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PubMed2000

Optic properties of bile liquid crystals in human body.

AIM:To further study the properties of bile liquid crystals, and probe into the relationship between bile liquid crystals and gallbladder stone formation, and provide evidence for the prevention and treatment of cholecystolithiasis.METHODS:The optic properties of bile liquid crystals in human body were determined by the method of crystal optics under polarizing microscope with plane polarized light and perpendicular polarized light.RESULTS:Under a polarizing microscope with plane polarized light, bile liquid crystals scattered in bile appeared round, oval or irregularly round. The color of bile liquid crystals was a little lighter than that of the bile around. When the stage was turned round, the color of bile liquid crystals or the darkness and lightness of the color did not change obviously. On the border between bile liquid crystals and the bile around, brighter Becke-Line could be observed. When the microscope tube is lifted, Becke-Line moved inward, and when lowered, Becke-Line moved outward. Under a perpendicular polarized light, bile liquid crystals showd some special interference patterns, called Malta cross. When the stage was turning round at an angle of 360(o), the Malta cross showed four times of extinction. In the vibrating direction of 45(o) angle of relative to upper and lower polarizing plate, gypsum test-board with optical path difference of 530nm was inserted, the first and the third quadrants of Malta cross appeared to be blue, and the second and the fourth quadrants appeared orange. When mica test-board with optical path difference of 147nm was inserted, the first and the third quadrants of Malta cross appeared yellow, and the second and the fourth quadrants appeared dark grey.CONCLUSION:The bile liquid crystals were distributed in bile in the form of global grains. Their polychroism and absorption were slight, but the edge and Becke-Line were very clear. Its refractive index was larger than that of the bile. These liquid crystals were uniaxial positive crystals. The interference colors were the first order grey-white. The double refractive index of the liquid crystals was n = 0.011-0.015.

Journal Article↗
PubMed1999

Role of fibronectin during biological apatite crystal nucleation: ultrastructural characterization.

The role of adhesion molecules like osteopontin and bone sialoprotein, both containing the Arg-Gly-Asp sequence have been shown to have a role in mineral formation, whereas fibronectin (FN), another adhesive protein, was never studied during the mineralization processes. The formation and maturation of biological apatite crystals are under matrix control, and one of the roles of specific crystal proteins is to control the nucleation and growth of biological apatite during the mineralization process (promotion or inhibition). In the case of calcium phosphate ceramic used as a bone substitute, a dissolution-precipitation process occurs after implantation before the bone ingrowth and bone mineralization. The early precipitation consists of common biological apatite crystals. These crystals are the result of secondary nucleation and a heteroepitaxic growth process on synthetic residual crystals. In in vivo studies, hydroxyapatite crystals were implanted subcutaneously into mice for 1 or 2 weeks. Fibronectin immunogold labeling of the newly formed crystals on surfaces of high-resolution transmission electron microscopy sections of retrieved implants revealed the close association of these precipitated crystals with FN. In in vitro experiments using a solution of human FN incubated in the presence of calcium phosphate crystals, we obtained apatite crystal precipitation. The fibronectin network observed in high-resolution transmission electron microscopy showed numerous clusters of very small particles (1 nm in diameter and 2 nm in length), whereas the same experiment realized as control on albumin revealed no crystal precipitation. These results demonstrate for the first time the role of FN in early biological crystal nucleation. This process could have important biological significance in accounting for ectopic calcification, primary nucleation in calcified tissue, and bone ingrowth on calcium phosphate ceramics.

Animals↗
PubMed1985

Crystal-induced endogenous pyrogen production. A further look at gouty inflammation.

We found previously that crystals of sodium urate and silicon dioxide (silica) can stimulate the production of endogenous pyrogen (EP), now called interleukin-1 (IL-1), the polypeptide mediator of fever and other aspects of inflammation. We have confirmed and extended the work with urate crystals and have examined 2 other crystals associated with joint problems, hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD). The crystals were added to suspensions of human blood leukocytes (2.5 X 10(6) monocytes/dose, with 10% fresh autologous plasma); after 18 hours of incubation, the EP content of the supernatants was assayed in the rabbit pyrogen test. HA and CPPD crystals neither induced EP production nor reduced the amount of staphylococci-induced EP. Presized (10 - 40 micron) urate crystals were pyrogenic, but less so than the unsized and aggregated urate crystals investigated previously and reexamined here. On ultrasonication, the aggregated urate crystals became first more pyrogenic and then less so as the crystals were dispersed and broken down. Ultrasound did not impart pyrogenicity to HA or CPPD crystals: their failure to stimulate EP/IL-1 production from leukocytes in vitro indicates a difference in their phlogistic properties, compared with crystals of urate or silica. The results with urate crystals have pathogenetic implications in a number of areas of gouty inflammation: initiation of the acute attack, other aspects of the acute-phase response, polyarticular involvement, and the inflammatory consequences of chronic stimulation by tophaceous material.

Calcium Pyrophosphate↗
PubMed2006

Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals.

OBJECTIVE: Triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface molecule that was recently identified on monocytes and neutrophils. TREM-1 has been implicated in the early inflammatory responses induced by microbes, but its pathophysiologic role in nonmicrobial inflammation remains unknown. In the present study, we investigated the role of TREM-1 in acute inflammation induced by monosodium urate monohydrate (MSU) crystals. Induction of TREM-1 expression by MSU crystal-stimulated murine resident peritoneal macrophages and infiltrating leukocytes in a murine air-pouch model of crystal-induced acute inflammation was determined. The biologic role of TREM-1 in crystal-induced cytokine production by resident peritoneal macrophages was also investigated. METHODS: TREM-1 expression by resident peritoneal macrophages and infiltrating leukocytes in a murine air-pouch model was determined by quantitative real-time polymerase chain reaction, Western blot analysis, and flow cytometry. Cytokine production by resident peritoneal macrophages after incubation with MSU crystals in the presence or absence of an anti-TREM-1 agonist antibody was determined by enzyme-linked immunosorbent assay. RESULTS: TREM-1 expression by resident peritoneal macrophages was significantly induced after stimulation with the crystals. Maximum expression of TREM-1 transcripts and protein occurred at 1 and 4 hours after exposure to the crystals, respectively. Costimulation of resident peritoneal macrophages with MSU crystals and an anti-TREM-1 agonist antibody synergistically increased the production of both interleukin-1beta and monocyte chemotactic protein 1 compared with stimulation with the crystals alone. MSU crystals also induced TREM-1 expression in infiltrating leukocytes in a murine air-pouch model of crystal-induced acute inflammation. CONCLUSION: These findings suggest that rapid induction of TREM-1 expression on resident peritoneal macrophages and neutrophils by MSU crystals may contribute to the development of acute gout through enhancement of inflammatory responses.

Acute Disease↗
PubMed2002

Surface structure study of biological calcium phosphate apatite crystals from human tooth enamel.

A surface-structure study of human tooth enamel crystals has been carried out by high-resolution electron microscopy (HREM). The surfaces of several crystals have been examined and the surfaces of a single crystal are described here. The observations made on this crystal are similar to the observations made on the other crystals, although the difference of morphology in the crystals observed indicates that the growth control by matrix proteins takes only place on the [011 macro 0] surfaces of the crystals. The crystal described here is oriented along the [112 macro 0] direction, and the following surfaces have been analyzed: (11 macro 00), (011 macro 1), (01 macro 11) and (0001). A subsurface reconstruction is observed just below the surface of the crystal not bonded to the matrix and lying above the supporting film. Observation of the matrix surrounding the crystal shows the existence of poorly crystalline phases with a structure close to that of hydroxyapatite (OHAP). Finally, a comparison of the images of the (011 macro 0) surface with computer-simulated images calculated for several models of the OHAP surface structure shows that the surface itself is stoichiometric and contains both calcium and phosphate groups. The knowledge of the binding sites of proteins on biomineral crystals such as the ones found in human tooth enamel is of prime importance for the understanding of their growth process. In this study, the first structure determination of the surface of human tooth enamel crystals is presented.

Apatites↗
PubMed1992

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.

Crystallization↗
PubMed1994

Identification of a molecular switch that selects between two crystals forms of bovine pancreatic trypsin inhibitor.

Two crystals forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2-fold axis in form III crystals, the Lys 41 side-chain amino nitrogens from 2 symmetry-related molecules are only 2.72 A apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side-chain amino group is protonated and forms a salt bridge with a solvent-derived phosphate group. For the Lys 41 side-chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional intermolecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals.

Animals↗
PubMed1990

Effects of urinary macromolecules on the crystallization of calcium oxalate.

The macromolecular fraction of urine with a molecular weight above 3,000 was isolated by dialysis. In the dialysed urine the rate of calcium oxalate (CaOx) crystallization was reduced both in the presence and absence of CaOx seed crystals. There was a clear relationship between crystallization and the relative concentration of the dialysed urine, with the highest crystallization propensity at the lowest concentration of macromolecules. Dilution of dialysed urine also affected crystal size distribution, with a predominance of small (2.8-4.5 microns) crystals in 100% dialysed urine and of large (5.6-14.0 microns) crystals in 5% dialysed urine. This is consistent with a macromolecular inhibition of both crystal growth and aggregation. Analysis of the crystal size distribution 120 min after supersaturation of whole urine to a level at which approximately 100 crystals in the size interval 3.5-5 microns were detected in a Coulter counter surprisingly disclosed a higher mean crystal volume in urine samples from normal subjects than from stone formers. This gives support to the assumptions that macromolecules might be of importance during the initial phase of CaOx crystallization and that urine from stone formers and normal subjects might be different in this respect.

Calcium Oxalate↗
PubMed2006

Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells.

Calcium phosphates (CaPs) have been investigated as substrates to promote bone formation both in vitro and in vivo. The aim of this study was to examine the proliferation and differentiation of rat bone marrow stromal cells (BMSCs) cultured on three-dimensional (3D) octacalcium phosphate (OCP) crystal assemblies. The cytotoxicity of OCP crystal assemblies was evaluated by measuring the lactate dehydrogenase (LDH) release from BMSCs during 10h of incubation with OCP crystal assemblies. The proliferation of BMSCs on OCP crystal assemblies in medium with or without osteogenic supplements was also investigated using the MTT assay with tissue culture treated plastic (TP) as the control. The tissues formed by BMSCs cultured on OCP crystal assemblies for 24 days were examined following staining with haematoxylin and eosin (H&E), alkaline phosphatase (ALP) and Van Gieson's techniques. The influence of OCP crystal assemblies on mRNA expression of alpha chain of collagen type I (Coll-Ia), ALP and osteocalcin (OC), osteonectin (ON), osteopontin (OP), lumican, Cbfa1, EST317 and EST350 by the BMSCs were also investigated using semi-quantitative RT-PCR. Although OCP crystals were relatively cytotoxic compared with TP, proliferation of BMSCs occurred when seeded onto OCP crystal assemblies. BMSCs cultured on OCP demonstrated similar proliferation rates as found on the control and no significant difference (P<0.05) in the number of cells cultured in medium supplemented with or without osteogenic additives on TP and OCP. The deposition of collagen and ALP were detected in tissue synthesised by BMSCs cultured on OCP crystals assemblies. OCP crystal assemblies down-regulated basal bone ECM proteins, including Coll-Ia, ON and lumican, in the first week of culture, whilst up-regulation of the same genes was observed after 24 days of culture. The observed down-regulation of Cbfa1 on OCP substrates was consistent with the negative effect of OCP crystal assemblies on the genes encoding bone ECM proteins. The up-regulation of OC mRNA expression by OCP crystal assemblies could be related to the requirement for synthesis of more OC proteins to control the concentration of calcium ions in culture medium.

Animals↗
PubMed2006

Factors determining types and morphologies of calcium oxalate crystals: molar concentrations, buffering, pH, stirring and temperature.

BACKGROUND: Calcium oxalate (CaOx) can be crystallized in several forms and morphologies. We evaluated factors that determine differential types and shapes of CaOx crystals generated in vitro. METHODS: CaCl2 and Na2C2O4 solutions at various molar concentrations were mixed in different conditions (with or without Tris-HCl buffer and varying pH, temperature and speed of stirring) and incubated overnight. RESULTS: A total of 78 conditions were evaluated. The most frequently observed type of CaOx crystals was calcium oxalate monohydrate (COM). In 18.2 MOmega.cm water, typical monoclinic prismatic form of COM was found when 0.5-1 mmol/l CaCl2 and 0.5-1 mmol/l Na2C2O4 were mixed, whereas the COM dendrites were found when higher concentrations were used. Calcium oxalate dihydrate (COD) crystals were observed when 5 mmol/l CaCl2 and 0.5 mmol/l Na2C2O4 were employed. With the same molar concentrations of CaCl2 and Na2C2O4, the sequence of adding these 2 chemicals into the chamber had some effects on crystal types and morphologies. The presence of Tris-HCl buffer in the solution enhanced COM crystal growth and aggregation. The pH greater than 5.0 was associated with the presence of weddellite COD. Magnetic stirring of the supersaturated solution resulted to reduction in size of all crystal forms; the higher speed provided the smaller crystals. Finally, crystallization of CaOx at 4 degrees C was more efficient than performing the experiment at 25 and 37 degrees C. CONCLUSIONS: Molar concentrations, order of adding the substrates, buffering, pH, stirring and temperature have significant effects on CaOx crystal formation, types and morphologies. Cataloging these differential forms of crystals generated in different conditions will be useful for further study on modulations of CaOx crystals and kidney stone disease.

Buffers↗
PubMed2006

Influence of 4-cyano-4'-biphenylcarboxylic acid on the orientational ordering of cyanobiphenyl liquid crystals at chemically functionalized surfaces.

We report two methods that involve tailoring of the chemical composition of the nematic liquid crystal 4-cyano-4'-pentylbiphenyl to achieve control over the orientational ordering of the liquid crystal on chemically functionalized surfaces. The first method involves the direct addition of 4-cyano-4'-biphenylcarboxylic acid to 4-cyano-4'-pentylbiphenyl. The second method involves exposure of 4-cyano-4'-pentylbiphenyl to ultraviolet light and photochemical generation of a range of products, including 4-cyano-4'-biphenylcarboxylic acid. The addition of the acid or exposure to ultraviolet light accelerated the rate at which the liquid crystal exhibited an orientational transition from planar to perpendicular (homeotropic) alignment on surfaces presenting ammonium groups. The appearance of the homeotropic orientation of the UV-treated 4-cyano-4'-pentylbiphenyl on ammonium-terminated surfaces was dependent on the thickness of the film of liquid crystal (13-50 mum), consistent with a dipolar coupling between the liquid crystal and the electric field associated with an electrical double layer generated at the ammonium surface. Although the addition of 4-cyano-4'-biphenylcarboxylic acid or UV treatment of the liquid crystal also promoted homeotropic orientations on surfaces presenting hydroxyl groups, the orientations of the UV-treated liquid crystal on the hydroxyl-terminated surface did not change with thickness of the film of liquid crystal in the manner observed on the ammonium-terminated surfaces. The latter result indicates that the mechanism leading to homeotropic anchoring on hydroxyl-terminated surfaces is distinct from that on ammonium-terminated surfaces. Measurements performed using polarization modulation infrared reflection-absorption spectroscopy suggest that hydrogen bonding between the 4-cyano-4'-biphenylcarboxylic acid and the hydroxyl-terminated surface is responsible for the homeotropic anchoring on the surface. Finally, the orientation of the liquid crystal on methyl-terminated surfaces was not influenced by the addition of 4-cyano-4'-biphenylcarboxylic acid nor UV treatment. These results illustrate how the chemical composition of liquid crystals can be manipulated to achieve control over their ordering on surfaces that possess chemical functionality relevant to the development of liquid crystal-based sensors and diagnostic tools. We illustrate the utility of this approach by using the tailored liquid crystal to amplify and optically transduce the presence of proteins arrayed on ammonium-terminated surfaces.

Biphenyl Compounds↗
PubMed1990

Influence of chondroitin sulfate, heparin sulfate, and citrate on Proteus mirabilis-induced struvite crystallization in vitro.

Struvite crystals were produced by Proteus mirabilis growth in artificial urine, in the presence of a number of naturally occurring crystallization inhibitors. The use of phase contrast light microscopy enabled the effects of added chondroitin sulfate A, chondroitin sulfate C, heparin sulfate, or sodium citrate, on struvite crystal growth rates to be rapidly monitored as changes in crystal habit. Struvite crystals formed as a consequence of the urease activity of P. mirabilis under all chemical conditions. In the absence of inhibitor, early crystal development was marked by large quantities of amorphous precipitate, followed immediately by the appearance of rapidly growing X-shaped or planar crystals. Addition of the glycosaminoglycans, chondroitin sulfate A, chondroitin sulfate C, or heparin sulfate to the artificial urine mixture had no effect on the rate of crystal growth or appearance. When sodium citrate was present in elevated concentrations, crystal appearance was generally slowed, and the crystals assumed an octahedral, slow growing appearance. None of the added compounds had any influence on bacterial viability, pH, or urease activity. It is therefore likely that the inhibitory activity displayed by sodium citrate might be related to its ability to complex magnesium or to interfere with the crystal structure during struvite formation. From these experiments it would appear that citrate may be a factor in the natural resistance of whole urine to struvite crystallization.

Chondroitin Sulfates↗
PubMed1997

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile.

BACKGROUND/AIMS: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstone pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. METHODS: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. RESULTS: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. CONCLUSIONS: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

Apoproteins↗
PubMed2003

Strong magnetic field effect on the dissolution process of tetragonal lysozyme crystals.

Either a homogeneous or inhomogeneous magnetic field has been known to dampen the protein crystal growth. To date the mechanism is not clear. However, it was generally proposed that the magnetic field may dampen the convection in the solution, resulting in a reduced crystal growth rate and possibly a good crystal quality, similar to the case of protein crystal growth in space. To understand the mechanism of the magnetic field effect on protein crystal growth, further explorations on the magnetic field effect on protein solution, on the processes of crystal growth and dissolution, and on different crystallization (solution) systems, should be valuable. In this paper we present our recent efforts to study magnetic field effects on the dissolution processes of tetragonal lysozyme crystals under a strong magnetic field. A layer of oriented tetragonal lysozyme crystals was prepared under a temperature gradient and magnetic field, after that the crystals were dissolved by increasing the temperature of the solution. The lysozyme molecules will diffuse upwards due to the steep concentration gradient at the lower side of the cell caused by the dissolution. The evolution of the concentration in the solution was measured in-situ using a Mach-Zehnder interferometer. The results confirmed that the dissolution process of the crystals was slowed by the magnetic field. Judging from the concentration evolution versus time at different positions in the solution, we concluded that the apparent diffusion coefficient of lysozyme molecules was decreased by the magnetic field. The results were discussed using a suspended crystal model in the initial dissolution stage.

Crystallization↗
PubMed2003

Screening of nucleation conditions using levitated drops for protein crystallization.

The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-microL volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the viscous alcohol 2-methyl 2,4-pentanediol. From a digitized picture of the levitated drop volume, calculations are performed giving the concentrations of all components in the drop at any time during a "precipitation experiment". Supersaturation is the prerequisite for crystal nucleation, and protein precipitation indicates high supersaturation. A light source illuminates the levitated drop, and protein precipitation is monitored using right-angle light scattering. On the basis of these intensity measurements and the volume determination, precipitation diagrams for each crystallizing agent are constructed that give the protein/crystallizing agent concentration boundaries between the minimum and the maximum detectable protein precipitation. Guided by the concentration values obtained from such plots, when approaching the supersaturation region, separate crystallization drops are mixed and allowed to equilibrate under paraffin oil. At conditions in which microcrystals can be observed, the nucleation tendency of the macromolecule is confirmed. Optimization of crystallization conditions can then follow. Proteins tested include alcohol dehydrogenase and D-serine dehydratase. Alcohol dehydrogenase, known to crystallize easily, was used to evaluate whether the ultrasonic field inhibits nucleation. Details are given for the screening procedure of D-serine dehydratase, an enzyme earlier found to be difficult to crystallize reproducibly. The time and material-saving qualities of this method are emphasized, since a range of conditions can quickly be screened using small amounts of protein to roughly determine solubility characteristics of a protein before crystallization trials are initiated.

Alcohol Dehydrogenase↗
PubMed1996

Effect of change in shape factor of a single crystal on its dissolution behavior.

PURPOSE: To study the effect of change in the shape factor of real crystals on their dissolution behavior using a potassium dichromate crystal as a model for particulates in general. METHODS: A model geometry (parallelepiped) has been suggested for a dissolving particle. Single crystals of potassium dichromate which are monoclinic prisms were grown individually from supersaturated solutions at 40 degrees C. Dissolution studies were carried out on five such crystals in 0.1N H2SO4 at 25 degrees C and a stirrer speed of 50 +/- 1 rpm. The five crystals had different degrees of non-isometricity. Initial dimensions of the crystals were measured using image analysis techniques. The shape factor of the dissolving crystal as a function of time was obtained indirectly from the dissolution data. RESULTS: The shape factor of a single crystal changed significantly after about 50% dissolution. The nature of this change depended on the degree of non-isometricity of the crystal. The change in shape factor of the dissolving crystal was accounted for in the Hixson-Crowell cube root law, and a modified form of the cube root equation was developed. This equation for dissolution explained the observed upward curvature in the cube root law plot. CONCLUSIONS: The shape factor for any non-isometric particle cannot be considered to be constant over the dissolution event, as is commonly assumed. This change has an appreciable effect on the dissolution behavior of crystals. This study is particularly of significance for elongated shapes like needles and platelets. By the methodology described here, it was possible to determine the initial shape factor of the crystal and the intrinsic dissolution rate constant.

Crystallization↗
PubMed2003

Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride.

PURPOSE: The purpose of this work was to study the possibility of preventing mannitol crystallization in frozen solutions by using pharmaceutically acceptable additives. METHODS: Differential scanning calorimetry (DSC) and low-temperature X-ray diffractometry (LTXRD) were used to characterize the effect of additives on mannitol crystallization. RESULTS: DSC screening revealed that salts (sodium chloride, sodium citrate, and sodium acetate) inhibited mannitol crystallization in frozen solutions more effectively than selected surfactants, alpha-cyclodextrin, polymers, and alditols. This finding prompted further studies of the crystallization in the mannitol-NaCl-water system. Isothermal DSC results indicated that mannitol crystallization in frozen solutions was significantly retarded in the presence of NaCl and that NaCl did not crystallize until mannitol crystallization completed. Low-temperature X-ray diffractometry data showed that when a 10% w/v mannitol solution without additive was cooled at 1 degrees C/min, the crystalline phases emerging after ice crystallization were those of a mannitol hydrate as well as the anhydrous polymorphs. In the presence of NaCl (5% w/v), mannitol crystallization was suppressed during both cooling and warming and occurred only after annealing and rewarming. In the latter case however, mannitol did not crystallize as the hydrate, but as the anhydrous delta polymorph. At a lower NaCl concentration of 1% w/v, the inhibitory effect of NaCl on mannitol crystallization was evident even during annealing at temperatures close to the Tg' (-40 degrees C). A preliminary lyophilization cycle with polyvinyl pyrrolidone and NaCl as additives rendered mannitol amorphous. CONCLUSION: The effectiveness of additives in inhibiting mannitol crystallization in frozen solutions follows the general order: salts > alditols > polyvinyl pyrrolidone > alpha-cyclodextrin > polysorbate 80 approximately polyethylene glycol approximately poloxamer. The judicious use of additives can retain mannitol amorphous during all the stages of the freeze-drying cycle.

Calorimetry, Differential Scanning↗
PubMed1999

The effects of human urine on the adhesion of calcium oxalate crystal to Madin-Darby canine kidney cells.

OBJECTIVE: To determine the effect of human urine on the adhesion of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells in a model system in vitro. MATERIALS AND METHODS: MDCK cells were exposed to COM crystals in the presence of various human urine samples. COM crystals adhering on MDCK cells were quantified by measuring the calcium concentration using atomic absorption analysis. The inhibitory activities were estimated individually for various urine samples from healthy subjects and recurrent stone formers. RESULTS: Human urine inhibited the adhesion of COM crystals to MDCK cells, with some variations between individual samples. The most potent inhibition of crystal adhesion was expressed by the macromolecular fraction of the urine. Pretreatment of crystals with human urine before exposure to the cells significantly reduced crystal adhesion, suggesting that human urine altered the properties of the crystal surface but not the cell surface. CONCLUSION: Coating of the crystals by some component(s) of human urine might be an important physiological event in preventing adhesion or retention of crystals in the renal tubules. Although the mechanisms by which crystal adhesion is prevented are unknown, a low potential for inhibiting adhesion may be a risk factor in stone formation because it permits crystal adhesion and retention in the tubules.

Animals↗