2 to 8mol/L) as a chaotropic agent. The serum dilution 1:50 and 1:100 and the concentrations of urea 6mol/L and 8mol/L seemed to be suitable for the determination of aCL avidity. The avidity of antibodies specifically bound to the cardiolipin and those bound to the cardiolipin-free wells significantly differed in their avidity. The simultaneous determination of higher-avidity aCL and lower-avidity polyspecific antibodies, whose presence complicates the ELISA methods by an increase of the background signal, might limit the shortcomings of some ELISA methods.

Adaptive regulation of amino acid metabolism on early parenteral lipid and high-dose amino acid administration in VLBW infants – A randomized, controlled trial

An anabolic state can be achieved upon intravenous amino acid administration during the immediate postnatal phase despite a low energy intake. The optimal dosing of amino acid and energy intake has yet to be established. The aim was to quantify the efficacy of early initiation of parenteral lipids and increased amounts of amino acids on metabolism and protein accretion in very low birth weight infants. 28 very low birth weight infants were randomized to receive parenteral nutrition with glucose and either 2.4gamino acids/(kg·d) (control group), 2.4gamino acids/(kg·d) plus 2-3glipid/(kg·d) (AA+lipid group), or 3.6gamino acids/(kg·d) plus 2-3glipid/(kg·d) (high AA+lipid group) from birth onward. On postnatal day 2, we performed a stable isotope study with [1-(13)C]phenylalanine, [ring-D4]tyrosine, [U-(13)C6,(15)N]leucine, and [methyl-D3]α-ketoisocaproic acid to quantify intermediate amino acid metabolism. The addition of lipids only had no effect on phenylalanine metabolism, whereas the addition of both lipids and additional amino acids increased the amount of phenylalanine used for protein synthesis. In addition, high amino acid intake significantly increased the rate of hydroxylation of phenylalanine to tyrosine, increasing the availability of tyrosine for protein synthesis. However, it also increased urea concentrations. Increasing energy intake from 40 to 60kcal/(kg·d) did not increase protein efficiency as measured by phenylalanine kinetics. The leucine data were difficult to interpret due to the wide range of results and inconsistency in the data between the phenylalanine and leucine models. High amino acid and energy intakes from birth onwards result in a more anabolic state in very low birth weight infants, but at the expense of higher urea concentrations, which reflects a higher amino acid oxidation. Long-term outcome data should reveal whether this policy deserves routine implementation. This trial was registered at www.trialregister.nl, trial number NTR1445, name Nutritional Intervention for Preterm Infants-2.

Urea, Guanidine Hydrocloride and 2,2,2-Trifluoroethanol Can Change the Amyloid Fibril Formation of Model Proteins: A Spectroscopic Study

The influence of external agents on proteins function and structure is essential to elucidate the unfolding pathways and the protein self-assemble properties. The knowledge of the protein amyloid fibril formation process is important due to the fields that this subjected is related, in particular for the neurodegenerative disorders as Parkinson's and Alzheimer's diseases. In the present study we evidenced the influence of different external agents on the Bovine Serum Albumin (BSA) and Lysozyme amyloid fibril formation by means of UV-Vis and Fluorescence spectroscopy. Concerning BSA, the presence of urea (< 3M) was able to induce the formation of amyloid fibrils at 328 K and increasing urea concentration the amount of protein in the amyloid form also increases. Moreover, a different effect was evidenced by the presence of TFE, where BSA underwent an amorphous aggregation process, leading even to the flocculation of BSA. Interestingly the presence of both urea and TFE up to 5% induces the appearance of amyloid fibrils, instead of amorphous aggregation. Regarding GndHCl, it was not able to induce the formation of amyloid fibrils in neither BSA nor Lysozyme. It is interesting that GndHCl and Urea are well-known as protein denaturant agents, however, their interaction in the protein surface is quite different, such a difference could lead the protein to different final conformations, including the amyloid fibril one. This study indicates that the hydration shell can play an important role in the amyloid aggregation process.Acknowledgements: FAPESP (grant # 2012/01953-9) CNPq (grant # 479229/2011-2, # 303048/2012-3).

Screening diagnostics to identify triggers in 21 cases of steroid‐responsive meningitis‐arteritis

To evaluate whether screening tests used to identify infectious and neoplastic triggers for immune-mediated haemolytic anaemia, in particular a complete blood count and differential, serum biochemistry profile, urine analysis (including culture), abdominal ultrasound and thoracic radiographs, can identify triggers for steroid-responsive meningitis-arteritis. Retrospective descriptive review. Twenty-one steroid-responsive meningitis-arteritis cases were identified in which all screening tests had been performed. All cases had changes in complete blood count (including neutrophilia, monocytosis, lymphocytosis, eosinopenia or anaemia); 19 had changes in biochemistry (including hypoalbuminaemia, hyperglobulinaemia, increased alkaline phosphatase activity, hyperphosphataemia, increased total calcium concentration, hypercholesterolaemia, hyperkalaemia, increased urea concentration and increased alanine aminotransferase activity); two cases had an elevated urine protein to creatinine ratio but none had positive urine culture results; no cases had abnormalities on orthogonal radiographs of the thorax; four cases had abnormalities identified on abdominal ultrasound, which following cytological examination suggested inflammation in the absence of pathological organisms. Screening tests used to identify infectious and neoplastic triggers in immune-mediated haemolytic anaemia did not isolate triggers for steroid-responsive meningitis-arteritis in the population of dogs under investigation.

Iron-dependent modifications of the flower transcriptome, proteome, metabolome, and hormonal content in an Arabidopsis ferritin mutant

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe–haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions.

Structural and hyperfine properties of Mn and Co-incorporated akaganeites

The structural and hyperfine properties of pure and substituted akaganeites prepared in the presence of Mn, Co and urea are presented and discussed. In all samples, the chloride content increased with the increase in the urea concentration of the parent solution, and a small Mn-for-Fe or Co-for-Fe substitution occurred. In pure akaganeites, the increase of urea concentration provoked an enlargement of the unit cell volume and a decrease of the crystallinity of the synthesised oxides. The incorporation of Mn and Co provoked changes in cell parameters and an increase in the crystallinity of the samples. The hyperfine parameters for both iron sites of the akaganeites remained practically unchanged, and the spectral areas of the iron sites located close to the chlorides decreased for the doped samples. The recoilless f-factor increased for the substituted akaganeites, indicating an increase in the strength of the atomic bonding of the iron ions.

Treatment of Severe Atopic Dermatitis with Cyclosporine: A Review of Eight Patients

AbstractAtopic dermatitis is most frequently well controlled with topical therapeutic agents, but based on several studies, 10-20% of patients need systemic therapy. The most common systemic treatment for atopic dermatitis in everyday practice includes systemic corticosteroids, although there are insufficient valid data to support this. Cyclosporine is the treatment of choice for severe atopic dermatitis resistant to other commonly used treatment options, since its favorable therapeutic risk/benefit ratio is well documented in randomized placebo controlled trials, and also in uncontrolled trials. However, approximately 10% of patients with atopic dermatitis with indication for cyclosporine treatment are actually treated with this modality in Serbia, and there are no published case series on its use in this region so far. In this article, we evaluated the treatment efficacy and safety of cyclosporine microemulsion in patients with severe atopic dermatitis hospitalized at the Military Medical Academy in Belgrade from 2009 to 2012. This restrospective analysis included patients with severe forms of atopic dermatitis treated at the Department of Dermatology of the Military Medical Academy from 2009 - 2012. The hospital database was used to retrieve patients’ medical records. Approximately 200 patients were treated for atopic dermatitis and 20 patients were admitted to the hospital, 17 due to severe forms of disease. In total, 8 of 17 (47.05%) hospitalized patients with severe forms or erythroderma due to atopic dermatitis were treated with cyclosporine microemulsion with an initial dose of 4-5 mg/kg. Laboratory tests were done before treatment, 7 days later, and/or at the end of hospitalization. Therapeutic efficacy was evaluated based on the percentage of reduction of skin lesions from baseline to the end of hospital treatment (early efficacy), and at the end of follow-up (late efficacy). Duration of therapy, adverse events, treatment efficacy and reasons for treatment cessation were recorded during the follow-up period. There were five male and 3 female patients, with an average age of 36.8 years (15 - 60 years). Previous treatment modalities in all patients included emollients, topical and systemic corticosteroids and PUVA therapy. The average dose of cyclosporine was 4.5±0,5 mg/kg. Median reduction of skin lesions at discharge was 60%. There was no need for further hospitalization after an average of 10±3.2 days. Mean duration of treatment was 16 months (3 - 24), with an average reduction of skin lesions of 75% during follow-up. Arterial blood pressure increased in 3/8 (37.5%) patients, regardless of their age, with and average increase of systolic blood pressure of 11.9±11.6 mm Hg (median 7.5, 0-30 mm Hg) and diastolic blood pressure of 5.6±12.9 mm Hg (median 0, -10-20). Mean increase in urea concentration was 0.3 mmol/L (11.8%) and creatinine increased only in three patients by 4.2% (median increase 4 mmol/L). Hypertension was found in three patients during follow-up, and there were no other adverse events. In conclusion, based on previous studies and this small case series of hospitalized patients with severe forms of atopic dermatitis including erythroderma, cyclosporine can be regarded as a safe and effective treatment modality and it can be recommended as first line therapy in severe forms of atopic dermatitis refractory to topical therapy and phototherapy. Long term therapy, however, should be avoided and a maximum 1-2 year therapy is recommended.

Theoretical study on the structures and properties of mixtures of urea and choline chloride

In this work, we investigated in detail the structural characteristics of mixtures of choline chloride and urea with different urea contents by performing molecular dynamic (MD) simulations, and offer possible explanations for the low melting point of the eutectic mixture of choline chloride and urea with a ratio of 1:2. The insertion of urea molecules was found to change the density distribution of cations and anions around the given cations significantly, disrupting the long-range ordered structure of choline chloride. Moreover, with increasing urea concentration, the hydrogen bond interactions between choline cations and Cl(-) anions decreased, while those among urea molecules obviously increased. From the hydrogen bond lifetimes, it was found that a ratio of 1:2 between choline chloride and urea is necessary for a reasonable strength of hydrogen bond interaction to maintain the low melting point of the mixture of choline chloride with urea. In addition, it was also deduced from the interaction energies that a urea content of 67.7 % may make the interactions of cation-anion, cation-urea and anion-urea modest, and thus results in the lower melting point of the eutectic mixture of choline chloride and urea. The present results may offer assistance to some extent for understanding the physicochemical properties of the eutectic mixture of choline chloride and urea, and give valuable information for the further development and application of deep eutectic solvents.

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant ka increased 20-fold when the concentration of urea was raised from 0M to 6M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the ka value reached its maximum (ka,max) in all urea concentrations. Interestingly, the ka,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.

Honey-Induced Protein Stabilization as Studied by Fluorescein Isothiocyanate Fluorescence

Protein stabilizing potential of honey was studied on a model protein, bovine serum albumin (BSA), using extrinsic fluorescence of fluorescein isothiocyanate (FITC) as the probe. BSA was labelled with FITC using chemical coupling, and urea and thermal denaturation studies were performed on FITC-labelled BSA (FITC-BSA) both in the absence and presence of 10% and 20% (w/v) honey using FITC fluorescence at 522 nm upon excitation at 495 nm. There was an increase in the FITC fluorescence intensity upon increasing urea concentration or temperature, suggesting protein denaturation. The results from urea and thermal denaturation studies showed increased stability of protein in the presence of honey as reflected from the shift in the transition curve along with the start point and the midpoint of the transition towards higher urea concentration/temperature. Furthermore, the increase in ΔG D H2O and ΔG D 25°C in presence of honey also suggested protein stabilization.

Urea Induced DNA Denaturation

Urea destabilizes (and at high concentrations, denatures) protein and nucleic acid secondary and tertiary structures. DNA and RNA secondary and tertiary structure stability has typically been studied by monitoring the attenuation of the unfolding transition temperature with increasing urea concentration. In this work, we have used uv-absorbance of four DNA dodecamer duplexes (with guanine-cytosine content ranging between 17-42%) at or near room temperature to determine the unfolding equilibrium constant dependence on urea concentration. We have found that the dependence of the unfolding equilibrium constant on urea concentration depends only weakly on duplex guanine-cytosine content, despite different urea concentration ranges where the duplexes unfold. The near constant dependence of the unfolding equilibrium constant on urea concentration for the duplexes indicate the interaction of urea with the newly exposed surface area during unfolding is nearly the same for all duplexes. Additionally, dln Kobs/d[urea] values agree favorably in magnitude with those determined via thermal denaturation, suggesting urea interactions at the newly exposed DNA surface area depend only minimally on temperature.

Denaturation of RNA secondary and tertiary structure by urea: simple unfolded state models and free energy parameters account for measured m-values.

To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analogue, we derived average interaction potentials (per square angstrom of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single-strand segments. These estimates, combined with the three interaction potentials and a term to account for interactions of urea with released ions, yield calculated m-values that are in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A-form conformation. The primary driving force for urea-induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though interactions of urea with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNAs are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with an increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states.

Unfolding of Cytochrome c upon Interaction with Azobenzene-Modified Copolymers

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome c (cyt c) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] of 9-10 M, which was analyzed under the framework of the equilibrium between two states (native-unfolded). In the presence of polymers, the native folding of cyt c was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.