Kinetics of phase transformations in a model with metastable fluid-fluid separation: A molecular dynamics study

By molecular dynamics (MD) simulations we study the crystallization process in a model system whose particles interact by a spherical pair potential with a narrow and deep attractive well adjacent to a hard repulsive core. The phase diagram of the model displays a solid-fluid equilibrium, with a metastable fluid-fluid separation. Our computations are restricted to fairly small systems (from 2592 to 10368 particles) and cover long simulation times, with constant energy trajectories extending up to 76x10(6) MD steps. By progressively reducing the system temperature below the solid-fluid line, we first observe the metastable fluid-fluid separation, occurring readily and almost reversibly upon crossing the corresponding line in the phase diagram. The nucleation of the crystal phase takes place when the system is in the two-fluid metastable region. Analysis of the temperature dependence of the nucleation time allows us to estimate directly the nucleation free energy barrier. The results are compared with the predictions of classical nucleation theory. The critical nucleus is identified, and its structure is found to be predominantly fcc. Following nucleation, the solid phase grows steadily across the system, incorporating a large number of localized and extended defects. We discuss the relaxation processes taking place both during and after the crystallization stage. The relevance of our simulation for the kinetics of protein crystallization under normal experimental conditions is discussed. (C) 2002 American Institute of Physics.

An enantio-selective chromatographic stationary phase for S-ibuprofen prepared by stoichiometric molecular imprinting

Molecularly Imprinted Polymers (MIPs) against S-ibuprofen were synthesised using a tailor made functional monomer, 2-acrylamido-4-methylpyridine, following extensive pre-polymerisation studies of template-monomer complexation. An apparent association constant of 340 +/- 22 M-1 was calculated that was subsequently corrected to account for dimerisation of ibuprofen (K-dim = 320 +/- 95 M-1) resulting in an intrinsic association constant of 715 +/- 16 M-1, consistent with previously reported values. Using the synthesised imprinted polymer as a stationary phase, complete resolution of a racemic mixture of ibuprofen was achieved in predominantly aqueous mobile phases. An imprinting factor of 10 was observed, and was found to be in agreement with the difference in the average number of binding sites between MIP and blank polymers, calculated by staircase frontal chromatography. The imprinted polymers exhibited enhanced selectivity for the templated drug over structurally related NSAIDs. When applied as sorbents in solid-phase extraction of ibuprofen from commercial tablets, urine and blood serum samples, recoveries up to 92.2% were achieved. © The Royal Society of Chemistry 2012

The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Towards An Acoustic Model-Based Poroelastic Imaging Method: I. Theoretical Foundation

The ultrasonic measurement and imaging of tissue elasticity is currently under wide investigation and development as a clinical tool for the assessment of a broad range of diseases, but little account in this field has yet been taken of the fact that soft tissue is porous and contains mobile fluid. The ability to squeeze fluid out of tissue may have implications for conventional elasticity imaging, and may present opportunities for new investigative tools. When a homogeneous, isotropic, fluid-saturated poroelastic material with a linearly elastic solid phase and incompressible solid and fluid constituents is subjected to stress, the behaviour of the induced internal strain field is influenced by three material constants: the Young's modulus (E(s)) and Poisson's ratio (nu(s)) of the solid matrix and the permeability (k) of the solid matrix to the pore fluid. New analytical expressions were derived and used to model the time-dependent behaviour of the strain field inside simulated homogeneous cylindrical samples of such a poroelastic material undergoing sustained unconfined compression. A model-based reconstruction technique was developed to produce images of parameters related to the poroelastic material constants (E(s), nu(s), k) from a comparison of the measured and predicted time-dependent spatially varying radial strain. Tests of the method using simulated noisy strain data showed that it is capable of producing three unique parametric images: an image of the Poisson's ratio of the solid matrix, an image of the axial strain (which was not time-dependent subsequent to the application of the compression) and an image representing the product of the aggregate modulus E(s)(1-nu(s))/(1+nu(s))(1-2nu(s)) of the solid matrix and the permeability of the solid matrix to the pore fluid. The analytical expressions were further used to numerically validate a finite element model and to clarify previous work on poroelastography.

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples

Abstract An HPLC method has been developed and validated for the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 µl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30–1000 ng/ml for spironolactone and 25–1000 ng/ml for 7a-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7a-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Synthesis, Kinetic Evaluation and Utilization of a Biotinylated Dipeptide Proline Diphenyl Phosphonate for the Disclosure of Dipeptidyl Peptidase IV-like Serine Proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Thermodynamic Studies of Ionic Interactions in Aqueous Solutions of Imidazolium-Based Ionic Liquids [Emim][Br] and [Bmim][Cl]

Experimental measurements of density at different temperatures ranging from 293.15 to 313.15 K, the speed of sound and osmotic coefficients at 298.15 K for aqueous solution of 1-ethyl-3-methylimidazolium bromide ([Emim][Br]), and osmotic coefficients at 298.15 K for aqueous solutions of 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) in the dilute concentration region are taken. The data are used to obtain compressibilities, expansivity, apparent and limiting molar properties, internal pressure, activity, and activity coefficients for [Emim][Br] in aqueous solutions. Experimental activity coefficient data are compared with that obtained from Debye-Hückel and Pitzer models. The activity data are further used to obtain the hydration number and the osmotic second virial coefficients of ionic liquids. Partial molar entropies of [Bmim][Cl] are also obtained using the free-energy and enthalpy data. The distance of the closest approach of ions is estimated using the activity data for ILs in aqueous solutions and is compared with that of X-ray data analysis in the solid phase. The measured data show that the concentration dependence for aqueous solutions of [Emim][Br] can be accounted for in terms of the hydrophobic hydration of ions and that this IL exhibits Coulombic interactions as well as hydrophobic hydration for both the cations and anions. The small hydration numbers for the studied ILs indicate that the low charge density of cations and their hydrophobic nature is responsible for the formation of the water-structure-enforced ion pairs.

Detection of synthetic glucocorticoid residues in cattle tissue and hair samples after a single dose administration using LC-MS/MS

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the detection of several synthetic glucocorticoids in kidney, muscle and hair samples of cattle after a single intramuscular injection is described. After a dichloromethane wash of the hair samples, analytes were released from the hair matrix by enzymatic digestion. Muscle samples were also digested enzymatically using proteinase, while kidney samples were deconjugated by Helix pomatia juice. These preliminary steps were followed by a methanol extraction and a solid phase extraction (SPE) clean up step for all matrices. Chromatographic separation was achieved on a Hypersil Hypercarb column and MS/MS data were obtained in the multiple reaction monitoring mode using negative electrospray ionization. The developed protocols were evaluated by assessing residue concentrations in muscle, kidney and hair samples of thirteen calves, treated with a particular intramuscular injection of glucocorticoid. The lowest residue levels were found in muscle samples (approximately 5% of the residue levels in kidney), while high residue levels were obtained in hair samples. Hair is an interesting matrix since the sampling is non-invasive and the drugs may stay incorporated for a longer period of time. (C) 2004 Elsevier B.V. All rights reserved.

Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4 +/-5.4%; P

Liquid structure of the ionic liquid 1,3-dimethylimidazolium bis{(trifluoromethyl)sulfonyl}amide

Neutron diffraction has been used to determine the liquid structure of 1,3-dimethylimidazolium bis{( trifluoromethyl) sulfonyl} amide ([dmim][NTf2]). Significantly smaller charge ordering is found in this liquid compared with analogous chloride and hexafluorophosphate salts due to the diffuse charge density and size of the [NTf2](-) anion. This is manifested in a much larger cation-cation and cation-anion separation and an overlap of the cation-cation and cation-anion shells. Comparison of the liquid structure with the crystal structure reported by Holbrey et al. ( Dalton Trans. 2004, 2267) indicates little correlation, for example, the [NTf2](-) anion adopts a trans orientation predominantly in the liquid whereas a cis orientation is found in the solid phase.

Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors

We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide containing dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC 3.4.24.26). The aim of the study was to derive an inhibitor profile for LasB with regard to mapping the S´1 binding site of the enzyme. Consequently, a focussed library of 160 members has been synthesised, using standard Fmoc-solid phase methods (on a Rink-amide resin), in which a subset of amino acids including examples of those with basic (Lys, Arg), aromatic (Phe, Trp), large aliphatic (Val, Leu) and acidic (Asp, Glu) side-chains populated the P´2 position of the inhibitor sequence and all 20 natural amino acids were incorporated, in turn, at the P´1 position. The study has revealed a preference for aromatic and/or large aliphatic amino acids at P´1 and a distinct bias against acidic residues at P´2. Ten inhibitor sequences were discovered that exhibited sub to low micromolar Ki values.

COD removal from textile industry effluent: pilot plant studies

This work involved the treatment of industrial waste water from a nylon carpet printing plant. As dyeing of nylon is particularly difficult, acid dyes, fixing agents, thickeners, finishing agents, are required for successful colouration and cause major problems with the plant's effluent disposal in terms of chemical oxygen demand (COD). Granular activated carbon (GAC) Filtrasorb 400 was used to treat a simulated process plant effluent containing all the pollutants. Equilibrium isotherm experiments were established and experimental data obtained showed good empirical correlation with Langmuir isotherm theory. Column experimental data, in terms of COD were correlated using the bed depth service time (BDST) model. Solid phase loading in the columns were found to approach that in equilibrium studies indicating an efficient use of adsorbent. The results from the BDST model were then used to design a pilot adsorption rig at the plant. The performance of the pilot plant column were accurately predicted by scale-up from the bench scale columns. (C) 2001 Elsevier Science BN. All rights reserved.

Drum granulation of NPK fertilizers

Nitrate-based NPK fertilizer was granulated in a bench scale drum granulation unit. The initial fertilizer possessed a particle size distribution similar to those in industrial granulation units. In this work, three factors are identified affecting the degree of fertilizer granulation, these are solution to solid phase ratio, the binder viscosity and the optimal rotation speed of the drum. Experimental results indicate that a critical solution to solid phase ratio is required for an increase in granulation in terms of mass median diameter. The saturated solution viscosity in this system was measured and correlated well to binder viscosity granulation theory with the critical Stokes number calculated at 700. The optimum rotation speed for flighted and unflighted drums correlated with the Froude number relationship for full scale granulation units. (C) 2000 Elsevier Science S.A. All rights reserved.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 mu l of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17 alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r >0.994). Accuracy (% RE) and precision (% CV) values for within and between day were