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PubMed2005

Protein nanocrystallography: growth mechanism and atomic structure of crystals induced by nanotemplates.

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant alpha-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation.

Animals↗

PubMed2005

Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli.

AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 A resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 92.2, c = 51.9 A. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (VM = 2.6 A3 Da(-1)) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering.

2-Propanol↗

PubMed2006

Structure of Bacillus halmapalus alpha-amylase crystallized with and without the substrate analogue acarbose and maltose.

Recombinant Bacillus halmapalus alpha-amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallization of BHA at room temperature in the presence of acarbose and maltose; data were collected at cryogenic temperature to a resolution of 1.9 A. It was found that the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 47.0, b = 73.5, c = 151.1 A. A maltose molecule was observed and found to bind to BHA and previous reports of the binding of a nonasaccharide were confirmed. The second crystal form was obtained by pH-induced crystallization of BHA in a MES-HEPES-boric acid buffer (MHB buffer) at 303 K; the solubility of BHA in MHB has a retrograde temperature dependency and crystallization of BHA was only possible by raising the temperature to at least 298 K. Data were collected at cryogenic temperature to a resolution of 2.0 A. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.6, b = 59.0, c = 209.8 A. The structure was solved using molecular replacement. The maltose-binding site is described and the two structures are compared. No significant changes were seen in the structure upon binding of the substrates.

Acarbose↗

PubMed1988

Occurrence of cholesterol monohydrate crystals in gallbladder and hepatic bile in man: influence of bile acid treatment.

The occurrence of cholesterol monohydrate crystals was examined and related to the degree of cholesterol saturation in gallbladder bile and hepatic bile of gallstone (GS) patients (n = 34), gallstone-free (GSF) subjects (n = 33) and GS patients treated with chenodeoxycholic acid (CDCA (n = 7) or ursodeoxycholic acid (UDCA) (n = 11) for 3 weeks prior to cholecystectomy. Twenty-five untreated GS patients (74%) and four UDCA-treated patients (40%) displayed cholesterol crystals in the gallbladder bile. Only two GSF subjects (6%) and none of the CDCA-treated patients had crystals. Half of the patients with crystals in the gallbladder bile had crystals also in the hepatic bile. Cholesterol saturation of the gallbladder bile was higher in GS (142 +/- 15%, mean +/- SEM) than in GSF patients (74 +/- 5%). Saturation was also higher in GS patients with crystals (157 +/- 20%) than in those without crystals (99 +/- 12%). Gallbladder bile was unsaturated in all CDCA- and UDCA-treated patients. The results underline the importance of the degree of cholesterol saturation for the formation of cholesterol crystals. The data also give further support to the concept that the mechanism for inducing gallstone dissolution is different for CDCA and UDCA.

Adult↗

PubMed1996

The pathogenesis of polychromatic cholesterol crystals in the anterior chamber.

PURPOSE: Polychromatic cholesterol crystal of the anterior chamber are an interesting and unusual finding. This paper examines the different pathogenetic mechanisms leading to the formation of these clinically detectable anterior chamber crystals. METHODS: Three aetiologically different cases which exhibited polychromatic crystals in the anterior chamber were reviewed. Aqueous samples were examined by wet field microscopy in all cases and additionally by electron microscopy in one of these. One enucleated globe was available for histopathology. RESULTS: Typical highly refringent cholesterol crystals were identified in the aqueous of all cases. In the first case, the cholesterol crystals developed following the breakdown of vitreous and anterior chamber haemorrhage. In the second case, the cholesterol appeared to derive from the subretinal fluid of a chronic total retinal detachment in the absence of any intraocular haemorrhage. The cholesterol crystals of the final case resulted from phacolysis and were associated with a marked neutrophil response and the presence of proteinaceous crystals consistent with the crystallins. CONCLUSIONS: Anterior chamber cholesterolosis is a secondary phenomenon that always occurs as a result of an ocular disease process. Although the prognosis is dismal for chronically diseased eyes displaying cholesterol crystals in the anterior chamber, the prognosis for eyes with phacolysis may be excellent.

Adult↗

PubMed1991

Reproducibility of synovial fluid examination for crystals.

A quality assurance survey of synovial fluid examination for crystals was performed at six teaching hospitals. Aliquots of 12 different fluids (three with no crystals, one with betamethasone crystals, four with calcium pyrophosphate dihydrate (CPPD) crystals and four with monosodium urate monohydrate [urate] crystals) were examined by all six laboratories. Four laboratories performed well (10 or more correct out of 12) but two did poorly (50% or less correct). False positive crystal identification occurred in eight of 72 samples, but all false positive reports were from two laboratories. CPPD and urate crystals were missed in seven of 24 (29%) and five of 24 (21%) samples respectively. The standard of synovial fluid examination for crystals in Sydney teaching hospitals is not uniform and in some cases appears unsatisfactory.

Betamethasone↗

PubMed1989

Protein crystal growth in microgravity.

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.

Animals↗

PubMed1968

Dry heat or gaseous chemical resistance of Bacillus subtilis var. niger spores included within water-soluble crystals.

Inclusion of spores of Bacillus subtilis var. niger in water-soluble crystals increased the resistance of the spores to dry heat and to a gaseous mixture of methyl bromide and ethylene oxide. Resistance of spores in glycine crystals to dry heat at 125 C was increased 5 to 24 times compared to unprotected spores. There appeared to be a positive correlation between the size of the crystal and the degree of resistance. The resistance to dry heat of spores included in sodium chloride crystals was about six times greater than unprotected spores. A gaseous mixture of methyl bromide (964 mg/liter) and ethylene oxide (642 mg/liter) at 37% relative humidity was ineffective in sterilizing spores enclosed within these water-soluble crystals, as was ethylene oxide alone. However, if the relative humidity was sufficiently high to dissolve the crystals during exposure to the vapor, viable-spore counts were drastically reduced or were negative. The surfaces of crystals grossly contaminated with dry spores were sterilized by exposure to gaseous ethylene oxide. Sterilization of heat-labile or moisture-labile materials with a critical requirement for sterility, as in planetary probes or drugs, may be complicated by the presence of spores in naturally occurring water-soluble crystals. This phenomenon is similar to the protection afforded spores entrapped in solid plastics.

Bacillus subtilis↗

PubMed1992

Architecture of peroxisomal alcohol oxidase crystals from the methylotrophic yeast Hansenula polymorpha as deduced by electron microscopy.

The architecture of alcohol oxidase crystalloids occurring in vivo in the peroxisomes of methylotrophic yeasts was deduced from electron micrographs of similar crystals of the Hansenula polymorpha enzyme grown in vitro. Three characteristic views of the crystal are observed, as well as single layers in the very early stages of crystal formation. The crystal is concluded to be cubical, with every octameric molecule making the same contacts with four neighbors in one plane, at right angles to its fourfold axis. The unit cell contains six octamers, in three mutually orthogonal orientations, and two large holes, which can accommodate other peroxisomal proteins involved in methanol metabolism. The crystal contains channels, connecting the holes, which allow the diffusion of relatively large molecules through the crystal. Crystal formation depends on just one contact per subunit, which may explain the fragility of the crystals.

Alcohol Oxidoreductases↗

PubMed1968

Immunological homology between crystal and spore protein of Bacillus thuringiensis.

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.

Bacillus↗

PubMed1974

Occurrence of cholesterol crystals in human bile.

The occurrence of cholesterol crystals was studied in 20 consecutive gallstone patients with functioning gallbladders. The frequency with which crystals were found rose sharply with the number of stones. Gallbladder bile was found more often to contain cholesterol crystals than hepatic bile of the same individual. Such crystals were absent in T tube drain bile from 10 consecutive choledochostomy patients, studied after the reestablishment of the enterohepatic circulation for at least five days. In gallstone patients in whom the gallbladder was visualized at cholecystography the hepatic bile contained cholesterol crystals more often than in patients with gallbladders not so visualized. In the latter patients the crystals tended to disappear after prolonged fasting. Bile analysis showed hepatic bile of patients with non-functioning gallbladders to be less lithogenic than bile in cases with functioning gallbladders. In the former group bile contained relatively more chenodeoxycholic acid than in the latter. The composition of bile with cholesterol crystals did not differ significantly from that of bile without crystals. In the final analysis it is important to identify possible factors responsible for the precipitation of cholesterol from supersaturated bile.

Bile↗

PubMed1996

Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism.

The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.

Antibodies, Monoclonal↗

PubMed2000

Regulation of renal epithelial cell affinity for calcium oxalate monohydrate crystals.

The binding and internalization of calcium oxalate monohydrate (COM) crystals by tubular epithelial cells may be a critical step leading to kidney stone formation. Exposure of MDCK cells to arachidonic acid (AA) for 3 days, but not oleic or linoleic acid, decreased COM crystal adhesion by 55%. Exogenous prostaglandin PGE(1) or PGE(2) decreased crystal binding 96% within 8 h, as did other agents that raise intracellular cAMP. Actinomycin D, cycloheximide, or tunicamycin each blocked the action of PGE(2), suggesting that gene transcription, protein synthesis, and N-glycosylation were required. Blockade of crystal binding by AA was not prevented by the cyclooxygenase inhibitor flurbiprofen, and was mimicked by the nonmetabolizable AA analog eicosatetryanoic acid (ETYA), suggesting that generation of PGE from AA is not the pathway by which AA exerts its effect. These studies provide new evidence that binding of COM crystals to renal cells is regulated by physiological signals that could modify exposure of cell surface molecules to which the crystals bind. Intrarenal AA, PGs, and/or other agents that raise the intracellular concentration of cAMP may serve a protective function by preventing crystal adhesion along the nephron, thereby defending the kidney against crystal retention and stone formation.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed2000

Adhesion of uric acid crystals to 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, 12% of which contain uric acid (UA) either alone or admixed with calcium oxalates or calcium phosphates. UA crystals bind 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. The urinary glycoproteins osteopontin, nephrocalcin, and Tamm-Horsfall glycoprotein had no effect on binding of UA crystals to the cell surface, whereas other polyanions including specific glycosaminoglycans blocked UA crystal adhesion. Specific polycations also inhibited adhesion of UA crystals and appeared to exert their inhibitory effect by coating cells. However, removal of anionic cell surface molecules with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal binding, and sialic acid-binding lectins had no effect. These observations suggest that hydrogen bonding and hydrophobic interactions play a major role in adhesion of electrostatically neutral UA crystals to renal cells, unlike the interaction of calcium-containing crystals with negatively charged molecules on the apical cell surface via ionic forces. After adhesion to the plasma membrane, subsequent cellular events could contribute to UA crystal retention in the kidney and the development of UA or mixed calcium and UA calculi.

3T3 Cells↗

PubMed2001

Methods for measuring crystallization in urolithiasis research: why, how and when?

Whereas crystalluria does not distinguish between kidney stone formers and healthy people and thus can be considered a physiologic event, kidney stone formation is a pathologic incident and reflects a specific form of biomineralization. Both single urinary crystals as well as whole kidney stones form under exquisite control of organic macromolecules. Simple crystal formation in the urinary tract is distinguished from stone formation in the kidney by the process of particle retention. The latter occurs either because nucleated crystals strongly aggregate to particles too large to pass freely through the tubules ('free particle' theory), or because crystals become abnormally adherent to tubular cell surfaces ('fixed particle' theory). Since it is impossible to mimic all the processes involved in stone formation in vitro, it is highly important to carefully chose a specific crystallization process for in vitro studies, and to select the most appropriate experimental conditions for measuring the chosen process as reliably as possible. This overview aims at critically reviewing the principles of currently available assay systems for studying crystallization processes involved in stone formation. Consensus is reached by the experts that no in vitro system really mimics what happens in renal stone formation, but that carefully designed in vitro studies will always play an important part in urolithiasis research. For such studies, it is highly important to exactly control the appropriate experimental conditions that are relevant to a specific crystallization process under investigation. Practical guidelines for researchers working with crystallization systems are provided, and it is concluded that international efforts should be made to standardize the terminology, to agree on a set of basic experimental parameters (temperature, pH, artificial urine composition), and to adopt simple tests or conditions are reference points for quality and comparative control.

Calcium Oxalate↗

PubMed2003

Pericellular matrix formation by renal tubule epithelial cells in relation to crystal binding.

BACKGROUND/AIM: Retention of crystals in the kidney ultimately leads to renal stone formation. Hyaluronan (HA) has been identified as binding molecule for calcium oxalate monohydrate crystals. The association of high molecular mass (M(r)) HA with cell surface receptors such as CD44 gives rise to pericellular matrix (PCM) formation by many eukaryotic cells in culture. Here, we study the ability of several renal tubular cell lines to assemble PCMs and to synthesize high-M(r) HA during proliferation in relation to crystal retention. METHODS: PCM assembly by MDCK-I, MDCK-II, and LLC-PK1 cells was visualized by particle exclusion assay. Metabolic labeling studies were performed to estimate the cellular production of HA. The expression of CD44 and HA was studied using fluorescent probes, and crystal binding was quantified with radiolabeled calcium oxalate monohydrate. RESULTS: PCMs were formed, and HA was expressed by most MDCK-I and some MDCK-II, but not by LLC-PK1 cells. All cell types expressed CD44 at their apical surface. MDCK-I and MDCK-II cells secreted, respectively, 14.7 +/- 1.6 and 0.5 +/- 0.2 pmol [3H]glucosamine incorporated in high-M(r) HA, whereas LLC-PK1 cells did not secrete HA. Streptomyces hyaluronidase treatment significantly decreased crystal binding (microg/cm2) to MDCK-I cells (from 8.6 +/- 0.4 to 3.9 +/- 0.9), but hardly to MDCK-II cells (from 10.2 +/- 0.2 to 9.6 +/- 0.1) or LLC-PK1 cells (from 10.2 +/- 0.8 to 9.9 +/- 0.3). CONCLUSIONS: There are various forms of crystal binding to renal tubular cells in culture. Crystal attachment to MDCK-I and some MDCK-II cells involves PCM assembly that requires high-M(r) HA synthesis. HA production and PCM formation do not play a role in crystal binding to LLC-PK1 and the majority of MDCK-II cells. It remains to be determined which form of binding is involved in renal stone disease.

Animals↗

PubMed2005

Expression profiling of crystal-induced injury in human kidney epithelial cells.

BACKGROUND: Deposition of crystals within tubular lumens is a feature of many kidney stone diseases, including crystals of calcium oxalate monohydrate (COM) in primary hyperoxaluria and of 2,8-dihydroxyadenine (DHA) in adenine phosphoribosyltransferase deficiency. Crystals are injurious to renal epithelial cells, but the molecular bases of cell injury have not been well characterized. METHODS: We used a cDNA microarray to identify the time-dependent changes in gene expression associated with the interaction of COM or DHA crystals with primary cultures of normal human kidney cortical epithelial cells. RESULTS: We observed gene expression changes that were common to both crystal types, as well as a number of crystal-specific responses. A subset of genes known to be aberrantly expressed in kidney tissue from stone formers also showed an altered expression in COM- or DHA-treated normal human kidney cortical epithelial cells. CONCLUSIONS: Our results show that cultured epithelial cells exposed to COM or DHA crystals demonstrate cellular responses that may be physiologically relevant, thus suggesting that this experimental system may be useful for elucidating the mechanisms of crystal-induced renal cell injury.

Adenine↗

PubMed1987

Role of magnesium in the growth of calcium oxalate monohydrate and calcium oxalate dihydrate crystals.

Since about 85% of synthesized calcium oxalate dihydrate (COD) crystals proved not to have changed into calcium oxalate monohydrate (COM) crystals at 30 min of incubation time at 37 degrees C when our evaluation method of the COD-to-COM ratio was being used, we made a comparative study of the inhibitory effects of magnesium, one of the well-known inhibitors of calcium oxalate stone formation, on the growth of seeded COM and COD crystals. The results demonstrated that magnesium in identical concentrations might have stronger inhibitory effects on the growth of COM crystals than on that of COD crystals and suggested that these different effects of magnesium on the growth of COM and COD crystals might arise not only from the difference between the specific surface areas of COM and COD crystals, but also from that between the direct inhibitory effects of magnesium on these two types of calcium oxalate crystal growth.

Calcium Oxalate↗