Conformational and hydration effects of site-selective sodium, calcium and strontium ion binding to the DNA Holliday junction structure d(TCGGTACCGA)(4)

The role of metal ions in determining the solution conformation of the Holliday junction is well established, but to date the picture of metal ion binding from structural studies of the four-way DNA junction is very incomplete. Here we present two refined structures of the Holliday junction formed by the sequence d(TCGGTACCGA) in the presence of Na+ and Ca2+, and separately with Sr2+ to resolutions of 1.85 Angstrom and 1.65 Angstrom, respectively. This sequence includes the ACC core found to promote spontaneous junction formation, but its structure has not previously been reported. Almost complete hydration spheres can be defined for each metal cation. The Na+ sites, the most convincing observation of such sites in junctions to date, are one on either face of the junction crossover region, and stabilise the ordered hydration inside the junction arms. The four Ca2+ sites in the same structure are at the CG/CG steps in the minor groove. The Sr2+ ions occupy the TC/AG, GG/CC, and TA/TA sites in the minor groove, giving ten positions forming two spines of ions, spiralling through the minor grooves within each arm of the stacked-X structure. The two structures were solved in the two different C2 lattices previously observed, with the Sr2+ derivative crystallising in the more highly symmetrical form with two-fold symmetry at its centre. Both structures show an opening of the minor groove face of the junction of 8.4degrees in the Ca2+ and Na+ containing structure, and 13.4degrees in the Sr2+ containing structure. The crossover angles at the junction are 39.3degrees and 43.3degrees, respectively. In addition to this, a relative shift in the base pair stack alignment of the arms of 2.3 Angstrom is observed for the Sr2+ containing structure only. Overall these results provide an insight into the so-far elusive stabilising ion structure for the DNA Holliday junction. (C) 2003 Elsevier Science Ltd. All rights reserved.

Hydration effects on the structural properties and haem-haem interaction in haemoglobin

Direct and simultaneous measurements of hydration water content and protein conformation have been performed using quartz crystal microbalance and visible absorption spectroscopy. Equilibrium and kinetics of methaemoglobin/haemichrome transition induced by the alteration of the degree of hydration was investigated in thin films exposed to controlled humidity. The kinetics experiment show that the conversion of species achieve the equilibrium more rapidly that the amount of sorbed water by the protein. The transition shows a sigmoid behaviour and suggest cooperative phenomena manifested by haem-haem interaction. The water hydration network contributing to the haem haem interaction advise that water acts as allosteric effectors for the conversion between species. Irreversible changes produced by complete drying are clearly shown.

In vivo skin effects of a dimethylaminoethanol (DMAE) based formulation

Dimethylaminoethanol (DMAE) has been used in anti-aging formulations but few scientifically based data address its efficacy. The aim of this study was to evaluate the effects of DMAE-based formulations on hairless mice and human skin. Formulations containing with or without DMAE were applied to the dorsum of hairless mice. Histopathological and histometric evaluations were carried out after seven days. Formulations were also applied to the ventral forearm and the lateral periocular area of human volunteers. Stratum corneum water content and skin mechanical properties were analyzed using Corneometer and Cutometer, before and after a single and repeated application. Histometric evaluations showed that formulations with or without DMAE increased the viable epidermis thickness, but only the DMAE-supplemented formulation led to increased dermal thickness. DMAE also induced increase in collagen fiber thickness, which was observed in the histopathological study. After the single and the 8-week period application on human skin, formulations with and without DMAE enhanced the stratum corneum water content in the forearm skin. Mechanical properties were not significantly modified. So, we can suggest that DMAE action is related to its effects on the dermis as observed in the histopathological and histometric studies and showed hydration effects on skin.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.

Study of water and dimethylformamide interaction by computer simulation

Monte Carlo simulations of water-dimethylformamide (DMF) mixtures were performed in the isothermal and isobaric ensemble at 298.15 K and 1 atm. The intermolecular interaction energy was calculated using the classical 6-12 Lennard-Jones pairwise potential plus a Coulomb term. The TIP4P model was used for simulating water molecules, and a six-site model previously optimised by us was used to represent DMF. The potential energy for the water-DMF interaction was obtained via standard geometric combining rules using the original potential parameters for the pure liquids. The radial distribution functions calculated for water-DMF mixtures show well characterised hydrogen bonds between the oxygen site of DMF and hydrogen of water. A structureless correlation curve was observed for the interaction between the hydrogen site of the carbonyl group and the oxygen site of water. Hydration effects on the stabilisation of the DMF molecule in aqueous solution have been investigated using statistical perturbation theory. The results show that energetic changes involved in the hydration process are not strong enough to stabilise another configuration of DMF than the planar one.

Efficacy of Cosmetic Formulations Containing Dispersion of Liposome with Magnesium Ascorbyl Phosphate, Alpha-Lipoic Acid and Kinetin

The present study aimed to evaluate the photoprotective effects of cosmetic formulations containing a dispersion of liposome with magnesium ascorbyl phosphate (MAP), alpha-lipoic acid (ALA) and kinetin, as well as their effects on the hydration and viscoelastic skin properties. The photoprotection was determined in vitro (antioxidant activity) and in vivo on UV-irradiated hairless mouse skin. The hydration effects were performed with the application of the formulations under study on the forearm of human volunteers and skin conditions were analyzed before and after a single application and daily applications during 4 weeks in terms of transepidermal water loss (TEWL), skin moisture and viscoelastic properties. The raw material under study possessed free-radical scavenging activity and the formulation with it protected hairless mouse skin barrier function against UV damage. After 4 weeks of application on human skin, the formulation under study enhanced stratum corneum skin moisture and also showed hydration effects in deeper layers of the skin. Thus, it can be concluded that the cosmetic formulation containing a dispersion of liposome with MAP, ALA and kinetin under study showed photoprotective effects in skin barrier function as well as pronounced hydration effects on human skin, which suggests that this dispersion has potential antiaging effects.

Comparative effects of pimecrolimus cream vehicle and three commercially available moisturizers on skin hydration and transepidermal water loss

Background: Hydration and integrity of the horny layer is essential to normal skin function. Objective: Comparison of the hydrating properties of three moisturizers with pimecrolimus cream vehicle. Methods: Four test preparations (high-quality skin cream, cold cream emulsion, emollient oil, pimecrolimus cream vehicle) were applied to four different regions of the forearms and legs. Transepidermal water loss (TEWL) was assessed by evaporimetry at baseline, and 3 and 6 hours after arm application, and electrical capacitance was assessed by corneometry at baseline, and 1, 2, 3 and 6 hours after leg application. Results: Corneometry assessment - in terms of efficacy in moisturizing the skin, test preparations were ranked (best to worst): high-quality skin cream (45.9 arbitrary units versus 75.3; p < 0.001) > pimecrolimus vehicle cream (46.6 versus 61.5; p < 0.001) > emollient oil (43.5 versus 54.8; p = 0.006) > cold cream emulsion (44.8 versus 49.9; p = 0.738). Untreated skin (control) had a mean capacitance of 44.8 units at baseline and 48.5 units at endpoint. Evaporimetry (assessment of TEWL) revealed no significant differences between control and any test preparation at any timepoint. Conclusions: Pimecrolimus cream vehicle has skin hydration properties comparable with highly effective commercially available products. No test preparation had a significant effect on TEWL.

Effects of hydration status on cognitive performance and mood

Although it is well known that water is essential for human homeostasis and survival, only recently have we begun to understand its role in the maintenance of brain function. Herein, we integrate emerging evidence regarding the effects of both dehydration and additional acute water consumption on cognition and mood. Current findings in the field suggest that particular cognitive abilities and mood states are positively influenced by water consumption. The impact of dehydration on cognition and mood is particularly relevant for those with poor fluid regulation, such as the elderly and children. We critically review the most recent advances in both behavioural and neuroimaging studies of dehydration and link the findings to the known effects of water on hormonal, neurochemical and vascular functions in an attempt to suggest plausible mechanisms of action. We identify some methodological weaknesses, including inconsistent measurements in cognitive assessment and the lack of objective hydration state measurements as well as gaps in knowledge concerning mediating factors that may influence water intervention effects. Finally, we discuss how future research can best elucidate the role of water in the optimal maintenance of brain health and function.

The effects of anticalcification treatments and hydration on the molecular dynamics of bovine pericardium collagen as revealed by (13)C solid-state NMR

This article describes a solid-state NMR (SSNMR) investigation of the influence of hydration and chemical cross-linking on the molecular dynamics of the constituents of the bovine pericardium (BP) tissues and its relation to the mechanical properties of the tissue. Samples of natural phenetylamine-diepoxide (DE)- and glutaraldehyde (GL)-fixed BP were investigated by (13)C cross-polarization SSNMR to probe the dynamics of the collagen, and the results were correlated to the mechanical properties of the tissues, probed by dynamical mechanical analysis. For samples of natural BP, the NMR results show that the higher the hydration level the more pronounced the molecular dynamics of the collagen backbone and sidechains, decreasing the tissue`s elastic modulus. In contrast, in DE- and GL-treated samples, the collagen molecules are more rigid, and the hydration seems to be less effective in increasing the collagen molecular dynamics and reducing the mechanical strength of the samples. This is mostly attributed to the presence of cross-links between the collagen plates, which renders the collagen mobility less dependent on the water absorption in chemically treated samples. Copyright (C) 2010 John Wiley & Sons, Ltd.

Effects of Surface Hydration and Application Method on the Bond Strength of Self-Etching Adhesives to Dentin

The effect of application methods and dentin hydration on the bond strength of three self-etching adhesives (SEA) were evaluated; 195 extracted bovine incisors were used. The buccal surface was ground in order to expose the dentin, which remained 2-mm minimum thickness, measured by a thickness meter through an opening on the lingual surface. Adper Single Bond 2 (TM) was used for the control group. The SEA were applied following two modes of application: passive or active and two hydration states of the dentin surface-dry and wet. After light-curing, composite buildups were made using Grandio (TM) composite. The specimens were sectioned and tested with a microtensile bond strength test. The application method and the hydration state resulted in statistical differences (p = 0.000) making the values of active application for mu TBS to dentin higher than passive application. The wet surfaces showed higher mu TBS to dentin ratios than dry surfaces. There were no statistical differences in mu TBS among the SEA tested but there were differences regarding to control group.

Effects of Surface Hydration State and Application Method on the Bond Strength of Self-etching Adhesives to Cut Enamel

Purpose: To evaluate the effect of surface hydration state and application method on the microtensile bond strength of one-step self-etching adhesives systems to cut enamel.Materials and Methods: One hundred ninety-five bovine teeth were used. The enamel on the buccal side was flattened with 600-grit SiC paper. For the control group, 15 teeth received Adper Single Bond 2, applied according to manufacturer's recommendations. The other specimens were divided into three groups according to the adhesive system used: Futura Bond M (FM; Voco), Clearfil S-3 Bond (CS; Kuraray), and Optibond All in One (OA; Kerr). For each group, two hydration states were tested: D: blown dry with air; W: the excess of water was removed with absorbent paper. Two application methods were tested: P (passive): the adhesive was simply left on the surface; A (active): the adhesive was rubbed with an applicator point. A coat of Grandio composite resin (Voco) was applied on the surface. The teeth were sectioned to obtain enamel-resin sticks (1 x 1 mm), which underwent microtensile bond testing. The data in MPa were submitted to a three-way ANOVA and Tukey's test (alpha = 5%).Results: The ANOVA showed significant differences for application method and the type of adhesive, but not for hydration state. For the application method, the results of Tukey's test were: P: 31.46 (+/-7.09)a; A: 34.04 (+/-7.19)b. For the type of adhesive, the results were: OA: 31.29 (+/-7.05)a; CS: 32.28 (+/-7.14)a; FM: 34.68 (+/-7.17)b; different lower-case letters indicate statistically significant differences.Conclusion: Active application improved the bond strength to cut enamel. The adhesive Futurabond M showed the highest bond strength to cut enamel.

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O-2 binding

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O-2 affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the osmotic stress method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl- and of DPG concentration. We find that only similar to 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with similar to 72 that bind in the presence of more than 50 mM Cl- or more than 15 mu M DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl-, DPG, and H2O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.