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PubMed1980

Crystal-associated arthropathies: what's new in old joints.

Degenerative arthritis in the aged includes two major disease categories--osteoarthritis and the crystal-associated arthropathics. The crystals chiefly involved are monosodium urate (gout) and calcium pyrophosphate dihydrate (CPPD), although several others (e.g., cholesterol, brushite and apatite) have been implicated. This report illustrates how the newer diagnostic techniques such as polarized-light microscopy, analytical electron microscopy and x-ray microdiffraction have augmented knowledge concerning diseases associated with articular crystal deposition. For example, diffraction techniques are required for accurate identification of the apatite crystals found in synovial fluid effusions and in the matrix vesicles of degenerate cartilage. According to ultrastructural studies, monosodium urate crystals found in tophi, joint surfaces and effusions show a distinct morphology. Present in inactive joints, the crystal surfaces are bare; in acute gout, the crystals are covered with mucin, confirming the observation that protein binding to crystals is necessary for inflammation to proceed. CPPD disease is by far the most common crystal-associated arthropathy affeting the aged. The incidence of CPPD deposits in articular tissues increases with age but, in contrast to gout, affects both men and women. The pathogenesis of CPPD disease is a mystery, but factors under investigation include matrix abnormalities, ionic imbalances, and enzyme disorders.

Aged↗
PubMed1987

Crystal protein formed by Bacillus subtilis cells.

Electron microscopic investigation of ultrathin sections of Bacillus subtilis Cgr4 cells revealed the presence of crystal-like inclusions which were formed of spheric homogeneous subunits. The frequency of cells with a crystal-like inclusion in the culture approached 1%. The appearance of the crystal protein in cells coincided in time with spore morphogenesis. However, the process of crystal protein formation and sporulation are two alternatives: the cells either form the crystal protein or continue spore morphogenesis. Fractionation of cells in the stationary growth phase on a Percoll density gradient showed that the cells containing the crystal protein accumulated in the fraction corresponding to a 1.14-g/ml Percoll density. The cells were disintegrated by sonication, and alkaline-extracted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After sodium dodecyl sulfate-gel electrophoresis, the fraction enriched with crystal-containing cells showed practically a single band with a molecular weight of 47,000 that corresponded to the crystal-forming protein. The antigenic features and amino acid composition indicated certain similarities between the crystal-forming protein in B. subtilis Cgr4 cells and the spore coat protein.

Amino Acids↗
PubMed2007

Time required for disappearance of urate crystals from synovial fluid after successful hypouricaemic treatment relates to the duration of gout.

OBJECTIVES: To determine whether hypouricaemic treatment results in the disappearance of urate crystals from gouty joints and to define the time required. METHODS: In 18 patients with monosodium urate (MSU) crystal proven gout, and after the initiation of successful serum uric acid (SUA)-lowering treatment, an arthrocentesis of the asymptomatic signal joint (11 knees, 7 first metatarsophalangeal joints) was performed every 3 months to obtain a synovial fluid (SF) sample. The sample was then analysed for the presence of MSU crystals, and the number of crystals/400x field was noted. SUA levels and the duration of gout were also noted. RESULTS: MSU crystals disappeared from the SF of all 18 joints after reduction of SUA to normal levels. The time required for disappearance ranged from 3 to 33 months; disappearance time correlated with the duration of gout (r(s) = 0.71; p<0.01). The median number of MSU crystals in the SF samples before urate-lowering treatment was 7.5 (2.5-11) crystals/400x field, reducing to 3 (1-6.5) crystals/400x field (p<0.05) at 3 months. Crystal counts continued to decrease after 3 months. CONCLUSIONS: In gout, reduction of SUA to normal levels results in disappearance of urate crystals from SF, requiring a longer time in those patients with gout of longer duration. This indicates that urate crystal deposition in joints is reversible. Normalisation of SUA levels results in a decrease in the concentration of MSU crystals in SF in the asymptomatic gouty joints. This may partially explain the reduced frequency of gouty attacks when a patient has been treated with SUA-lowering drugs.

Adult↗
PubMed1990

Distribution of crystals in the superficial zone of elderly human articular cartilage of the femoral head in subcapital fracture.

Crystal distribution in articular cartilage of femoral heads resected at hip arthroplasty for fracture of the femoral neck was examined in 10 patients (77-91 years) by computerised image analysis of electron micrographs. Crystal content of the zenith did not differ from that of the infrafoveal region of the femoral head in the superficial 0.5 mm of the cartilage. Crystal area density (percentage area of the section occupied by crystal profiles) was higher in a zone 0-50 microns than in the subjacent zone 50-500 microns from the articular surface, particularly in the zenith of the femoral head. It was also higher on the superficial (towards the articular surface) aspect than on the deep aspect of cells in perilacunar matrix. Crystal area density was similar in lacunar matrix, perilacunar matrix, and matrix remote from cells. Matrix containing cell debris was more heavily impregnated with crystals than any other site. No difference in crystal content was observed in cartilage beneath articular surfaces which were ultrastructurally smooth or roughened. The diminishing gradient of crystal area density with depth from the articular surface, the absence of a marked spatial association with living cells, and the impregnation of sites containing cell debris suggest that crystal deposition in these cases is not due primarily to chondrocyte activity.

Aged↗
PubMed2001

Preservation of calcium pyrophosphate dihydrate crystals: effect of Mayer's haematoxylin staining period.

OBJECTIVE: To clarify the deleterious effects of Mayer's haematoxylin staining procedure which result in a decrease in, or complete loss of, the number of calcium pyrophosphate dihydrate (CPPD) crystals, and to determine the proper staining period for preserving the crystals in a histological paraffin section of articular tissues. METHODS: Paraffin sections of CPPD crystal-bearing articular tissues of six patients were stained with Mayer's haematoxylin for 3, 8, or 15 minutes, and subsequently with eosin for one minute. The specimens were examined with an Olympus BHS polarised light microscope. The pH of Mayer's haematoxylin solution was measured with a TOA pH meter. RESULTS: Positive birefringent CPPD crystals were seen clearly in all specimens stained with Mayer's haematoxylin for three minutes. The specimens stained for eight minutes showed a reduced number of crystals. No crystals were seen in the specimens stained for 15 minutes. Ordinary light microscopy showed no notable differences in the stainability of nucleus, cell membrane, and their surrounding tissues among specimens when stained with Mayer's haematoxylin for either 3, 8, or 15 minutes. The pH of Mayer's haematoxylin solution was 2.31. CONCLUSIONS: To find CPPD crystals in the paraffin sections of articular tissues, the staining period with Mayer's haematoxylin should be limited to three minutes. The longer the staining period, the greater the reduction in the number of crystals owing to the strong acidity of the haematoxylin solution. A staining period of 15 minutes causes a complete loss of CPPD crystals.

Aged↗
PubMed1999

New strategies for protein crystal growth.

Protein crystallization is the most difficult and time-consuming step in the determination of a protein's atomic structure. As X-ray diffraction becomes a commonly available tool in structural biology, the necessity for rational methodologies and protocols to produce single, high-quality protein crystals has come to the forefront. The basics of protein crystallization conform to the classical understanding of crystallization of small molecules. Understanding the effect of solution variables such as pH, temperature, pressure, and ionicity on protein solubility allows the proper evaluation of the degree of supersaturation present in protein crystallization experiments. Physicochemical measurements such as laser light scattering, X-ray scattering, X-ray diffraction, and atomic force microscopy provide a clearer picture of protein crystal nucleation and growth. This ever deepening knowledge base is generating rational methods to produce protein crystals as well as means to improve the diffraction quality of such protein crystals. Yet, much remains unclear, and the protein crystallization research community will be quite active for many years to come.

Biomedical Engineering↗
PubMed1997

Adhesion of hydroxyapatite crystals to anionic sites on the surface of renal epithelial cells.

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 20% of which contain hydroxyapatite (HA). HA crystals bound rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. Adhesion was blocked by diverse polyanions including heparin, pentosan polysulfate, polyaspartate, and polyglutamate, as well as many found in tubular fluid such as chondroitin sulfates A and B, heparan sulfate, citrate, nephrocalcin, and osteopontin. The polycations cetylpyridinium chloride and cationized ferritin, as well as the cationic dyes alcian blue, polyethylenimine, and brilliant blue R, also inhibited adhesion of HA crystals, as did specific lectins including Triticum vulgaris (wheat germ agglutinin). Anions that inhibited adhesion of crystals appeared to act on the crystal surface, whereas cations and lectins exerted their effect on the cell. Treatment of cells with neuraminidase inhibited binding of crystals, suggesting that anionic cell surface sialic acid residues function as HA crystal receptor sites that can be blocked by specific cations or lectins. Adherence of HA crystals to cells of another renal line (MDCK) and, to 3T3 fibroblasts was also inhibited by heparin, polyaspartate, alcian blue, and T vulgaris lectin, suggesting that these crystals bind to analogous molecules on the surface of different types of cells. These results suggests that the structure, quantity, and/or function of soluble anions in tubular fluid, as well as those anchored to the cell surface, could be critical determinants of HA crystal retention in the nephron and the subsequent formation of a renal stone.

Animals↗
PubMed2001

Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, NRK-52E.

Madin-Darby canine kidney (MDCK) cells have been used in research on crystal adhesion to epithelial cells. Recently, matrix proteins were identified, and studies of the genes and proteins expressed in renal epithelial cells have become active. The present study confirms the usefulness of the NRK-52E cell line, derived from the rat, in the study of attachment with calcium oxalate crystals. The calcium oxalate crystal suspension was distributed on top of the cells. After incubation, the monolayers were rinsed to remove non-associated crystals. After fixation, the association of crystals and NRK-52E cells was visualized using scanning electron microscopy. Calcium oxalate crystals were attached to the surface of NRK-52E cells. Under high magnification, many of the microvilli of the cells had elongated towards the crystals, and microvilli projections appeared to catch the crystals. The NRK-52E cell line is useful in the study of attachment between crystals and urinary epithelial cells in the kidney, especially for the regulation and analysis of genes and proteins.

Animals↗
PubMed1988

The effect of crystal structure on mouse lung inflammation and fibrosis.

In order to identify the physical and structural parameters that relate best to the membranolytic, inflammatory, and fibrotic potentials of different silicon dioxide (SiO2) and titanium dioxide (TiO2) crystals, we have studied the potential of four different SiO2 and two different TiO2 crystal structures to lyse human red blood cells and to induce pulmonary inflammation and fibrosis in mice. The crystals studied were quartz, tridymite, cristobalite, coesite, anatase, and rutile. Mice were injected intratracheally with each crystal at constant surface area. Inflammation and fibrosis were assessed 6 wk after crystal instillation by wet lung weight (lung index), protein concentration of lung lavage fluid, the level of hydroxyproline in the lung, and histologic examination. In vitro red blood cell (RBC) lysis was evaluated by incubating the crystals with 51Cr-labeled RBC and measuring the release of 51Cr into the medium. Known crystallographic data for each of the minerals were used to calculate the percent occupied volume. Biologic activity seemed to correlate with percent occupied volume, suggesting that surface molecular topology may be important in crystal-cell interactions. The crystals with more irregular surfaces and protruding oxygen atoms, which form surface pockets (quartz, tridymite, and cristobalite), showed a dramatic increase over saline controls for lung index (greater than 2 x), cell number and lavage protein concentration (greater than 4 x), and hydroxyproline level (greater than 2 x). The other more boxlike crystals (coesite, anatase, and rutile) displayed little change in these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis delta-endotoxins.

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectangular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.

Amino Acid Sequence↗
PubMed1985

Determination of the protein content of crystals formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 and modified human fibrinogen using an improved Ficoll density gradient method.

We demonstrated for several protein crystals of known protein content that the simple Ficoll density gradient method for crystal density determination as described by Westbrook (1976) often leads to quite erroneous results. In particular, the apparent density of loosely packed crystals can show a tremendous change within the first minutes of measurement. In order to derive the correct protein content the apparent crystal density must be followed as a function of time and has to be extrapolated back to the time of insertion of the crystal into the gradient. The packing densities of four novel protein crystals, formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 (two forms), and modified human fibrinogen have been determined and that of proteinase II of Crotalus adamanteus has been corrected. The C-phycocyanin crystals were found to contain (in contrast to earlier results reported by others) only one (alpha beta)-monomer, the phycocyanin-645 crystals two and three (alpha alpha' beta 2)-monomers, respectively, and the fibrinogen crystals one fibrinogen molecule per asymmetric unit.

Centrifugation, Density Gradient↗
PubMed2004

Crystallization mechanisms of hemoglobin C in the R state.

Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.

Crystallization↗
PubMed2005

The protonation-deprotonation kinetics of the protonated Schiff base in bicelle bacteriorhodopsin crystals.

In the recently published x-ray crystal structure of the "bicelle" bacteriorhodopsin (bbR) crystal, the protein has quite a different structure from the native and the in cubo bacteriorhodopsin (cbR) crystal. Instead of packing in parallel trimers as do the native membrane and the cbR crystals, in the bbR crystal the protein packs as antiparallel monomers. To date, no functional studies have been performed, to our knowledge, to investigate if the photocycle is observed in this novel protein packing structure. In this study, both Raman and time-resolved transient absorption spectroscopy are used to both confirm the presence of the photocycle and investigate the deprotonation-reprotonation kinetics of the Schiff base proton in the bbR crystal. The observed rates of deprotonation and reprotonation processes of its Schiff base have been compared to those observed for native bR under the same conditions. Unlike the previously observed similarity of the rates of these processes for cbR crystals and those for native bacteriorhodopsin (bR), in bbR crystals the rate of deprotonation has increased by 300%, and the rate of reprotonation has decreased by nearly 700%. These results are discussed in light of the changes observed when native bR is delipidated or monomerized by detergents. Both the change of the hydrophobicity of the environment around the protonated Schiff base and Asp85 and Asp96 (which could change the pKa values of proton donor-acceptor pairs) and the water structure in the bbR crystal are offered as possible explanations for the different observations.

Aspartic Acid↗
PubMed2006

Interactions between Ca2+-ATPase and the pentameric form of phospholamban in two-dimensional co-crystals.

Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase.

Animals↗
PubMed1992

Morphological evidence for biological control of urate crystal formation in vivo and in vitro.

Urate crystal formation, and subsequent gout, occurs in only a minority of hyperuricaemic subjects indicating that factors other than hyperuricaemia are involved. Biological substances (especially proteins) alter crystal growth in vitro independently of uric acid concentration. Physiological crystal formation, known to be under biological control, is characterised morphologically by uniformity of size and constraint of crystal shape. To determine whether pathological urate crystal formation is influenced by the surrounding biological milieu we examined, using scanning electron microscopy, the morphology of urate crystals formed either in vivo or in vitro. We found morphological evidence of biological control of in vivo urate crystal formation and demonstrated that those characteristics could be induced in vitro by the addition of serum, synovial fluid and certain serum proteins to the growth medium during crystal formation. Urate crystal formation is dependent, not only on the degree of hyperuricaemia, but also on the surrounding biological milieu.

Blood Proteins↗
PubMed1991

[The effect of magnesium on calcium oxalate crystal shapes generated with glycosaminoglycan].

Our previous report has shown that the shapes of calcium oxalate crystals formed in a supersaturated solution with glycosaminoglycan varied with glycosaminoglycan species. The present study was conducted to demonstrate the effect of magnesium on the shapes of calcium oxalate crystals with glycosaminoglycan by optical and scanning electron microscopy. In the presence of magnesium, the phenomenon of the plate- or sheet-like crystals parallel growing on the surface of other crystals was observed with each glycosaminoglycan and was enhanced with hyaluronic acid, chondroitin sulfate, heparan sulfate or heparin, more marked in this order. On the other hand, with heparan sulfate or heparin the stratification of the plate-like crystals was observed, whereas with hyaluronic acid or chondroitin sulfate it was not observed. Moreover, the stratification of the crystals with heparin was stimulated more markedly with increasing magnesium concentrations, and the lamella-shaped crystals at low heparin concentrations in the presence of magnesium was similar to the crystals at high heparin concentrations. These results suggest that magnesium stimulates the effect of glycosaminoglycan on calcium oxalate crystal shapes.

Calcium Oxalate↗
PubMed1999

No pathophysiologic relationship of soluble biliary proteins to cholesterol crystallization in human bile.

This study explores the pathophysiologic effects of soluble biliary glycoproteins in comparison to mucin gel and cholesterol content on microscopic crystal and liquid crystal detection times as well as crystallization sequences in lithogenic human biles incubated at 37 degrees C. Gallbladder biles from 13 cholesterol gallstone patients were ultracentrifuged and microfiltered (samples I). Total biliary lipids were extracted from portions of samples I, and reconstituted with 0.15 m NaCl (pH 7.0) (samples II). Portions of samples II were supplemented with purified concanavalin A-binding biliary glycoproteins (final concentration = 1 mg/mL) (samples III), or mucin gel (samples IV), respectively, isolated from the same cholesterol gallstone biles. Samples V consisted of extracted biliary lipids from uncentrifuged and unfiltered bile samples reconstituted with 0.15 m NaCl (pH 7.0). Analytic lipid compositions of samples I through IV were identical for individual biles but, as anticipated, samples V displayed significantly higher cholesterol saturation indexes. Detection times of cholesterol crystals and liquid crystals were accelerated in the rank order of samples: IV > V > I = II = III, indicating that total soluble biliary glycoproteins in pathophysiologic concentration had no appreciable effect. Crystallization sequences (D. Q-H. Wang and M. C. Carey. J. Lipid Res. 1996. 37: 606-630; and 2539-2549) were similar among samples I through V. Crystal detection times and numbers of solid cholesterol crystals were accelerated in proportion to added mucin gel and the cholesterol saturation of bile only. For pathophysiologically relevant conditions, our results clarify that mucin gel and cholesterol content, but not soluble biliary glycoproteins, promote cholesterol crystallization in human gallbladder bile.

Bile↗
PubMed1999

Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems.

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate>chenodeoxycholate>cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and beta-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.

Bile Acids and Salts↗