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PubMed2003

The role of osteopontin on calcium oxalate crystal formation.

OBJECTIVE: We evaluated whether osteopontin (OPN) and other proteins with the RGD sequence as in OPN (RGD family proteins) that are present in renal tubular cells (fibronectin [FN], Tamm-Horsfall glycoprotein [THP], vitronectin [VN], and laminin [LN]) inhibit the aggregation and growth of calcium oxalate (CaOx) crystals by a novel seed crystal method using collagen granules (CG) with and without OPN adhered on the surface. We also evaluated the effect of solid phase OPN, FN and THP in which the relationship between their proteins and CaOx crystallization was reported. Moreover, the state and time-course changes in CaOx crystals adhered to CG were observed under scanning electron microscopy (SEM). METHODS: The inhibitory activity (IA) on the aggregation and growth of CaOx crystals was measured in vitro by the conventional seed crystal method using isotopes. In this study, the following nine samples were used: OPN alone; FN alone; THP alone; VN alone; LN alone; CG alone; and CG with OPN, FN, or THP adhered on the surface (OPN/FN/THP-immobilized CG). In addition, the state and time-course changes in CaOx crystals adhered to CG were evaluated by SEM. RESULTS: Using the conventional seed crystal method, the following values of IA were obtained: 91.7% (37.5 micro g/ml) for OPN, 5.0% (100 micro g/ml) for FN, 2.0% (100 micro g/ml) for THP, 3.0% (100 micro g/ml) for VN, and 1.0% (100 micro g/ml) for LN. However, the value of IA obtained by our seed crystal method using CG was 92.1% (180cm(2)/5ml PBS) when CG alone was used. Although the value of IA was decreased by 33.6% when OPN-immobilized CG was used, it did not significantly change when FN/THP-immobilized CG was used. When CG alone was used, the evaluation of CaOx crystallization by SEM demonstrated mild adherence and aggregation of CaOx crystal suspension (seed crystals) on the CG surface, although newly formed crystals only slightly adhered to the CG surface. When OPN-immobilized CG was used, marked adherence and aggregation of seed crystals were observed, in addition to the relatively increased adherence of newly formed crystals. When FN/THP-immobilized CG was used, newly formed crystals only slightly adhered to the CG surface, although the degree of seed crystal adherence and aggregation did not significantly change. CONCLUSIONS: These findings suggest that the immobilization of OPN to the CG surface enhances the adherence and aggregation of seed crystals, as well as enhancing the adherence of newly formed crystals, resulting in decreased IA of CG (overall promotion of crystal deposition). Therefore, the results of this study clarified that OPN enhances the formation and aggregation of CaOx crystals in this experimental system.

Calcium Oxalate↗
PubMedDate not supplied

Metastability and transformation of polymorphic crystals in biodegradable poly(butylene adipate).

Polymorphism phenomenon of melt-crystallized poly(butylene adipate) (PBA) has been studied by wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). It has been found that the isothermal crystallization leads to the formation of PBA polymorphic crystals, simply by changing the crystallization temperature. The PBA alpha crystal, beta crystal, and the mixture of two crystal forms grow at the crystallization temperatures above 32 degrees C, below 27 degrees C, and between these two temperatures, respectively. The relationship between PBA polymorphism and melting behaviors has been analyzed by the assignments of multiple melting peaks. Accordingly, the equilibrium melting temperatures Tm degrees of both alpha and beta crystals were determined by Hoffman-Weeks and Gibbs-Thomson equations for the purpose of understanding the structural metastability. The Tm degrees of the PBA alpha crystal was found to be higher than that of the beta crystal, indicating that the PBA alpha crystal form is a structurally stable phase and that the beta crystal form is a metastable phase. The analysis of growth kinetics of PBA polymorphic crystals indicates that the metastable PBA beta crystal is indeed the kinetically preferential result. Based on the thermal and kinetic results, the phenomenon of stability inversion with crystal size in melt-crystallized PBA was recognized, in terms of the growth mechanisms of PBA alpha and beta crystals and the transformation of beta to alpha crystals. The PBA beta --> alpha crystal transformation takes place at a sufficiently high annealing temperature, and the transformation has been evident to be a solid-solid-phase transition process accompanied by the thickening of lamellar crystals. The molecular motion of polymer chains in both crystalline and amorphous phases has been discussed to understand the thickening and phase transformation behaviors.

Biocompatible Materials↗
PubMed1995

Structural and chemical characteristics and maturation of the calcium-phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture.

The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy. The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached.

Animals↗
PubMed1996

Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.

Using complementary physical-chemical techniques we defined five different crystallization pathways as functions of time (30 days) and increasing lecithin (egg yolk) content in pathophysiologically relevant model biles super-saturated (cholesterol saturation indices, 1.2 - 2.7) by dilution of approximately equal to 29 g/dl bile salt-lecithin-cholesterol micellar solutions. As evidenced by quasi-elastic light-scattering spectroscopy, supersaturation was heralded by the appearance of unilamellar vesicles. With the lowest lecithin contents, arc-like crystals with habit and density (d 1.030 g/mL) consistent with anhydrous cholesterol appeared first and evolved via helical and tubular crystals to form plate-like cholesterol monohydrate crystals (d 1.045 g/mL). With higher lecithin fractions, cholesterol monohydrate crystals appeared earlier than arc and other transitional crystals. With typical physiological lecithin contents, early liquid crystals (d 1.020 g/mL) were followed by cholesterol monohydrate crystals and subsequent appearances of arc and other intermediate crystals. With higher lecithin contents, liquid crystals were followed by cholesterol monohydrate crystals only, and at the highest lecithin mole fractions, liquid crystals appeared that did not generate solid crystals. Added calcium increased solid crystal number in proportion to its concentration (5 - 20 mM) but did not influence appearance times, crystallization pathways, or micellar cholesterol solubilities. Decreases in temperature (37 degrees --> 4 degrees C), total lipid concentration (7.3 --> 2.4 g/dL), and bile salt hydrophobicity (3 alpha, 12 alpha --> 3 alpha, 7 alpha, 12 alpha --> 3 alpha, 7 beta hydroxylated taurine conjugates) progressively shifted all crystallization pathways to lower lecithin contents, retarded crystallization, and decreased micellar cholesterol solubilities. The lecithin content of mother biles decreased markedly during crystallization especially where liquid crystals were a coexisting phase at equilibrium. This systematic study provides a framework for understanding cholesterol crystallization in human and animal biles and for examining factors that influence the kinetics of phase separation.

Bile↗
PubMed2000

A systematic approach to the large-scale production of protein crystals.

Crystallization has recently emerged as a suitable process for the manufacture of biocatalysts in the form of cross-linked enzyme crystals (CLECs) or for the recovery of proteins from fermentation broths. In both instances it is essential to define conditions which control crystal size and habit, and that yield a reliable recovery of the active protein. Experiments to define the crystallization conditions usually depend on a factorial design (either incomplete or sparse matrix) or reverse screening techniques. In this work, we describe a simple procedure that allows the effect of three factors, for example protein concentration, precipitant concentration and pH, to be varied simultaneously and smoothly over a wide range. The results are mapped onto a simple triangular diagram where a 'window of crystallization' is immediately apparent, and that conveniently describes variations either in the crystal features, such as their yield, size, and habit, or in the recovery of biological activity. The approach is illustrated with two enzymes, yeast alcohol dehydrogenase (ADH I) and Candida rugosa lipase. For ADH the formation of two crystal habits (rod and hexagonal) could be controlled as a function of pH (6.5-10) and temperature (4-25 degrees C). At pH 7, in 10 to 16% w/v polyethylene glycol (PEG) 4000, only rod-shaped crystals formed whereas at pH 8, in 10 to 14% w/v PEG, only hexagonal crystals existed. For both enzymes, catalyst recovery was greatest at high crystallization agent concentrations and low protein concentration. For ADH, the greatest activity recovery was 87% whereas for the lipase crystals, by using 45% v/v 2-methyl-2,4-pentanediol (MPD) as the crystallization agent, a crystal recovery of 250 crystals per µl was obtained. For the lipase system, the use of crystal seeding was also shown to increase the crystal recovery by up to a factor of four. From the crystallization windows, the original conditions based on literature precedent (35% v/v MPD, 1 mM CaCl(2), 1.8 mg protein/ml) were altered (47.5% v/v MPD, 2 mM CaCl(2), 3 mg protein/ml). This led to an improved recovery of the lipase under conditions that scale reliably from 0.5 ml to 500 ml with no change in size, shape or recovery of the crystals themselves. Finally, these crystals were crosslinked with 5% v/v glutaraldehyde and mass and activity balances were calculated for the entire process of CLEC production. Up to 35% of the lipase activity present in the crude solid was finally recovered in the lipase CLECs after propan-2-ol fractionation, crystallization, and crosslinking.

Journal Article↗
PubMed1984

Structural basis and dynamics of the fiber-to-crystal transition of sickle cell hemoglobin.

The kinetics of the assembly of structurally distinct, polymeric aggregates constituting the fiber-to-crystal transition of sickle cell hemoglobin in slowly stirred, deoxygenated solutions has been studied with the use of electron microscopy as a function of pH, as a function of the crystal structures of mutant forms of human deoxyhemoglobins employed as nucleating seeds, and as a function of hemoglobin S chemically modified at the Cys F9 (beta 93) position. The temporal order of appearance of fibers of approximately 210 A diameter, bundles of aligned fibers, macrofibers of greater than or equal to 650 A diameter, and microcrystals is observed. Microscopic fragments of end-stage crystals formed under slowly stirred conditions and introduced as nucleating seeds enhance the rate of crystallization only when added prior to the formation of large bundles of aligned fibers, while microscopic seed crystals added after the formation of bundles of aligned fibers do not alter the rate of crystallization. Over the pH range 6.3 to 7.1, the presence of macrofibers does not influence modulation of the kinetics of the transition with seed crystal fragments. Microscopic seed crystals of deoxyhemoglobin S and deoxyhemoglobin C formed under acidic conditions (pH less than 6.5) have a comparable influence on the kinetics of the fiber-to-crystal transition to that of end-stage crystals. Microscopic seed crystals of deoxyhemoglobin C formed under alkaline conditions (pH greater than 6.5) enhance the formation of macrofibers but do not alter the rate of crystallization. Under conditions associated with enhanced formation of macrofibers, metastable microscopic crystals having axial periodicities of approximately 64 A and approximately 210 A are observed in the intermediate phase of the transition, while end-stage crystals have axial unit cell dimensions identical to those of deoxyhemoglobin S crystallized from polyethylene glycol solutions of pH less than 6.5. Although the metastable crystals may arise from fragments of macrofibers, it is shown that they cannot be transformed directly into end-stage crystals under slowly stirred conditions without undergoing dissolution. These results stipulate that the pathway of the fiber-to-crystal transition proceeds according to the reaction: (Formula: see text) wherein the rate-limiting step is the alignment of fibers into large bundles, and macrofibers are not an intermediate of the fiber-to-crystal transition.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization↗
PubMed2003

Crystal attachment to injured renal collecting duct cells: influence of urine proteins and pH.

BACKGROUND: The attachment of crystals to injured kidney epithelium is thought to be a necessary event in the development of urolithiasis. In vivo, the crystals are coated with urinary macromolecules that define the surface properties of the crystals. The present study examines the influence of coating of calcium oxalate crystals with urinary macromolecules on their attachment to both healthy (polarized) and injured (nonpolarized) primary inner medullary collecting duct (IMCD) cells. METHODS: Calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals were coated with urine macromolecules by incubating the crystals in urine from normal healthy volunteers at pH 5, 6, and 7. The level of attachment of the coated crystals to IMCD cells was also determined at pH 5, 6, and 7. The adsorbed proteins were extracted from the crystal surfaces and separated by gel electrophoresis. RESULTS: The coating of calcium oxalate crystals with urine proteins greatly reduced the attachment of crystals to both control and injured IMCD cells. At pH levels below 6, the crystals readily attached to injured cells. Extraction and separation of the adsorbed proteins showed that both COM and COD crystals adsorbed a similar array of proteins. At pH 5 and 6, several trace proteins were adsorbed to the crystals and were not apparent at pH 7. CONCLUSION: The coating of crystals with urine macromolecules greatly reduces the attachment of the crystals to normal healthy epithelia. The coating and attachment of the crystals below pH 6 enhances the attachment to injured cells. The enhanced crystal attachment could possibly be associated with one or more proteins adsorbed to the crystal surface that are not adsorbed to the crystals at higher pH.

Animals↗
PubMed1997

Localization of Charcot-Leyden crystal protein in individual morphological phenotypes of human basophils stimulated by f-Met peptide.

BACKGROUND: Human basophils undergo anaphylactic degranulation, characterized by extrusion of membrane-free granules, and piecemeal degranulation, characterized by progressive removal of granule contents in the absence of granule extrusion. F-Met peptide stimulates a degranulation continuum in human basophils that includes both forms of secretion. Charcot-Leyden crystal protein is stored in the granules of unstimulated human basophils. OBJECTIVE: The objective of this study was to determine the subcellular localization of the Charcot-Leyden crystal protein in individual morphological basophil phenotypes that are stimulated by f-Met peptide and are associated with secretion. METHODS: A post-embedding immunogold analysis was used to detect changes in the subcellular sites of Charcot-Leyden crystal protein in human basophils stimulated with f-Met peptide. Human basophils from normal donors were purified by countercurrent centrifugal elutriation and Percoll density gradients, stimulated to degranulate with 1 micron f-Met peptide (or incubated in buffer controls), and recovered for histamine assay, electron microscopy and immunogold labelling. Specificity controls included omission of the primary antibody and substitution of the primary antibody with non-immune normal rabbit IgG or with Charcot-Leyden crystal protein-Sepharose-absorbed primary antibody. RESULTS: The results showed new sites of labelling and different densities of labelling for Charcot-Leyden crystal protein in distinctive basophil phenotypes stimulated by f-Met peptide. New sites for Charcot-Leyden crystal protein included nucleus, cytoplasm, degranulation channel, degranulation channel membrane, plasma membrane, and a newly recognized granule population similar to primary granules in eosinophils. These new sites, as well as previously documented sites of Charcot-Leyden crystal protein (granules, intragranular Charcot-Leyden crystals, cytoplasmic vesicles) showed variable labelling when analysed by phenotype. Other sites (besides intragranular Charcot-Leyden crystals) of formed Charcot-Leyden crystals included cytoplasm, degranulation channel, extracellular space and, rarely, nucleus. Analysis of cytoplasmic vesicles, total granules and altered granules, and gold particles in subcellular compartments in seven identifiable phenotypes revealed that f-Met peptide stimulated human basophils to empty their granules by transporting Charcot-Leyden crystal protein in vesicles to the plasma membrane in the absence of granule extrusion in cells exhibiting piecemeal degranulation. In cells exhibiting anaphylactic degranulation, gold-labelled Charcot-Leyden crystals were extruded to the cells' exterior in concert with granule particles and concentric dense membranes contained within granules. Completely degranulated cells had a high density of plasma membrane gold label that was associated with numerous gold-laden endocytotic cytoplasmic vesicles. Basophils reconstituted their main granule population, within which Charcot-Leyden crystals resided, in part by endocytosis of previously released plasma membrane-bound Charcot-Leyden crystal protein. Completely recovered cells displayed decreased Charcot-Leyden crystal protein labelling of the plasma membrane and vesicle compartments, the presence of a highly labelled new granule subset that resembled Charcot-Leyden crystal protein-containing primary granules in eosinophils, and the highest density of granule and intragranular Charcot-Leyden crystal gold labelling of all phenotypes that developed after stimulation. CONCLUSION: Seven individual f-Met peptide-activated human basophil phenotypes labelled by an ultrastructural immunogold method to detect subcellular sites of Charcot-Leyden crystal protein showed changing distributions of this protein which document the capability of human basophils to undergo complex release and recovery reactions that may be pertinent to the functions of Charcot-Leyden crystal protein and the capabilit

Anaphylaxis↗
PubMed2006

Surface crystallization of indomethacin below Tg.

PURPOSE: To study the surface crystallization of indomethacin (IMC) below T (g) and its effects on the kinetics of overall crystallization. METHODS: Crystal growth rates in liquid layers formed between microscope cover glasses were measured with the top cover glass in place and removed. Polymorphs were identified by powder X-ray diffraction, Raman microscopy, and melting-point determination by hot-stage microscopy. Surface crystals were identified by scratching the sample surface, by cutting the sample to expose its interior, and by analyzing the intensity of X-ray diffraction. Amorphous IMC particles of different sizes were stored at 40 degrees C (T (g)-2 degrees C) and analyzed at different times by differential scanning calorimetry to obtain the kinetics of crystallization. RESULTS: Crystal growth of IMC below T (g) at the free surface was approximately two orders of magnitude faster than that in the bulk, resulting in a surface layer of crystals around a slower-crystallizing interior. Surface crystallization yielded mainly the gamma polymorph. Amorphous IMC powders showed rapid initial crystallization at 40 degrees C, but the crystallization abruptly slowed down at "saturation levels" below 100%; the larger the particles, the lower the "saturation level." CONCLUSION: The faster surface crystallization of IMC than the bulk crystallization leads to unusual crystallization kinetics wherein a rapid initial increase of crystallinity is followed by an abrupt slowdown of crystallization. Surface crystallization should be distinguished from bulk crystallization in modeling and controlling the crystallization of amorphous solids.

Algorithms↗
PubMed2000

Crystallization of lactose from carbopol gels.

PURPOSE: To crystallize lactose under static conditions with a view to preparing crystals of well-defined morphology. METHODS: et-Lactose monohydrate was crystallized from neutralized Carbopol 934 gels. When the majority of crystals had grown to maturity, the gels were acidified using diluted hydrochloric acid and the crystals were harvested by filtration or centrifugation and washed with ethanol-water mixtures. RESULTS: Crystals prepared from the gel had a consistently narrower size distribution than control crystals, prepared from solution under constant stirring. If crystallization was effected in the gel without sedimentation of the crystals, then the resultant crystals had smooth surfaces without visually detectable surface roughness or asperities viewed by optical microscopy. The crystals from Carbopol gels also exhibited the uniform shape of an elongated tomahawk regardless of the crystallization conditions, in contrast to crystallization under constant stirring, where the crystal shape of lactose changed with crystallization conditions especially as a function of the initial concentration of lactose. All batches of lactose crystals prepared from Carbopol gels existed as alpha-lactose monohydrate, which showed better flowability than the controls of a similar particle size. CONCLUSIONS: Crystallization from Carbopol gel produces lactose crystals of uniform size, regular shape, smooth surface, and improved flowability.

Acrylic Resins↗
PubMed2006

Crystallization of helical oligomers with chirality selection. I. A molecular dynamics simulation for bare helix.

Helical polymers often exhibit pronounced chirality recognition during crystallization. By molecular dynamics simulation, we have already shown that the helical polymers crystallize with or without marked chirality selection depending on structural details of the polymer molecules. We have there classified the helical polymers into two categories: the bare helices made of only backbone atoms which show rather tolerant chirality selection, and the general helices with large side groups showing strict chirality recognition. Polymer crystallization is in general largely hampered and retarded by slow dynamics of the entangled chains, and therefore short helical oligomers are very suitable models for studying the chiral crystallization. We here report on molecular simulations of crystallization in the bare helical oligomer molecules by the use of Monte Carlo and molecular dynamics simulations. First we confirm the low temperature chiral crystal phase and the reversible order-disorder transition. We also observe frequent inversions of the helical sense, and the helix reversal defects propagating along the chains. Then we investigate crystallization from the melt into the chiral crystal phase. We find that the crystallization rate depends very sensitively on the degree of undercooling. The crystallization is found to be the first order transition that conforms well to the traditional picture of crystal growth in small molecules. Even when the crystallization directly into the chiral crystal phase is conducted, marked chirality selections are not observed at the early stage of crystallization; the chains adhere to the crystal surfaces selecting their helical senses rather at random resulting in racemic crystallites. The isothermal crystallization for a sufficiently long time, however, yields lamellar crystals composed of well-developed chiral domains, the growth of which seems to be accomplished through the transition back into the ordered chiral crystal phase.

Journal Article↗
PubMed2005

Structural consequences of hen egg-white lysozyme orthorhombic crystal growth in a high magnetic field: validation of X-ray diffraction intensity, conformational energy searching and quantitative analysis of B factors and mosaicity.

A novel method has been developed to improve protein-crystal perfection during crystallization in a high magnetic field and structural studies have been undertaken. The three-dimensional structure of orthorhombic hen egg-white (HEW) lysozyme crystals grown in a homogeneous and static magnetic field of 10 T has been determined and refined to a resolution of 1.13 angstroms and an R factor of 17.0%. The 10 T crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.54 (3), b = 73.86 (6), c = 30.50 (2) angstroms and one molecule per asymmetric unit. A comparison of the structures of the 0 T and 10 T crystals has been carried out. The magnitude of the structural changes, with a root-mean-square deviation value of 0.75 angstroms for the positions of all protein atoms, is similar to that observed when an identical protein structure is resolved in two different crystalline lattices. The structures remain similar, with the exception of a few residues e.g. Arg68, Arg73, Arg128 and Gln121. The shifts of the arginine residues result in very significant structural fluctuations, which can have large effects on a protein's crystallization properties. The high magnetic field contributed to an improvement in diffraction intensity by (i) the displacement of the charged side chains of Arg68 and Arg73 in the flexible loop and of Arg128 at the C-terminus and (ii) the removal of the alternate conformations of the charged side chains of Arg21, Lys97 or Arg114. The improvement in crystal perfection might arise from the magnetic effect on molecular orientation without structural change and differences in molecular interactions. X-ray diffraction and molecular-modelling studies of lysozyme crystals grown in a 10 T field have indicated that the field contributes to the stability of the dihedral angle. The average difference in conformational energy has a value of -578 kJ mol(-1) per charged residue in favour of the crystal grown in the magnetic field. For most protein atoms, the average B factor in the 10 T crystal shows an improvement of 1.8 angstroms(2) over that for the 0 T control; subsequently, the difference in diffraction intensity between the 10 T and 0 T crystals corresponds to an increase of 22.6% at the resolution limit. The mosaicity of the 10 T crystal was better than that of the 0 T crystal. More highly isotropic values of 0.0065, 0.0049 and 0.0048 degrees were recorded along the a, b and c axes, respectively. Anisotropic mosaicity analysis indicated that crystal growth is most perfect in the direction that corresponds to the favoured growth direction of the crystal, and that the crystal grown in the magnetic field had domains that were three times the volume of those of the control crystal. Overall, the magnetic field has improved the quality of these crystals and the diffracted intensity has increased significantly with the magnetic field, leading to a higher resolution.

Animals↗
PubMed2005

Analysis for crystals in synovial fluid: training of the analysts results in high consistency.

BACKGROUND: Identification of monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in synovial fluid samples is diagnostic of gout and CPPD crystal related arthropathy. Various studies have shown poor consistency in results of crystal analysis. OBJECTIVE: To determine whether training of the analysts increases the consistency. METHODS: An expert rheumatologist gave a course on crystal detection and identification. The four trained observers then blindly and independently examined synovial fluid samples previously classified by the expert which had been obtained from patients with both crystal arthropathies and other non-crystal related inflammatory joint conditions. RESULTS: 194 observations were made on 64 synovial fluid samples: 96 without crystals (49.4%), 55 with CPPD crystal (28.4%), and 43 with MSU crystals (22.2%). For crystal detection (presence or absence of crystals), sensitivity was 95.9% and specificity 86.5%. For identification of MSU crystals, sensitivity was 95.3% and specificity 97.2%. For identification of CPPD crystals, sensitivity was 92.7% and specificity 92.1%. The kappa index of agreement with the reference standard between the observers was 0.84 for any crystal detection, 0.93 for MSU crystal sample identification, and 0.79 for CPPD crystal sample identification. CONCLUSIONS: For trained observers, the detection and identification of crystals in synovial fluid is a consistent procedure.

Arthritis↗
PubMed2002

Quantitation of cholesterol crystallization from supersaturated model bile.

Cholesterol crystallization is an essential step in gallstone formation. Although spectrophotometry and nephelometry have been used for quantitation of crystallization, potential effects of crystal size and shape have not been evaluated. We determined crystallization in model biles [total lipid concentration 7.3 g/dl, egg yolk Phosphatidylcholine (EYPC)/(EYPC+taurocholate) molar ratio = 0.05, 0.15, or 0.30; cholesterol saturation index (CSI) = 1.2, 1.7, or 2.0; 37 degrees C] plotting in the central three-phase (micelles, vesicles, and crystals containing) zone or in the left two-phase (micelles and crystals containing) zone of the equilibrium ternary phase diagram. Extent of crystallization estimated by spectrophotometry and nephelometry was related to chemical determination of crystal mass and to crystal size or shape (by microscopy). With all methods, crystallization was less extensive when vesicles were present (central three-phase zone) and at lower CSIs. In the left two-phase zone, particularly at EYPC/(EYPC+taurocholate), ratio of 0.15, there were strong increases in spectrophotometric and nephelometric readings during the first days of incubation, but decreases at later stages, despite progressive increases in crystal mass by chemical measurement. Initially, there were large numbers of very small crystals (<10 microm) in these biles, which were subsequently replaced by large cholesterol monohydrate crystals. Decreasing sizes of harvested cholesterol monohydrate crystals by sonication increased spectrophotometric and nephelometric values despite identical crystal mass. When cholesterol crystal mass is assayed by indirect methods such as spectrophotometry or nephelometry, results are strongly influenced by crystal size.

Bile↗
PubMed2004

Modulation of proliferating renal epithelial cell affinity for calcium oxalate monohydrate crystals.

Adhesion of urinary crystals to distal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. Monolayer cultures of distal nephron-derived MDCKI cells were used as a model to study crystal-cell interactions. COM crystal adhesion reached a peak 2 d after plating and progressively fell thereafter. The decline in crystal binding was accelerated by prostaglandin E(2) (PGE(2)) supplementation and delayed by blockade of PG production. Crystals avidly adhered to cells that migrated in to repair a scrape wound made in the monolayer and after a transient hypoglycemic insult. Exposure of MDCKI cells to uric acid crystals and soluble uric acid was also associated with increased crystal adhesion. Treatment of physically or hypoglycemically injured cells with trypsin or neuraminidase reduced crystal binding to baseline levels, suggesting that increased exposure of cell surface glycoproteins mediated the effect, whereas PGE(2) treatment blunted crystal binding to regenerating cells. Furthermore, when cells were grown in the presence of synthetic d-mannosamine analogues that can modify the conformation of cell surface sialoglycoconjugates, crystal binding to proliferating cells was decreased, whereas blockade of N-glycosylation with tunicamycin increased crystal adhesion to these cells. Therefore, COM crystal binding is enhanced to growing renal cells, synthesis of N-glycosylated cell surface proteins is essential to downregulate crystal binding to cells, and this response is modulated by physiologic signals such as PGE(2). Sialic acid residues seem to mediate crystal adhesion to growing cells, either directly or via linkage to other crystal-binding molecules. Subtle renal injury and subsequent nephron repair could be a factor promoting crystal adhesion and favoring calculus formation.

Animals↗
PubMed2003

Physical and structural studies on the cryocooling of insulin crystals.

Reflection profiles were analyzed from microgravity-grown ( micro g) and earth-grown insulin crystals to measure mosaicity (eta) and to reveal mosaic domain structure and composition. The effects of cryocooling on single-domain and multi-domain crystals were compared. The effects of cryocooling on insulin structure were also re-examined. Microgravity crystals were of larger volume, were more homogeneous and were of higher quality than earth crystals. Several micro g crystals contained a single mosaic domain which encompassed all or nearly all of the crystal with an eta(avg) of 0.005 degrees. The earth crystals varied in quality and all contained multiple domains with an eta(avg) of 0.031 degrees. Cryocooling caused a 43-fold increase in eta for micro g crystals (eta(avg) = 0.217 degrees ) and an eightfold increase for earth crystals (eta(avg) = 0.246 degrees ). These results indicate that very well ordered crystals are not completely protected from the stresses associated with cryocooling, especially when structural perturbations occur. However, there were differences in the reflection profiles. For multi-mosaic domain crystals, each domain individually broadened and separated from the other domains upon cryocooling. Cryocooling did not cause an increase in the number of domains. A crystal composed of a single domain retained this domain structure and the reflection profiles simply broadened. Therefore, an improved signal-to-noise ratio for each reflection was measured from cryocooled single-domain crystals relative to cryocooled multi-domain crystals. The improved signal from micro g crystals, along with the increase in crystal size, facilitated the measurement of the weaker high-resolution reflections. The observed broadening of reflection profiles indicates increased variation in unit-cell parameters, which may be linked to cryocooling-associated structural changes and disorder.

Crystallization↗
PubMed2000

The influence of crystallization conditions on the morphology of lactose intended for use as a carrier for dry powder aerosols.

Lactose has been widely used as a carrier for inhalation aerosols. The carrier morphology is believed to affect the delivery of the drug. The aim of this study was to investigate the effects of crystallization conditions on the morphology of alpha-lactose monohydrate intended for use as the carrier for dry powder aerosols. The crystallization of lactose was carried out from aqueous solutions at different supersaturations, temperatures, different stages of crystallization and in the presence of different water-miscible organic solvents. The majority of lactose crystals were found to be either tomahawk-shaped or pyramidal after crystallization at an initial lactose concentration between 33-43% w/w, but these became prismatic if the lactose concentration was increased to 50% w/w. A further increase in the lactose concentration to 60% w/w led to the preparation of elongated cuboidal crystals. Higher initial lactose concentrations tended to result in the crystallization of more elongated particles. Crystallization at 40 degrees C was shown to prepare lactose crystals with a more regular shape and a smoother surface than those crystallized at 0 degrees C. Lactose particles generated during the later stage of crystallization were found to be more regular in shape with a smoother surface than those prepared in the earlier stage. The addition of 10% (v/v) methanol or ethanol or acetone to the mother liquor increased the growth rate of lactose particles whereas addition of propanol or glycerine inhibited the rate of crystal growth. Lactose crystals prepared in the presence of glycerine were more regularly shaped with a smoother surface than those prepared in the presence of ethanol or acetone. All the resultant crystals were shown to comprise alpha-lactose monohydrate. Lactose crystals could be prepared with a precisely defined morphology by means of carefully controlling the crystallization conditions.

Aerosols↗
PubMed1984

Intermittency of cholesterol crystals in duodenal bile from gallstone patients.

The detection of cholesterol crystals in duodenal bile is of clinical value in the diagnosis of cholesterol gallstone disease; however, not all patients with cholesterol gallstones have crystals detected in their duodenal bile, thus limiting the value of examination of duodenal bile. The aims of this study were to (i) determine whether the lack of crystals in some patients with cholesterol gallstones was due to (a) the intermittent presence of crystals, (b) spontaneous crystal dissolution, or (c) changes in dietary cholesterol intake; (ii) determine whether incubation of duodenal bile for 24 h would result in crystal formation. Sixteen patients with radiolucent gallstones each underwent three duodenal biliary drainages. Thirty-one percent of patients had crystals in all three bile specimens, 12% in two specimens, 25% in one specimen, and 32% in no specimen. One of 31 specimens with small numbers of crystals initially had no crystals at 24 h, and five specimens initially devoid of crystals developed crystals by 24 h. Despite a significant increase in biliary cholesterol saturation index with increasing cholesterol intake, the prevalence of crystals in bile did not increase in gallstone patients. No crystals were identified in 18 specimens from normal subjects examined initially or in the 15 specimens that were examined after 24 h. We conclude that the intermittent presence of cholesterol crystals in duodenal bile is probably not due to dissolution of crystals or varying dietary cholesterol intake and that the frequency with which crystals are found increases with incubation. Determination of the diagnostic value of multiple duodenal biliary drainages or incubation of bile in patients with normal oral cholecystograms or gallbladder ultrasonograms, however, will require examinations of large numbers of patients.

Bile↗