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PubMed1994

Investigations on protein crystal growth by the gel acupuncture method.

In this work we explore the possibilities of the gel-acupuncture technique, proposed previously for the growth of protein single crystals [García-Ruiz, Moreno, Viedma & Coll (1993). Mater. Res. Bull. 28, 541-546]. The main advantage of the technique is that the crystals are obtained inside an X-ray capillary and, unlike classical microdiffusion techniques, it involves a very simple and accurate technical arrangement that permits the continuous monitoring of the crystals in their growth environment. In particular, we describe the growth of single crystals of lysozyme, concanavalin A and ribonuclease A. Different starting conditions have been used to grow single crystals of these proteins into different types of capillaries at several protein and precipitating-agent concentrations. It is demonstrated that the technique works for a wide range of precipitating agents commonly used in protein crystal growth, such as large polymers (PEG 4000 and PEG 6000), organic solvents (from methanol to butanol) and salts [NaCl, (NH(4))(2)SO(4)]. The range of inner diameter of the capillaries for which the technique works correctly has been also studied. The growth process and possible crystal movement was followed by video microscopy. Lysozyme crystals up to 3.1 mm were obtained but the average maximum linear crystal sizes were 2.0 mm for lysozyme, 0.4 mm for concanavalin A and 1.2 mm for ribonuclease, respectively. The waiting times to reach such a size, measured from the set-up of the experiments, were 72 h, 10 d and 5 d, respectively. Gels of tetramethoxysilane, tetraethoxysilane, sodium silicate, agar, high-strength agar and gel-gro have been tested in relation to their mechanical properties and their chemical interaction with the reactants. Finally, we discuss briefly the advantages of the gel-acupuncture technique and plausible applications other than crystal growth.

Journal Article↗
PubMed1995

Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization.

A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation, etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non-covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure-stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure-stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have 'structure-ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure-stabilizing agents will find useful application in the field of protein crystal growth.

Journal Article↗
PubMed1997

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

Journal Article↗
PubMed1997

Crystallization of chicken egg-white lysozyme from ammonium sulfate.

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Journal Article↗
PubMed2006

Use of crystal methamphetamine among gay men in London.

AIM: To examine the use of crystal methamphetamine (crystal meth) and its association with high-risk sexual behaviour among gay men in London. DESIGN: Cross-sectional surveys using self-administered questionnaires. SETTINGS: National Health Service (NHS) out-patient human immunodeficiency virus (HIV) treatment clinic in London; NHS HIV testing/sexual health clinics in London; central London gyms. PARTICIPANTS: HIV-positive gay men attending the HIV treatment clinic in 2002-03 (n = 388); HIV-negative gay men attending the HIV testing/sexual health clinics in 2002-03 (n = 266); gay men using the gyms between January and March 2003 (n = 445), 2004 (n = 653) and 2005 (n = 494). MEASUREMENTS: Percentage of gay men who reported: (i) using crystal meth in the previous 12 months; (ii) using other recreational drugs (e.g. cocaine, ecstasy, ketamine); (iii) high-risk sexual behaviour in the previous 3 months. FINDINGS: The percentage of gay men who had used crystal meth in the previous year varied by sample (HIV treatment clinic, 12.6%; HIV testing/sexual health clinics, 8.3%; gyms, 19.5%; P < 0.001) but did not change over time in the gyms (19.8%, 20.7%, 17.8%; P = 0.5). In all samples, the majority of men used crystal meth only once or twice a year. Most crystal meth users (> 80%) had taken other recreational drugs. Crystal meth and other drug users were more likely to report high-risk sexual behaviour than other men, e.g. HIV treatment clinic sample: crystal meth users, 34.7%; other drug users, 18.9%, non-users, 10.6%, P < 0.001. Cause and effect could not be established. CONCLUSION: Among gay men in London surveyed in clinics, approximately one in 10 reported using crystal meth in the previous 12 months (HIV-positive men 12.6%; HIV-negative men 8.3%). Most men used it infrequently--only once or twice a year. There was no evidence of increasing use of crystal meth between 2003 and 2005.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Fibronectin inhibits endocytosis of calcium oxalate crystals by renal tubular cells.

BACKGROUND: Fibronectin (FN; 230 kDa) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. We recently reported that FN has a protective effect against injury of renal tubular cells by exposure to oxalate and calcium oxalate (CaOX) crystals and inhibits the adhesion of CaOX crystals to renal tubular cells. In the study presented here, we investigated whether FN has inhibitory effect on crystal endocytosis by renal tubular cells. METHODS: The inhibitory effect of FN on endocytosis of CaOX crystals by MDCK cells was examined by using a radioactivity uptake assay. Also, crystal endocytosis by cells was morphologically assessed by means of transmission electron microscopy (TEM). RESULTS: FN had inhibitory effects on CaOX crystal endocytosis by MDCK cells. The morphological TEM study showed that few crystals were taken into cells when FN was added compared to the number of crystals when FN was not added. CONCLUSION: We found that FN had the inhibitory effects on the interaction between crystals and renal tubular cells, including the adhesion or endocytosis of crystals by cells.

Animals↗
PubMed1994

Comparison of Disulfide Contents and Solubility at Alkaline pH of Insecticidal and Noninsecticidal Bacillus thuringiensis Protein Crystals.

We compared two insecticidal and eight noninsecticidal soil isolates of Bacillus thuringiensis with regard to the solubility of their proteinaceous crystals at alkaline pH values. The protein disulfide contents of the insecticidal and noninsecticidal crystals were equivalent. However, six of the noninsecticidal crystals were soluble only at pH values of >/=12. This lack of solubility contributed to their lack of toxicity. One crystal type which was soluble only at pH >/=12 (strain SHP 1-12) did exhibit significant toxicity to tobacco hornworm larvae when the crystals were presolubilized. In contrast, freshly prepared crystals from the highly insecticidal strain HD-1 were solubilized at pH 9.5 to 10.5, but when these crystals were denatured, by either 8 M urea or autoclave temperatures, they became nontoxic and were soluble only at pH values of >/=12. These changes in toxicity and solubility occurred even though the denatured HD-1 crystals were morphologically indistinguishable from native crystals. Our data are consistent with the view that insecticidal crystals contain distorted, destabilized disulfide bonds which allow them to be solubilized at pH values (9.5 to 10.5) characteristic of lepidopteran and dipteran larval midguts.

Journal Article↗
PubMed2001

Diff Quik staining method for detection and identification of monosodium urate and calcium pyrophosphate crystals in synovial fluids.

OBJECTIVE: To evaluate whether the Diff Quik (DQ) staining method might prove useful in identifying monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals on permanent mounted stained slides. METHODS: 27 synovial fluid (SF) samples obtained from the knees of 21 patients with acute CPPD disease and 6 with acute gout were studied. Wet analysis for crystal detection and identification was performed within one hour of joint aspiration. In addition, 16 inflammatory synovial effusions obtained from patients with knee arthritis induced by non-crystalline inflammatory diseases were studied. For each SF, a DQ stained slide was analysed by two of the authors trained in SF analysis. The observers were blinded to the type of crystals present in the SF. Each slide was analysed by compensated polarised as well as transmitted light microscopy. An SF was considered positive if intracellular and/or extracellular crystals were clearly identified. In addition, the observer was asked to identify the type of the crystals using compensated polarised light microscopy. Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the DQ staining method were determined. RESULTS: 51 true positive and 28 true negative cases were correctly classified (39 CPPD samples, 12 MSU samples, 28 samples of crystal unrelated arthropathies). Overall, four false positive and three false negative cases were reported. In all the false positive cases, extracellular CPPD crystals were erroneously identified, whereas CPPD crystals present in the SF were not identified in the three false negative cases. All MSU specimens were correctly diagnosed. The overall specificity, sensitivity, and accuracy using DQ stained slides for crystal confirmation were respectively 87.5%, 94.4%, and 91.9%. The PPV was 92.7% and the NPV 90.3%. In particular, the specificity, sensitivity, and accuracy for CPPD detection were 90.9%, 92.9%, and 91.9%, with a PPV of 90.7 and an NPV of 93.0%. All the MSU specimens were correctly identified, providing 100% sensitivity, specificity, accuracy, PPV, and NPV. CONCLUSIONS: Stained preparations of SF, including DQ stained smears, could provide a useful tool for delayed SF analysis suitable for quality controls, including cytological examination and crystals detection and identification.

Benchmarking↗
PubMed2001

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

OBJECTIVE: To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes. METHODS: Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from chondrocytes or HOAS treated with BCP crystals. MMP-13 secretion was identified by immunoprecipitation and MMP-1 secretion by western blot of conditioned media. RESULTS: BCP crystals caused a 4.5-fold increase in [3H]thymidine incorporation by HOAS within 20 hours compared with untreated control cultures (p< or =0.05). BCP crystals induced MMP-13 mRNA accumulation and MMP-13 protein secretion by articular chondrocytes. In contrast, in HOAS, MMP-13 mRNA induced by BCP crystals was detectable only by RT-PCR, and MMP-13 protein was undetectable. BCP crystals induced MMP-1 mRNA accumulation and MMP-1 protein secretion by HOAS. MMP-1 expression was further augmented when HOAS were co-incubated with either BCP and tumour necrosis factor alpha (TNFalpha; threefold) or BCP and interleukin 1alpha (IL1alpha; twofold). CONCLUSION: These data confirm the ability of BCP crystals to activate HOAS, leading to the induction of mitogenesis and MMP-1 production. MMP-13 production in response to BCP crystals is substantially more detectable in porcine articular chondrocytes than in HOAS. These data support the active role of BCP crystals in osteoarthritis and suggest that BCP crystals act synergistically with IL1alpha and TNFalpha to promote MMP production and subsequent joint degeneration.

Animals↗
PubMed2005

Reconstruction of the concentration field around a growing KDP crystal with schlieren tomography.

Salt concentration distribution around a potassium dihydrogen phosphate (KDP) crystal growing from its aqueous solution has been experimentally determined using a laser schlieren technique. The growth process is initiated by inserting a KDP seed into its supersaturated solution, followed by slow cooling of the solution. Fluid convection leads to a distribution of concentration around the growing crystal. The pattern and strength of convection are important factors for the determination of the crystal growth rate and quality. Experiments have been conducted in a beaker with a diameter of 16.5 cm and a height of 23 cm. A monochrome schlieren technique has been employed to image the concentration field from four view angles, namely, 0 degrees, 45 degrees, 90 degrees, and 135 degrees. By interpreting the schlieren images as projection data of the solute concentration, the three-dimensional concentration field around the crystal has been determined using the convolution backprojection algorithm. The suitability of the overall approach has been validated using a simulated convective field in a circular differentially heated fluid layer, where full as well as partial data are available. Experiments have been conducted in the convection-dominated regime of crystal growth. The noncircular shape of the crystal is seen to affect axisymmetry of the concentration field close to the crystal surface. The reconstructed concentration fields reveal symmetry of the flow field away from the growing crystal. The solute concentration contours show large growth rates of the side faces of the crystal in comparison with the horizontal faces. In this respect, the concentration profiles are seen to correlate with the crystal geometry.

Journal Article↗
PubMed2004

Effect of specific mutations in helix alpha7 of domain I on the stability and crystallization of Cry3A in Bacillus thuringiensis.

Insecticidal crystal (Cry) proteins of Bacillus thuringiensis crystallize after synthesis forming large inclusions that stabilize these toxins in the environment after cell lysis until eaten by an insect. Despite the biological importance of crystallization, little is known about the structural elements of Cry molecules that facilitate this process. We identified subdomains that affect Cry3A structure possibly through improper folding by chimeric-scanning mutagenesis, substituting short peptides of a truncated 70-kDa Cry1C molecule that does not crystallize into Cry3A, a wild-type 70-kDa molecule that crystallizes readily. Cry3A consists of three domains that contain five different blocks of conserved amino acids. Domain substitution and mutagenesis within these blocks suggested that the specific structure of block 2, which spans the junction between domains I and II, was important to the relative stability of Cry3A and subsequent crystallization. Amino acid sequences of particular importance to stability in Cry3A block 2 were identified using three substitution mutants, each spanning about a third of this block. One that consisted of Cry1C helix alpha7 yielded no detectable protein, whereas the other two produced characteristic Cry3A crystals. Specific mutations in this region showed tyrosine 268 was critical to normal stability of Cry3A and subsequent crystallization in that a mutant, Y268L, was less stable than wild-type Cry3A and failed to form a characteristic Cry3A crystal. Circular dichroism analysis showed a decrease in this mutant's alpha-helicity, indicating the importance of tyrosine 268 to the specific conformation of helix alpha7 that facilitates stability and normal crystallization.

Amino Acid Sequence↗
PubMed2003

The utility of alizarin red s staining in calcium pyrophosphate dihydrate crystal deposition disease.

OBJECTIVE: To determine the most suitable staining method for preservation and detection of calcium pyrophosphate dihydrate (CPPD) crystals in histological sections of patients with CPPD crystal deposition disease. METHODS: Paraffin sections of CPPD crystal-bearing tissues of 31 patients were stained with hematoxylin and eosin (H&E) and Alizarin red S (ARS). For H&E, the sections were treated with Mayer's hematoxylin (pH 2.3) for 5 min and with eosin alcohol (pH 4.1) for 1 min. For ARS, 1% ARS dissolved in distilled water was adjusted to pH 6.4 by adding 0.1% ammonia solution drop by drop while stirring. As controls, unstained sections were soaked in 1% citric acid monohydrate solution (CAMS, pH 2.3) for 5 or 10 min. The histological preparations were examined under a compensated polarized light using a first-order red compensator. We counted the number of weakly positive birefringent CPPD crystals in 3 high power fields (HPF, 0.272 mm2). RESULTS: CPPD crystals were seen clearly in most specimens stained with ARS, but were markedly reduced in tissue sections stained with H&E or CAMS. The number of CPPD crystals detected in sections stained by ARS (1723 +/- 683 per 3 HPF, mean +/- standard deviation) was significantly higher compared with H&E, CAMS (5 min), and CAMS (10 min) (401 +/- 374, 1022 +/- 616, and 494 +/- 636 per 3 HPF, respectively; p < 0.001, each). CONCLUSION: Standard H&E staining reduces the number of visible CPPD crystals, probably due to the strong acidity of both hematoxylin and eosin solutions, whereas the ARS stain seems to preserve a large number of CPPD crystals. The utility of ARS staining may improve the identification of CPPD crystals and contribute to a correct diagnosis of CPPD crystal deposition.

Aged↗
PubMed1983

Stone matrix as proteins adsorbed on crystal surfaces: a microscopic study.

All urinary concretions are composed of a crystalline or mineral phase and a non-crystalline phase. Both phases vary markedly in their chemical nature. There are a number of theories about the role and morphogenesis of the organic phase or matrix. In our opinion, at least a part of the matrix is formed by adsorption of urinary proteins onto crystal surfaces. It has already been shown that naturally occurring polymers have an affinity for calcium oxalate crystal surfaces and that spaces filled with amorphous substances exist between calcium oxalate monohydrate crystals of whewellite renal stones. We wanted to visualize these crystal surfaces with adsorbed organic material. We studied calcium oxalate monohydrate crystals from urinary stones, and crystal made in a crystallizer and incubated in gamma globulin or bovine serum albumin. We also studied calcium oxalate monohydrate crystals experimentally induced in rat renal tubules. They were studied using light, scanning and transmission electron microscopy with, as well as without, EDTA digestion. All crystals were surrounded by an amorphous coat which may have originated by adsorption of proteins on crystal surfaces.

Animals↗
PubMed1996

Interaction of urinary crystals with renal epithelial cells in the pathogenesis of nephrolithiasis.

Renal tubular fluid in the distal nephron is supersaturated with calcium and oxalate ions that nucleate to form crystals of calcium oxalate monohydrate (COM), the most common crystal in renal stones. It is not known how these nascent crystals are retained in the nephron to form calculi in certain individuals. Recent studies from this laboratory indicate that COM crystals can bind within seconds to anionic, sialic acid-containing glycoproteins on the apical surface of kidney epithelial cells in culture, suggesting one mechanisms whereby crystals could be retained in the tubule. Adherence of crystals to renal epithelial cells is inhibited by specific urinary anions such as glycosaminoglycans, uropontin, nephrocalcin, and citrate, each of which binds to the crystalline surface. Thus competition for the crystal surface between soluble anions in tubular fluid and anions on the apical cell surface could determine whether or not a crystal binds to the cell. Once bound, crystals are quickly internalized by renal cells in culture; reorganization of the cytoskeleton, alterations in gene expression, and initiation of proliferation can then ensue. Each of these cellular events appears to be regulated by a different set of extracellular factors. Identification of molecules in tubular fluid and on the cell surface that modulate crystal-cell interactions, as well as their mechanism of action, appears critical for understanding the pathogenesis of nephrolithiasis.

Calcium Oxalate↗
PubMed1998

Interaction of amelogenin with hydroxyapatite crystals: an adherence effect through amelogenin molecular self-association.

At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant amelogenins (rM179, rM166), the synthetic leucine-rich amelogenin polypeptide (LRAP), poly(L-proline), and phosvitin were used. It was shown that the rM179 amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin > LRAP > poly(L-proline) > rM179 > rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed.

Amelogenin↗
PubMed2002

The separation of racemic crystals into enantiomers by chiral block copolymers.

A series of chiral double hydrophilic block copolymers (DHBCs) was synthesized and employed as additives in the crystallization of calcium tartrate tetrahydrate (CaT). We found that appropriate polymers can slow down the formation of the thermodynamically most stable racemic crystals as well as the formation of one of the pure enantiomeric crystals so that chiral separation by crystallization occurs even when racemic crystals can be formed. In addition, the presence of DHBCs results in major modifications of crystal morphology, creating unusual morphologies of higher complexity. Our study demonstrates the potential application of chiral DHBCs in the control of chirality throughout crystallization, in particular for racemic crystal systems, and also shows that enantiomeric excess of one enantiomer can be maximized by the kinetic control of crystallization.

Amino Acids↗
PubMed2003

Rapid induction of peroxisome proliferator-activated receptor gamma expression in human monocytes by monosodium urate monohydrate crystals.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a member of the nuclear hormone receptor superfamily and functions as a key regulator of lipid and glucose metabolism, atherosclerosis, and inflammatory responses. This study was undertaken to evaluate the biologic role of PPAR gamma in self-limiting episodes of acute gouty arthritis. To do this, we investigated PPAR gamma expression by monosodium urate monohydrate (MSU) crystal-stimulated monocytes, and we studied the effects of PPAR gamma ligands on crystal-induced acute inflammation. METHODS: PPAR gamma expression by MSU crystal-stimulated human peripheral blood mononuclear cells was determined by reverse transcription-polymerase chain reaction and immunostaining. Expression of CD36 on monocytes was detected by flow cytometric analysis. The effects of PPAR gamma ligands on in vitro crystal-induced cytokine production and on in vivo cellular infiltration during crystal-induced acute inflammation were also investigated. RESULTS: MSU crystals rapidly and selectively induced PPAR gamma expression by monocytes. Gene expression was detected as early as 2 hours, and maximum expression was observed at 4 hours after stimulation. The induced PPAR gamma was functional, since a PPAR gamma ligand was able to up-regulate CD36 expression on monocytes. A natural ligand of PPAR gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15deoxy-PGJ(2)), significantly reduced the crystal-induced production of cytokines by monocytes. Indomethacin inhibited cytokine production only at high concentrations, and an antidiabetic thiazolidinedione (troglitazone) failed to exert significant effects. Administration of troglitazone and 15deoxy-PGJ(2) significantly prevented cellular accumulation in a mouse air-pouch model of MSU crystal-induced acute inflammation. CONCLUSION: Rapid induction of PPAR gamma expression on monocytes by MSU crystals may contribute, at least in part, to the spontaneous resolution of acute attacks of gout.

Acute Disease↗
PubMed1983

Plasma protein binding by monosodium urate crystals. Analysis by two-dimensional gel electrophoresis.

Using 2-dimensional O'Farrell gel electrophoresis, we have mapped the proteins from undiluted plasma and serum which bind to monosodium urate (MSU) crystals. More than 30 crystal-associated polypeptides were visualized, including anionic and cationic species. Proteins increased on the crystals relative to plasma included C1q, C1-r, C1-s, fibronectin, fibrinogen, and kininogen. Crystal-bound polypeptides derived from IgG, albumin, and transferrin were recovered in decreased amounts relative to plasma. Direct evidence for activation of the complement and coagulation systems in plasma was provided by the identification of crystal-associated activation fragments of C1 and kininogen. Plasmas deficient in selected proteins (e.g., C1q and IgG) were used to define the role of these proteins in such activation events and confirmed activation of C1 in immunoglobulin-deficient plasma by MSU crystals. In summary, we have described a high resolution, semiquantitative approach to analyze protein binding to crystals, have documented the complexity of crystal-plasma protein interaction, and have provided direct evidence for the binding of coagulation system proteins and binding and activation of complement by MSU crystals, in whole plasma and IgG-deficient plasma.

Blood Coagulation↗