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PubMed1996

Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences.

In model biles, five crystallization sequences are present as functions of bile salt/lecithin (egg yolk) ratio and their positions on phase diagrams are influenced by bile salt hydrophobicity, temperature, and total lipid concentration (D. Q-H. Wang and M.C. Carey. J. Lipid Res. 1996.37: 606-630). To determine whether the same pathways occur ex vivo during cholesterol precipitation from human gallbladder biles, we examined 22 cholesterol gallstone (CSI = 1.56 +/- 0.26), 4 pigment gallstone (0.69 +/- 0.06), and 4 control biles (0.85 +/- 0.22) by microscopy and lipid analytic techniques for 30 days. Temperature was varied (4-45 degrees C) to move relative compositions into adjacent pathways or supersaturated zones to test whether the same bile could be forced to crystallize in different sequences. Sequences in native bile were identical to those in model systems composed of mixed bile salts-lecithin-cholesterol mixtures, and three corresponding pathways (B, C, D; op. cit.) were observed at 37 degrees C. With increasing lecithin content, we found i) B: plate-like cholesterol monohydrate crystals appeared before arc-shaped (putatively anhydrous cholesterol) crystals which transformed via helices and tubules into plate-like crystals and no liquid crystals formed; ii) C: lamellar liquid crystals, typified by birefringent multilamellar vesicles, were detected before cholesterol monohydrate crystals, and subsequently arc, helical and tubular crystals appeared; and iii) D: precipitation of lamellar liquid crystals was followed by cholesterol monohydrate crystals and no arc crystals were detected. Added EDTA prevented calcium bilirubinate formation, but crystallization sequences in these biles were identical to those without EDTA. We conclude that i) cholesterol crystallization pathways and sequences in human gallbladder biles are identical to model biles matched for appropriate physical-chemical conditions; ii) three of the five sequences observed in model biles were found in native bile; and iii) calcium bilirubinates neither promote biliary cholesterol crystallization nor influence crystal growth.

Bile Acids and Salts↗
PubMed1984

Lipoproteins containing apoprotein B are a major regulator of neutrophil responses to monosodium urate crystals.

The inflammatory response to intraarticular urate crystals is known to be variable in gouty arthritis. One source of variability may be the modulation of cellular responses by crystal-bound proteins. We have identified three apolipoproteins among the polypeptides bound to urate crystals exposed to plasma. Identification was first based on their coelectrophoresis with polypeptides from isolated lipoproteins and diminution in the protein coat of crystals exposed to lipoprotein-depleted plasma. The apoproteins were immunochemically identified by the Western blotting technique as apoprotein A-I, apoprotein B (apo B), and apoprotein E. Because neutrophils play a central role in acute gout, we investigated the potential effects of lipoproteins on neutrophil-urate crystal interactions. Plasma profoundly inhibited urate crystal-induced neutrophil luminol-dependent chemiluminescence (CL). Lipoprotein depletion by KBr density gradient centrifugation completely abrogated the inhibitory effect of plasma on urate-induced CL. The inhibitory activity of lipoprotein-depleted plasma was restored by adding back the d less than or equal to 1.25 g/cm3 lipoprotein fraction. Plasma also inhibited urate crystal-induced neutrophil superoxide generation and cytolysis (lactic dehydrogenase loss). This inhibition was significantly diminished by lipoprotein depletion, indicating that the lipoprotein effect was not limited to CL. Lipoprotein-depleted plasma reconstituted with very low, intermediate, and low density lipoproteins (LDL) inhibited crystal-induced CL. High density lipoprotein reconstitution was without effect. Immunodepletion from plasma of all apo B lipoproteins by agarose-bound apo B-specific antibody also removed all inhibitory activity for urate-induced CL. Thus, apo B lipoproteins were shown to be the inhibitory species in plasma. Binding of apo B lipoproteins to urate crystals and inhibition of CL was also seen in the absence of other plasma proteins. In addition, the binding of whole lipoprotein particles to the crystals was verified by detection of crystal-associated cholesterol in addition to the apoprotein. The effects of LDL on urate crystal-induced CL were stimulus specific. Coincubation of urate crystals and neutrophils in the presence of 10 micrograms/ml LDL resulted in 83% inhibition. In contrast, CL responses to a chemotactic hexapeptide, opsonized zymosan, and Staphylococcus aureus were not inhibited by LDL. The effects of depletion of apo B lipoproteins on plasma suppression of urate crystal-induced CL appeared to be unique. Plasma or sera depleted of other urate crystal-binding proteins including fibrinogen, fibronectin, C1q, and IgG retained virtually all their CL inhibitory activity. Lipoproteins containing apo B are thus a major regulator of neutrophil responses to urate crystals. These lipoproteins are present in variable concentration in synovial fluid and may exert an important influence on the course of gout.

Antibodies, Monoclonal↗
PubMed2006

Precipitants and additives for membrane crystallization of lysozyme.

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.

Biotechnology↗
PubMed2006

Crystallization behaviors of poly(3-hydroxybutyrate) and poly(l-lactic acid) in their immiscible and miscible blends.

By adjusting the molecular weight of the poly(l-lactic acid) (PLLA) component in poly(3-hydroxybutyrate) (PHB)/PLLA blends, we investigated the crystallization behaviors of the two components in their immiscible and miscible 50:50 blends by real time infrared (IR) spectroscopy. In the immiscible PHB/PLLA blend, the stepwise crystallization of PHB and PLLA was realized at different crystallization temperatures. PLLA crystallizes first at a higher temperature (120 degrees C). Its crystallization mechanism from the immiscible PHB/PLLA melt is not affected by the presence of the PHB component, while its crystallization rate is substantially depressed. Subsequently, in the presence of crystallized PLLA, the isothermal melt-crystallization of PHB takes place at a lower temperature (90 degrees C). It is interesting to find that there are two growth stages for PHB. At the early stage of the growth period, the Avrami exponent is 5.0, which is unusually high, while in the late stage, it is 2.5, which is very close to the reported value (n approximately 2.5) for the neat PHB system. In contrast to the stepwise crystallization of PHB and PLLA in the immiscible blends, the almost simultaneous crystallization of PHB and PLLA in the miscible 50:50 blend was observed at the same crystallization temperature (110 degrees C). Detailed dynamic analysis by IR spectroscopy has disclosed that, even in such apparently simultaneous crystallization, the crystallization of PLLA actually occurs faster than that of PHB. It has been found that, both in the immiscible and miscible blends, the crystallization dynamics of PHB are heavily affected by the presence of crystallized PLLA.

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray crystallographic studies of wild-type human ornithine transcarbamylase and two naturally occurring mutants at position 277.

Wild-type human ornithine transcarbamylase (OTCase) and two mutants (R277Q and R277W) that cause 'late-onset' hyperammonemia were crystallized and a preliminary structure determination was carried out. The unliganded wild-type enzyme crystallizes in the cubic space group I23, with unit-cell parameters a = b = c = 203.4 A. R277Q crystallizes in two crystal forms under the same crystallization conditions. One crystal form is isomorphous to that of unliganded wild-type crystals, with unit-cell parameters a = b = c = 202.2 A. The second form also belongs to a cubic space group, P4(3)32, but has unit-cell parameters a = b = c = 139.8 A. R277W crystals are isomorphous to the second crystal form of R277Q, with unit-cell parameters a = b = c = 138.7 A. None of these crystal forms is isomorphous to other crystal forms of OTCase that have been studied. The structures in both crystal forms have been solved using molecular replacement. In the first crystal form there are two monomers in the asymmetric unit, corresponding to a solvent content of 75%. Because of its high molecular and crystal symmetry and the presence of non-crystallographic symmetry, this structure could not be solved with AMoRe or X-PLOR, but was solved successfully with COMO. There is only one monomer in the asymmetric unit in the second crystal form, corresponding to a solvent content of 62%. This structure was successfully solved with AMoRe.

Crystallization↗
PubMed2005

Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.

BACKGROUND: The interaction between renal tubular cells and crystals generated in the tubular fluid could play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. Crystals are expected to form in the renal collecting ducts, but not in the proximal tubule. In the present investigation, we studied the damaging effect of calcium oxalate crystals on renal proximal and collecting tubule cells in culture. METHODS: Studies were performed with the renal proximal tubular cell lines, porcine proximal tubular cells (LLC-PK(1)) and Madin-Darby canine kidney II (MDCK-II) and the renal collecting duct cell lines, RCCD(1) and MDCK-I. Confluent monolayers cultured on permeable growth substrates in a two-compartment culture system were apically exposed to calcium oxalate monohydrate crystals, after which several cellular responses were studied, including monolayer morphology (confocal microscopy), transepithelial electrical resistances (TER), prostaglandin E(2) (PGE(2)) secretion, DNA synthesis ([(3)H]-thymidine), total cell numbers, reactive oxygen species [hydrogen peroxide (H(2)O(2))] generation, apoptotic (annexin V and DNA fragmentation), and necrotic (propidium iodide influx) cell death. RESULTS: Crystals were rapidly taken up by proximal tubular cells and induced a biphasic response. Within 24 hours approximately half of the cell-associated crystals were released back into the apical fluid (early response). Over the next 2 weeks half of the remaining internalized crystals were eliminated (late response). The early response was characterized by morphologic disorder, increased synthesis of PGE(2), H(2)O(2), and DNA and the release of crystal-containing cells from the monolayers. These released cells appeared to be necrotic, but not apoptotic cells. Scrape-injured monolayers generated even higher levels of H(2)O(2) than those generated in response to crystals. During the late response, crystals were gradually removed from the monolayers without inflammation-mediated cell death. Crystals did not bind to, were not taken up by, and did not cause marked responses in collecting tubule cells. CONCLUSION: This study shows that calcium oxalate crystals cause acute inflammation-mediated necrotic cell death in renal proximal tubular cells, but not in collecting tubule cells. The crystal-induced generation of reactive oxygen species by renal tubular cells is a general response to tissue damage and the increased levels of DNA synthesis seem to reflect regeneration rather than growth stimulation. As long as the renal collecting ducts are not obstructed with crystals, these results do not support an important role for crystal-induced tissue injury in the pathophysiology of calcium oxalate nephrolithiasis.

Animals↗
PubMed1998

[The correlation between tissue injury and calcium oxalate crystal production in rat's lung with experimental Aspergillus niger infection].

It is well known that calcium oxalate crystals (crystal) are commonly found in Aspergillus niger (A. niger) infection site. However, there have been no pathological reports of crystal production and tissue injury using an animal model. I experienced a case with pulmonary aspergilloma and infiltration shadow of the lung that I guessed resulted in crystal-induced inflammation. I performed animal experiments to investigate the possibility of crystals to cause tissue injury. Two different types of immunosuppressed rat models; i.e. cortisone acetate (CA) and CA + cyclophosphamide (CPM) induced-immunodeficiency models (CA model, CA + CPM model), were used for the experiments. In both models, rats were infected with A. niger. Then, the lungs were removed and examined histopathologically. The crystals were rarely found in both models on the third day after inoculation and it increased as time passed. However, more growth of hyphae and more production of crystals were observed in the CA + CPM model. In the CA model, crystal depositions in the bronchial epithelium and erosion of the bronchial epithelium adjacent to the crystals were observed. Crystal depositions in the bronchial epithelium were also seen in the CA + CPM model. In this lesion, severe erosions of the bronchial epithelium and invasion of A. niger mycelium into the tissue were observed. These findings suggest, the crystals in the bronchial epithelium may expand and destroy the cells around them. Therefore, I think that the crystals can induce tissue injury. However, in alveolar portions, depositions of crystals were seen mainly in the margin rather than in the central part of mycelial growth, but no findings indicating injury of the alveolar epithelial cells by crystals were seen. Therefore, possibility of crystals to cause tissue injury in the alveolar portion is not yet clear.

Animals↗
PubMed1998

[The effects of human urine on the adhesion of calcium oxalate crystal to renal tubular cells].

PURPOSE: We examined the effects of human urine on the adhesion of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells in vitro. METHODS: Quantitative assay of COM crystal adhesion to MDCK cells: MDCK cells were exposed to COM crystal suspension for 5 minutes. Various urine samples were added in the COM crystal suspension. The adherent COM crystals on the MDCK cells were dissolved by 5 N hydrochloric acid. Calcium concentration of the solution was measured by atomic absorption analysis to quantify the volume of adherent COM crystals. This assay was applied for the experiments as follows. (1) Effect of human urine on COM crystal adhesion. (2) To investigate whether human urine inhibited COM crystal adhesion by acting on the crystal surface or on the cell surface. (3) Isolation of the substance from human urine that inhibits COM crystal adhesion. (4) Comparison of the inhibitory activities of the urine between from stone formers and healthy controls. RESULTS AND CONCLUSION: (1) Human urine had a strong inhibitory effect on COM crystal adhesion to MDCK cells. (2) The capacity of human urine to inhibit adhesion of COM crystals to MDCK cells was shown to be mediated by their ability to act on the crystal surface. (3) We isolated a macromolecular fraction (MW 60,000) that had strong capacity to inhibit cellular adhesion of COM crystals through ion exchange and gel filtration chromatography. (4) Nine urine samples from stone formers demonstrated extreme low inhibitory activities, which supposed us that the inhibitory capacity of the urine against crystal adhesion might be one of the risk factors in kidney stone formation.

Animals↗
PubMed1998

The effect of protein impurities on lysozyme crystal growth.

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ¿110¿ and ¿101¿ lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.

Animals↗
PubMed1994

Quantitative analysis in the characterization and optimization of protein crystal growth.

Protein crystal growth often depends on the combination of many different factors. Some affect protein solubility directly; others may act indirectly by causing conformational changes. Systematic characterization of these factors can be important for generating good crystals. It can also provide useful insight into the biochemical behavior of the protein being crystallized. Here we focus on statistical methods to achieve these two objectives. (1) Characterization of a protein system by analyzing patterns of crystal polymorphism under different levels of biochemical parameters, such as ligands and pH. Tests of the reproducibility of crystal growth experiments indicate that quantitative scales of crystal quality can be statistically significant. Analysis of variance for a replicated, full-factorial design in which four factors were tested at two levels has been used to demonstrate highly significant, biochemically relevant, two-factor interactions strongly implicating pH and ligand-dependent conformational changes. (2) Optimization of crystal growth via response-surface methods. 'Minimum predicted variance' designs provide for efficient response-surface experiments aimed at constructing quadratic models in several dimensions. We have used such models to improve crystal size and quality significantly for three forms of Bacillus stearothermophilus tryptophanyl-tRNA synthetase. In one case we can now avoid having to increase the size by repeated seeding, a difficult procedure that also produces unwanted growth of satellite crystals. Graphs of two-dimensional level surfaces reveal a number of ridges, where the same result is obtained for many combinations of the factors usually varied when trying to improve crystals. An important inference is that it may be better to sample simultaneously for the effects of protein concentration and supersaturation. For a system involving only one crystallizing agent, supersaturation can be approximated as the product of protein and precipitant concentrations. Use of this search direction significantly improves the performance of response-surface experiments. Advantages of growing crystals at stationary points of their response surfaces include better crystals and higher reproducibility, since crystal growth at stationary points is insulated from the deleterious effects of experimental fluctuations. This arises because the derivatives of the response are by definition zero with respect to the experimental variables. Quantitative analysis of appropriately designed crystal growth experiments can thus be a powerful way to characterize complex and interacting biochemical dependencies in macromolecular systems and optimize parameters important to the crystallography.

Journal Article↗
PubMed1997

Histological observations of the adhesion and endocytosis of calcium oxalate crystals in MDCK cells and in rat and human kidney.

Adhesion and/or endocytosis of calcium oxalate crystals to the three kinds of tubular cells (Madin-Darby canine kidney (MDCK) cells, rat and human kidney) were demonstrated morphologically to presume the initial formation of kidney stone. After removal of the nonadhesion crystals, the cells were subsequently recultured in the vertical position. At various times thereafter, the interactions between COM crystals and MDCK cells were evaluated morphologically by SEM. COM crystals adhered to the surface of MDCK cells immediately, and the crystals were then endocytosed. The microvilli of the cells appeared to play an important role in these processes. At later times, some complexes that consist of aggregated calcium oxalate crystals and cell debris were observed sporadically. Kidney tissues were obtained from male Sprague-Dawley rats which were injected with sodium oxalate intraperitoneally. Experimentally induced calcium oxalate crystals were evaluated histologically using polarized light microscopy. Some crystals in the cortical portion were attached to the tubular epithelium or internalized into the luminal membrane. Whereas in the papilla, the aggregated crystals were observed lying free from the degenerated tubular lumen along with the cell debris. Human kidney tissues were obtained from 38 patients with calcium oxalate nephrolithiasis who underwent nephrolithotomy or partial nephrectomy before the era of ESWL. The specimens were examined for calcium crystals within the tubular lumen, attached to the tubular walls or internalized into the tubular cells, by polarized light microscopy. Approximately 50% of the specimens observed crystals attached to the tubular cell epithelium and some of them were seen inside the tubular cells. In conclusion, crystal-cell interaction resulted in movement of crystals from the lumen into the cells by an action of microvilli from the results of MDCK cells. However, it was not clear from the results in rats or human kidney tissue that crystal adhesion and/or endocytosis might be vital in the crystal growth in the kidney.

Animals↗
PubMed1996

Behavior of various cholesterol crystals in bile from patients with gallstones.

Besides classical plate-like cholesterol monohydrate crystals, a variety of crystal shapes have recently been described in model biles but their relevance for human gallstone formation is unknown. We therefore studied crystallization behavior in gallbladder bile from cholesterol stone patients (54 untreated, 13 ursodeoxycholate-treated) and 6 pigment stone patients. Bile preparation by ultrafiltration or ultracentrifugation left biliary lipid composition unchanged but plates and their aggregates, and arcs and needles crystallized more extensively while spirals and tubules crystallized less extensively in ultra-centrifuged bile than in ultrafiltered bile. Plates, aggregates, and arcs/needles were seen in 90 percent, 36 percent, and 18 percent of the cases respectively of fresh unfiltered biles of untreated cholesterol stone patients, while spirals and tubules were always absent. In ultrafiltered biles arcs/needles, plates and aggregates progressively developed as persistent forms. Spirals and tubules occurred transiently and were associated with increased deoxycholic acid (+41 percent, P = .039) and with more extensive cholesterol crystallization. Rate/extent of crystallization of all crystal forms was higher (P < .0001) for multiple than solitary cholesterol stone patients. Ursodeoxycholate-treated patients had atypical platelike cholesterol crystals in fresh unfiltered biles that decreased in size at prolonged observation and in 2 cases even dissolved after 15 and 20 days. No crystals ever developed in ultra-filtered bile of ursodeoxycholic acid (UDCA)-treated patients during 21 days. Pigment stone patients seldom developed crystals. Thus, plates, aggregates and arcs/needles are persistent forms with high crystallization rate in multiple cholesterol stone patients. Tubules and spirals are transient forms that are associated with more extensive crystallization. Patients treated with ursodeoxycholate often have atypical crystals in their fresh bile.

Adult↗
PubMed1993

Stabilization of protein crystals by electrostatic interactions as revealed by a numerical approach.

We developed a novel algorithm to solve numerically the Poisson-Boltzmann equations under a periodic boundary condition. By employing this algorithm to calculate the electrostatic potentials in two different types of protein crystals, a bovine pancreatic trypsin inhibitor (BPTI) orthorhombic crystal and a pig-insulin cubic crystal, the energy contributions of the electrostatic interactions to the crystals' stability were evaluated. At a high ionic strength, the condensed state of proteins in the crystal was stabilized electrostatically compared with that isolated in dilute solution because the attractive electrostatic interactions between neighboring protein molecules overcame the repulsive forces that originated from the same net charges of the equivalent protein molecules. On the other hand, at a low ionic strength the electrostatic interactions destabilized the crystalline state of both proteins, although a different dependence on the ionic strength was found between them. Here, the insulin crystal was more stable than the BPTI one because of the higher charge density in the BPTI crystal. In all of the solvent ionic strengths investigated, the attractive electrostatic interactions between charge pairs separated by less than 5 A on the respective protein molecules prominently stabilize the protein crystals. Therefore, two protein molecules in the crystals are oriented to compensate each other for their opposite charges on the surfaces. We also found a specific role for bound phosphate ions in the stabilization of the BPTI crystal, based on comparison of the electrostatic energies of the two crystals with and without the ions. By determining the contribution of each atomic charge in the crystals to the electrostatic energy, it was revealed that several electrostatic pairs specifically contributed to the crystal's stability. On the basis of our numerical calculation results, we propose a new method to design protein molecules that adopt stable crystals by replacing destabilizing residues with stabilizing ones and by introducing specific hydrogen bonds or salt bridges between neighboring protein molecules.

Algorithms↗
PubMed1994

Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Animals↗
PubMed1998

A general module for RNA crystallization.

Crystallization of RNA molecules other than simple oligonucleotide duplexes remains a challenging step in structure determination by X-ray crystallography. Subjecting biochemically, covalently and conformationally homogeneous target molecules to an exhaustive array of crystallization conditions is often insufficient to yield crystals large enough for X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemically informative structures. We reasoned that a well-folded RNA molecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed contacts are made. We have employed the GAAA tetraloop/tetraloop receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 A resolution.

Base Sequence↗
PubMed1993

Bovine gallbladder mucin accelerates cholesterol monohydrate crystal growth in model bile.

BACKGROUND: Gallbladder mucin accelerates cholesterol crystal nucleation, an early step in the pathogenesis of gallstones. To examine the role of gallbladder mucin in postnucleation gallstone maturation, the influence of mucin on cholesterol monohydrate crystal growth was studied in a novel model system. METHODS: Cholesterol crystals of a uniform size were incubated in model biles at 37 degrees C with varying cholesterol saturation indices. Crystal size was quantitated by measuring the width and length of individual crystals under polarizing light microscopy and calculating average crystal area. RESULTS: Crystal growth was dependent on the degree of cholesterol supersaturation of bile. Bovine gallbladder mucin (0.5-8 mg/mL) accelerated crystal growth in supersaturated model bile in a concentration- and time-dependent fashion compared with control incubations with bovine serum albumin or model bile alone (P < 0.05). Cholesterol crystal growth was accompanied by a progressive decrease in cholesterol saturation and an increase in total cholesterol crystal mass. Crystal growth was also accompanied by a decrease in total crystal number, suggesting net transfer of cholesterol to larger crystals. CONCLUSIONS: The acceleration of cholesterol crystal growth by gallbladder mucin may be of pathophysiological importance in the postnucleation maturation of cholesterol gallstones.

Animals↗
PubMed1998

Biliary secretory immunoglobulin A is a major constituent of the new group of cholesterol crystal-binding proteins.

BACKGROUND & AIMS: Recently we described a new group of lectin-bound biliary proteins that bind to cholesterol crystals, modify crystal morphology, and inhibit cholesterol crystallization. The aim of the current study was to characterize and identify individual members of this group of cholesterol crystal-binding proteins. METHODS: Crystal-binding proteins were purified from human gallbladder bile by lectin affinity chromatography and preparative gel electrophoresis. Purified crystal-binding proteins were characterized by using cholesterol crystal-growth assays, immunoblotting, and amino acid analysis. For comparison, identified biliary proteins were isolated from gallbladder bile by lectin affinity and immunoaffinity chromatography. RESULTS: The individual crystal-binding proteins with molecular weights of 74, 63, and 28 kilodaltons inhibited cholesterol crystallization in a dose-dependent manner (2.5-10 micrograms/mL). Immunoblotting with specific antibodies and N-terminal amino acid sequences revealed that the 74-kilodalton crystal-binding protein is the secretory component, the 63-kilodalton protein is the heavy chain, and the 28-kilodalton protein is the light chain of human secretory immunoglobulin (Ig) A. Isolated biliary IgA showed a potent inhibitory effect on cholesterol crystallization in model bile even at levels less than physiological concentrations (1-100 micrograms/mL). CONCLUSIONS: Biliary secretory IgA is a major constituent of the previously described group of cholesterol crystal-binding proteins. Crystal-binding IgA may be an important modulator of crystal agglomeration into stones and stone growth in vivo.

Amino Acid Sequence↗
PubMed1997

Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation.

BACKGROUND/AIMS: Cholesterol crystallization in a dilute, bile salt-rich model bile is a multiphase process in which early filamentous crystals gradually transform to classical cholesterol monohydrate plates. The pertinence of similar transformations in more complex model systems or native bile is, however, unclear. The aim of the present study was to characterize and monitor cholesterol crystallization in a model bile of physiological relevance. METHODS: A supersaturated model bile was prepared with a lipid composition (18 mM cholesterol, 37 mM lecithin, 120 mM taurocholate) that was derived from analyzing 10 gallbladder biles from cholesterol gallstone patients. Cholesterol crystallization was followed by light and electron microscopy, and sequential density gradient analysis of cholesterol-containing precipitates. RESULTS: During cholesterol crystallization a reproducible sequence of events was recorded. First (T<18 h), cholesterol-rich vesicular and multilamellar structures (density 1.005-1.015 g/ml) were observed. Later, (T>60 h) filamentous, helical, tubular (density 1.015-1.04 g/ml) and plate-like (density 1.04-1.06 g/ml) cholesterol crystals appeared. The concentration of crystals increased gradually, while bilayer structures became desaturated with cholesterol and disappeared, and early crystal forms were replaced by plates. Eventually (T>25 days) only classical plate-like cholesterol monohydrate crystals were present. Exposure of cholesterol-containing precipitates to micellar (100 mM) deoxycholate dissolved the bilayer structures but not the crystals. CONCLUSIONS: These data demonstrate that cholesterol crystallization in a physiologically relevant model bile is a multiphase process consisting of a sequence of transitions from vesicular and multilamellar structures to early crystal forms and to classical plate-like cholesterol monohydrate crystals. These transitions are associated with increasing density and decreasing phospholipid content of cholesterol precipitates. We suggest that time-lapse density gradient ultracentrifugation is a useful method for investigating and quantitating the process of cholesterol crystallization and factors that influence this process in bile.

Animals↗