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PubMed2003

Modeling of the separation of two enantiomers using a microbore column.

A microbore column packed with Chiralcel OB (cellulose tribenzoate coated silica) was used for the measurement of the single and competitive equilibrium-isotherm data of the 1-indanol enantiomers by frontal analysis. The amount of sample needed for the isotherm data acquisition was about 20 times less than that required with a conventional column. The data obtained were fitted to different single and competitive isotherm models. Both the single and the competitive data sets fitted best to the same Bilangmuir (BL) isotherm model with small differences in the numerical values of the parameters. The best fitted Bilangmuir single and competitive isotherm models were used to predict the overloaded experimental profiles of both pure enantiomers, of the racemic mixture, and of different enantiomeric mixtures. All the calculated profiles were in excellent agreement with the experimental ones. This agreement confirms that in many chiral separations, the competitive isotherms can be derived from data acquired from the mere racemic mixture with a sufficient accuracy for a correct prediction of the band profiles of all kinds of enantiomer mixtures, making possible the computer-assisted optimization of the experimental conditions.

Chromatography↗
PubMed2006

Thermophysical properties of aqueous and frozen states of BSA/water/Tris systems.

In the modelling and the optimization of pharmaceutical protein freeze-drying processes, thermophysical properties values of the formulation in frozen or in liquid states are necessary in order to determine the optimal operating conditions (temperature, pressure) of the two steps (sublimation, desorption) drying diagramme and the optimal storage conditions of the final freeze-dried product. The most important thermophysical properties of BSA/water/Tris system buffered with Tris-HCl (5%, w/w) at pH 7, a standard formulation largely used in industrial freeze-drying process of pharmaceutical proteins, are reported in this paper. The state diagram of this formulation was determined by modulated temperature differential scanning calorimetry (MTDSC) and, then the vitreous transition temperatures were interpreted as a function of water content by the Gordon-Taylor equation. The same technique was used to experimentally determine the heat capacity of the BSA/water frozen system. Moreover, the transient hot wire probe method was used to measure the thermal conductivity of the frozen system as a function of temperature. It proved that the thermal conductivity and the apparent heat capacity values for this dilute formulation were reasonably close to the values for the pure water/ice system. Sorption isotherms data were also measured by two different methods-the equilibrium with saturated salts solutions and also the controlled humidity oven. Water vapour sorption data were finally correlated by the three parameters Guggenheim, Anderson, De Boer (GAB) equation.

Adsorption↗
PubMed2006

High-pressure studies on protein aggregates and amyloid fibrils.

High hydrostatic pressure (HHP) modulates protein-protein and protein-solvent interactions through volume changes and thereby affects the equilibrium of protein conformational species between native and denatured forms as well as monomeric, oligomeric, and aggregated forms without the addition of chemicals or use of high temperature. Because of this unique property, HHP has provided deep insights into the thermodynamics and kinetics of protein folding and aggregation, including amyloid fibril formation. In particular, HHP is a useful tool to stabilize and populate specific folding intermediates, the characterization of which provides thorough understanding of protein folding and aggregation pathways. Furthermore, recent application of HHP for dissociation of protein aggregates, such as inclusion bodies (IBs), into native proteins in a single step facilitates protein preparation for structural and functional studies. This chapter overviews recent HHP studies on the population and characterization of folding intermediates associated with protein aggregation and protein refolding from protein aggregates of amyloid fibrils and IBs. Finally, we describe overall experimental procedures of HHP-mediated protein refolding and provide a detailed discussion of each operating parameter to optimize the refolding.

Amyloid↗
PubMed1995

HLA -A, -B, -DR haplotype frequencies in France--implications for recruitment of potential bone marrow donors.

We have undertaken a study of the haplotypes among French potential bone marrow donors in order to define the geographical regions of France with the maximum of polymorphism and also to develop a strategy for optimal donor recruitment. A maximum likelihood estimator was used to calculate haplotype frequencies and their support limits for each region and for the whole of France. The observed differences between the regions were statistically significant. For each region, the minimum number of haplotypes necessary to explain 50% of the total frequency was calculated and compared with the equivalent values, and confidence intervals, obtained by repeated random samplings from the overall file. This approach shows that some regions (e.g., Provence) appear to be richer in terms of the numbers of haplotypes observed, and others (e.g., Bretagne) poorer. In the latter case, however, the frequencies of the most common haplotypes are greater. The haplotype frequencies of the whole sample were used to calculate the probability of finding a match for the next potential recipient for given sizes of the donor file, assuming random selection of donors. They were also used to calculate expected numbers of the major phenotypes, assuming Hardy-Weinberg equilibrium, and these were compared with those observed in the real data file. In this way, a large number of under-represented and nonrepresented phenotypes were identified. For each of these phenotypes, the most probable haplotypes and the regions in which these have the greatest frequencies have been identified. A search for donors with such particular phenotypes would be much more fruitful if directed towards these regions.

Alleles↗
PubMed2002

Search for the optimal linker in tandem hairpin polyamides.

In order to target specific DNA sequences >or=10 base pairs in size by minor groove binding ligands, a search for the optimal linker in dimers of hairpin polyamides was initiated. Two series of tandem polyamides ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-L](HN)gamma-PyPyPy-beta-Dp (1a-e), where L represents a series of 4-8 carbon long aliphatic amino acid linkers, and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-L](HN)gamma-PyPyPy-beta-Dp (2a-e), where L represents a series of 2-6 carbon long aliphatic amino acid linkers, were synthesized and characterized by quantitative DNase I footprinting. beta, gamma and Dp represents beta-alanine, gamma-aminobutyric acid, and 3-(dimethylamino)propylamine, respectively. It was found that the five-carbon 5-aminovaleric acid (delta), is suitable to span one base-pair (bp) of DNA when incorporated into a tandem polyamide. ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-delta](HN)gamma-PyPyPy-beta-Dp (1b) binds the 10 bp binding-site 5'-AGTGAAGTGA-3' with equilibrium association constant K(a)=3.2 x 10(10) M(-1) and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-delta](HN)gamma-PyPyPy-beta-Dp (2d) binds the 11 bp binding-site 5'-AGTGATAGTGA-3' with K(a)=9.7 x 10(9) M(-1). Tandem 1b also bind the 11 bp site but with lower affinity affording a 15-fold specificity for the shorter binding site. Replacing a methylene group in the amino acid linker with an oxygen atom to form tandem polyamide ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-E](HN)gamma-PyPyPy-beta-Dp (4) where E represents the ether linker, resulted in that an 80-fold specificity for the 10 bp binding site over the 11 bp site.

Amino Acids, Neutral↗
PubMed1992

Logistical and ergonomic transportation capacity for refuse collection workers: a work physiology field study.

In a work physiology field study, the work flow and the electrocardiogram were recorded throughout whole shifts for six male refuse (garbage) collection workers who transported and emptied 1.1 m3 refuse containers. The work rate (WR), indicated by the number of 1.1 m3 containers emptied per unit of time, and the work pulse rate (WPR) were determined in the data evaluation. The work pulse rate increases with the work rate. The functional relationship can be approximated by a linear regression function (WPR = 20.9 + 35.8.WR). The work pulse rate reaches such high values that it must be assumed that the work cannot be performed continuously throughout the whole working day. Consequently, regular breaks should be provided. When determining the necessary duration of the breaks, it was assumed that an equilibrium between fatigue and recovery should be maintained during the working day. The recovery breaks should be at least long enough for the heart rate to return to the resting level. The necessary duration of the breaks was determined on the basis of the present field study and the laboratory investigations described in the literature. A minimum duration of 10 min per working hour results from the calculations. The breaks should be taken regularly at about hourly intervals. A transportation-capacity model is provided for the planning of the deployment of refuse workers. It permits calculation of the number of 1.1 m3 containers a three-man crew, comprising the refuse collection truck driver and two loaders, can empty during one shift. Both the logistical and ergonomic transportation capacities can be determined using the model. The logistical transportation capacity (TCL) indicates the number of containers which can be emptied per shift by a crew when only criteria such as the optimal utilization of the working time are considered. The ergonomic transportation capacity (TCE) is understood as the number of containers which can be emptied per shift taking into consideration the workers' strain and the recovery breaks they require. TCE amounts to between about 140 and 160 containers per shift depending on the type of city district. TCL is higher by between 20 and 30 containers per shift. In order to meet the demand of protecting workers' health, the number of containers to be emptied per shift by a three-man crew should not exceed the ergonomic transportation capacity.

Adult↗
PubMed2006

Information theory in living systems, methods, applications, and challenges.

Living systems are distinguished in nature by their ability to maintain stable, ordered states far from equilibrium. This is despite constant buffeting by thermodynamic forces that, if unopposed, will inevitably increase disorder. Cells maintain a steep transmembrane entropy gradient by continuous application of information that permits cellular components to carry out highly specific tasks that import energy and export entropy. Thus, the study of information storage, flow and utilization is critical for understanding first principles that govern the dynamics of life. Initial biological applications of information theory (IT) used Shannon's methods to measure the information content in strings of monomers such as genes, RNA, and proteins. Recent work has used bioinformatic and dynamical systems to provide remarkable insights into the topology and dynamics of intracellular information networks. Novel applications of Fisher-, Shannon-, and Kullback-Leibler informations are promoting increased understanding of the mechanisms by which genetic information is converted to work and order. Insights into evolution may be gained by analysis of the the fitness contributions from specific segments of genetic information as well as the optimization process in which the fitness are constrained by the substrate cost for its storage and utilization. Recent IT applications have recognized the possible role of nontraditional information storage structures including lipids and ion gradients as well as information transmission by molecular flux across cell membranes. Many fascinating challenges remain, including defining the intercellular information dynamics of multicellular organisms and the role of disordered information storage and flow in disease.

Computational Biology↗
PubMed1985

Investigation of operating parameters in high-performance displacement chromatography.

The effect of operational parameters of displacement chromatography was examined in the separation of various mixtures such as that of the main hydrolysis products of methylfurylbutyrolactone, a potential anticancer drug, the diastereoisomers benzoyl-D- and benzoyl-L-phenylalanyl-L-alanyl-L-proline, as well as polyethylene glycol homologues containing 1-10 ethylene oxide units. The chromatograph was assembled from modules generally used in analytical high-performance liquid chromatography (HPLC) and the column effluent was analyzed by an on-line HPLC unit at 30-sec intervals. Octadecyl-silica was used throughout as the stationary phase. Derivatives of ethylene glycol and propylene glycol as well as tetrabutylammonium bromide and n-butanol were used as displacers. The throughput was used as the measure of efficiency. In the absence of axial dispersion, for a given separation various displacers are expected to yield the same efficiency if the slope of the operating line is kept the same by appropriate adjustment of displacer concentrations. In practice, however, the optimum slope of the operating line has to be determined experimentally as most available chromatographic systems depart from ideal behavior. The dependence of the throughput on the flow-rate and feed load also indicated the presence of non-equilibrium phenomena and the optimum value of these parameters was established experimentally. In most cases water was used as the carrier solvent but the separation of poorly soluble peptides required the use of hydro-organic carriers. Results obtained with octadecyl-silicas of different origin and a given displacer were found to vary significantly suggesting that even for stationary phases of the same type the selection of displacer requires special consideration. Most experiments were carried out with columns having dimensions customary in analytical HPLC. Increasing the inner diameter of the column did not result in the expected increase in throughout probably due to poor distribution of the sample at the column entrance. Therefore scaling-up the process requires careful engineering of inlet conditions. Throughput can be increased by connecting a small inner diameter column to the outlet of a large diameter preparative column. As theoretical predictions for ideal displacement chromatography do not hold in practice when axial dispersion is significant, optimization of the process requires experimental support. The results obtained in the separation of a variety of mixtures shed light on the most important operational aspects of displacement chromatography and suggest approaches to find optimum conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Ascorbic Acid↗
PubMed1996

Modeling DNA hydration: comparison of calculated and experimental hydration properties of nuclic acid bases.

Hydration properties of individual nucleic acid bases were calculated and compared with the available experimental data. Three sets of classical potential functions (PF) used in simulations of nucleic acid hydration were juxtaposed: (i) the PF developed by Poltev and Malenkov (PM), (ii) the PF of Weiner and Kollman (WK), which together with Jorgensen's TIP3P water model are widely used in the AMBER program, and (iii) OPLS (optimized potentials for liquid simulations) developed by Jorgensen (J). The global minima of interaction energy of single water molecules with all the natural nucleic acid bases correspond to the formation of two water-base hydrogen bonds (water bridging of two hydrophilic atoms of the base). The energy values of these minima calculated via PM potentials are in somewhat better conformity with mass-spectrometric data than the values calculated via WK PF. OPLS gave much weaker water-base interactions for all compounds considered, thus these PF were not used in further computations. Monte Carlo simulations of the hydration of 9-methyladenine, 1-methyluracil and 1-methylthymine were performed in systems with 400 water molecules and periodic boundary conditions. Results of simulations with PM potentials give better agreement with experimental data on hydration energies than WK PF. Computations with PM PF of the hydration energy of keto and enol tautomers of 9-methylguanine can account for the shift in the tautomeric equilibrium of guanine in aqueous media to a dominance of the keto form in spite of nearly equal intrinsic stability of keto and enol tautomers. The results of guanine hydration computations are discussed in relation to mechanisms of base mispairing errors in nucleic acid biosynthesis. The data presented in this paper along with previous results on simulation of hydration shell structures in DNA duplex grooves provide ample evidence for the advantages of PM PF in studies of nucleic-acid hydration.

Base Composition↗
PubMed2006

Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD], 20 microM) and low-affinity (KD, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

Arginine↗
PubMed1998

Engineering the independent folding of the subtilisin BPN' pro-domain: correlation of pro-domain stability with the rate of subtilisin folding.

The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions. The instability of the isolated pro-domain suggests that there may be a thermodynamic linkage between the stability of the pro-domain and its ability to facilitate subtilisin folding. On the basis of the X-ray crystal structure of the pro-domain subtilisin complex, we have designed stabilizing mutations in three areas of the pro-domain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-helix 53-61 (K57E). These amino acid positions were selected because they do not contact subtilisin in the complex and because they appear to be in regions of the structure which are not well packed in the wild type pro-domain. Since none of the mutations directly contact subtilisin, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes subtilisin folding. By sequentially introducing the three stabilizing mutations, the equilibrium for folding the pro-domain was shifted from 97% unfolded to 65% folded. By measuring the ability of these mutants to fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin folding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined for the double mutant, Q40L-K57E, which is 50% folded: P + Su if (30 800 M-1 s-1, 0.04 s-1) PSI if (0.07 s-1, <0.005 s-1) PS. PSI is an intermediate complex which accumulates in the course of the reaction, and PS is the fully folded complex. The more stable the pro-domain, the faster the folding reaction up to the point at which the isomerization of the intermediate into the fully folded complex becomes the rate-limiting step in the folding process.

Cloning, Molecular↗
PubMed2001

Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence.

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.6 +/- 1.7 nM) and affinity for Rom (average K(D) = 60 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins. Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway. In contrast, dissociation of the complex was found to fit a single exponential decay (average k(off) = 8.9 x 10(-5) s(-1)). Consistent with these observations, a two-step mechanism for RNA loop-loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k(1) = 0.13 microM(-1)s(-1)) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k(2) = 0.09 s(-1)), where the helical stacking around the junctions is optimized.

2-Aminopurine↗
PubMed2001

A novel pH-sensitive membrane from chitosan--TEOS IPN; preparation and its drug permeation characteristics.

A novel organic-inorganic composite membrane was prepared, using tetra ethyl ortho silicate (TEOS) as an inorganic material and chitosan as an organic compound. Equilibrium and oscillatory swelling studies were conducted to investigate swelling behaviors of the membrane according to the pH of the swelling medium. Drug permeation experiments were also performed in phosphate buffer solution of the pH of 2.5 and 7.5, respectively. Lidocaine HCl, sodium salicylate and 4-acetamidophenol were selected as model drugs to examine the effect of ionic property of drug on the permeation behavior. The effects of membrane composition and the external pH on the swelling and the drug permeation behavior of IPN membrane could be summarized as follows; chitosan incorporated into TEOS IPN swelled at pH 2.5 while shrunk at pH 7.5. This swelling behavior was completely reversible and the membrane responded rapidly to the change in environmental pH condition. According to swelling behavior, an increase in pH from 2.5 to 7.5 yielded an increase in the rate of drug permeation because of the shrinking of the incorporated chitosan in TEOS IPN, while decrease in pH resulted in low permeation rate. The optimal TEOS-chitosan ratio for maximum pH-sensitivity existed and drug permeation was influenced not only with the external pH but also with the ionic interactions between the drug and membrane.

Chitin↗
PubMedDate not supplied

An ab initio computational molecular orbital study of the conformers of muconaldehyde, and the possible role of 2-formyl-2H-pyran in bringing about the conversion of a (Z,Z)-muconaldehyde structure into an (E,Z)-muconaldehyde structure.

To determine whether conformers of 2-formyl-2H-pyran (2F2HP) can serve as intermediates in the isomerization (Z,Z)-muconaldehyde-->(E,Z)-muconaldehyde--an essential step in the metabolic oxidation of benzene in which (E,E)-muconic acid is formed--we have carried out ab initio molecular orbital calculations at the MP2/6-31G* (frozen core, valence orbitals active) level with full geometry optimization at the RHF/6-31G* level. Taking account of the configuration about the carbon-carbon single bonds as well as that about the double bonds, the six conformers of (Z,Z)-muconaldehyde have been characterized, as well as the eight conformers of (E,Z)-muconaldehyde. Axial and equatorial conformers of 2F2HP have been identified, which exist in stable syn or anti forms depending on the relative position of the pyran and carbonyl O-atoms. Each step in the metabolic pathway is stereospecific. The initial ring opening in 2,3-epoxyoxepin gives (eZzZz)-muconaldehyde, which then undergoes ring closure to give anti-axial-2F2HP (step a). Inversion of the ring gives anti-equatorial-2F2HP, which upon ring opening gives (zEzZz)-muconaldehyde (step b). An alternative pathway links (zZzZz)-muconaldehyde with syn-axial-2F2HP (step a'), and syn-equatorial with (eEzZz)-muconaldehyde (step b'). Half-reaction times, taking into account the fractional representation of each reacting conformer in rapidly established equilibrium mixtures, suggest that the operative pathway is via step a, with little to choose between steps b and b'.

Aldehydes↗
PubMed1993

Effects of carbonic anhydrase inhibition on ventilation-perfusion matching in the dog lung.

Lung carbonic anhydrase (CA) permits rapid pH responses when changes in regional ventilation or perfusion alter airway and alveolar PCO2. These pH changes affect airway and vascular resistances and lung compliance to optimize the balance of regional ventilation (VA) and perfusion (Q) in the lung. To test the hypothesis that these or other CA-dependent mechanisms contribute to VA/Q matching, we administered acetazolamide (25 mg/kg intravenously) to six anesthetized and paralyzed dogs and measured VA/Q relationships before and after CA inhibition by the multiple inert gas elimination technique. Four other groups of dogs were studied to control for possible confounding effects of time under anesthesia and nonselective CA inhibition by acetazolamide: (a) saline placebo as a control for duration of anesthesia, (b) 4% CO2 inhalation to mimic systemic CO2 retention, (c) 1 mg/kg benzolamide (a selective renal CA inhibitor) or 0.5 meq/kg HCl to mimic systemic metabolic acidosis, and (d) 500 mg/kg 4,4'-dinitrostilbene-2,2'-disulfonate (an inhibitor of red cell band 3 protein) to mimic the respiratory acidosis arising from an intracapillary block to rapid mobilization of plasma HCO3- in CO2 exchange. Acetazolamide increased VA/Q mismatch and reduced arterial PO2 measured at equilibrium but these did not occur in the control group. There was no deterioration in VA/Q matching when systemic respiratory acidosis produced either by CO2 inhalation or 4,4'-dinitrostilbene-2,2'-disulfonate or metabolic acidosis (benzolamide or HCl) were imposed to mimic the effects of acetazolamide apart from its inhibition of lung CA. These results support the concept that lung CA subserves VA/Q matching in the normal lung.

Acetazolamide↗
PubMed2007

Screening method to identify preclinical liquid and semi-solid formulations for low solubility compounds: miniaturization and automation of solvent casting and dissolution testing.

We have developed an efficient screening method to identify liquid and semisolid formulations for low-solubility compounds. The method is most suitable for identifying dosing vehicles for compounds in lead optimization, where compound supply is limited and long-term stability is not a requirement. Dilute compound and excipient stock solutions are prepared in organic solvent and then dispensed and mixed in 96-well plates. The solvent is removed in a vacuum centrifuge evaporator, leaving neat formulation (e.g., 10-40 microg compound, 0.4 mg excipient) at the bottom of each well. After an aging step, an aqueous dilution medium is added and the plates are incubated (agitation by orbital shaking). The diluted formulations are then filtered and analyzed by ultraviolet (UV) absorbance or high-performance liquid chromatography (HPLC). To illustrate the method, two compounds (aqueous solubility </=1 microg/mL) were combined with eight solubilizing excipients, at four drug-loading levels (25, 50, 75, and 100 mg/g) and three incubation times (0.5, 2, and 24 h). This allowed identification of the excipients and loading levels, which generated the highest and most stable kinetic solubility upon dilution. The peak kinetic solubility was strongly correlated with the equilibrium solubility. Application of the method to screening compound/surfactant/oil formulations is also presented.

Chemistry, Pharmaceutical↗
PubMed2005

Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications.

Solvation-assisted pressure tuning has been employed to unravel unknown structural and kinetic aspects of the insulin aggregation and fibrillation process. Our approach, using fluorescence, Fourier transform infrared and atomic force microscopy techniques in combination with pressure and solvent perturbation, reveals new insights into the pre-aggregated regime as well as mechanistic details about two concurrent aggregation pathways and the differential stability of insulin aggregates. Pressure uniformly fosters the dissociation of native insulin oligomers, whereas the aggregation pathways at elevated temperatures are affected by pressure differently and in a cosolvent-dependent manner. Moderate pressures accelerate the amyloid pathway in the presence of EtOH (leading to essentially monomeric aggregating species) via relatively dehydrated transition states with negative activation volumes for nucleation and elongation. Alternatively, a novel, fast equilibrium pathway to distinct beta-sheet-rich oligomers with thioflavin T-binding capability is accessible to partially unfolded insulin monomers at pressures below approximately 200 bar in the absence of EtOH. These oligomers, probably off the normal fibrillation pathway, are stabilized mainly by electrostatic and hydrophobic interactions, lacking the precise packing of mature insulin fibrils, which renders them susceptible to quantitative pressure-induced dissociation. Due to a highly negative activation volume for dissociation (-70(+/-16)ml/mol), pressure dissociation is fast and technologically feasible at ambient temperatures and moderate pressures. Becoming kinetically very labile above 35 degrees C, the pressurized oligomers can re-enter the slower, ultimately irreversible, fibrillation pathway at higher temperatures. At pressures above approximately 1000 bar, the partial unfolding of insulin monomers, accompanied by a volumetric expansion, dominates the aggregation kinetics, which manifests in a progressive inhibition of the fibrillation. Unlike their precursors, the pressure-insensitivity of mature insulin fibrils demonstrates that an extensive hydrogen bonding network and optimized side-chain packing are crucial for their stability.

Animals↗
PubMed2006

Micro-anatomical response of cartilage-on-bone to compression: mechanisms of deformation within and beyond the directly loaded matrix.

The biomechanical function of articular cartilage relies crucially on its integration with both the subchondral bone and the wider continuum of cartilage beyond the directly loaded contact region. This study was aimed at visualizing, at the microanatomical level, the deformation response of cartilage including that of the non-directly loaded continuum. Cartilage-on-bone samples from bovine patellae were loaded in static compression until a near-equilibrium deformation was achieved, and then chemically fixed in this deformed state. Full-depth cartilage-bone sections, incorporating the indentation profile and beyond, were studied in their fully hydrated state using differential interference contrast microscopy. Morphometric measurements of the indented profile were used in combination with a force analysis of the tangential layer to investigate the extent to which the applied force is attenuated in moving away from the directly loaded region. This study provides microscopic evidence of a structure-related response in the transitional zone of the cartilage matrix. It is manifested as an intense chevron-type shear discontinuity arising from the constraints provided by both the strain-limiting articular surface and the osteochondral attachment. The discontinuity persists well into the non-directly loaded continuum of cartilage and is proposed as a force attenuation mechanism. The structural and biomechanical analyses presented in this study emphasize the important role of the complex microanatomy of cartilage, highlighting the interconnectivity and optimal recruitment of the load-bearing elements throughout the zonally differentiated cartilage depth.

Animals↗