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Face-selective adhesion of calcium oxalate dihydrate crystals to renal epithelial cells.

The interaction between the most common urinary crystal, calcium oxalate dihydrate (COD) and the surface of monkey renal epithelial cells of the BSC-1 line was investigated. The [100] face of exogenous COD crystals bound selectively and rapidly to the kidney cell surface. Cellular processes extended preferentially over the [100] face initially, and then progressively covered the crystal so that by 24 hours some crystals were observed beneath the plasma membrane. When nucleated from solution onto the surface of the cell monolayer, COD crystals oriented preferentially so that their [100] faces were in direct contact with the cell surface. In contrast, when siliconized glass was used as a substrate, nucleated COD crystals oriented randomly. Therefore, structures on the apical surface of renal tubular cells that mediate a stereospecific interaction with the molecular array presented by the [100] face of a COD crystal may be important determinants of crystal adhesion that could contribute to crystal retention and formation of kidney stones.

Animals↗

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects.

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-alpha-inhibitor (IalphaI) related proteins, alpha-1 microglobulin (alpha-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine.

Albumins↗

The effect of pH changes on the crystallization of calcium salts in solutions with an ion composition corresponding to that in the distal tubule.

The effect of pH changes on the crystallization in solutions with an ion composition assumed to correspond to that of urine in the distal part of the distal tubule was examined by recording the number and volume of crystals with a Coulter Multisizer and by studying the crystal morphology with scanning electron microscopy at different degrees of volume reduction. The experiments were carried out with 100 ml samples at different starting pH values without and with 20% of dialysed urine (dU). The number of crystals increased in response to the volume reduction. In solutions without dU, 100 or more crystals with a diameter in the range 2.4-45 microm were observed already at a volume reduction of 40% when the initial pH was 7.28. For solutions with a pH of 5.80 and 6.45 the corresponding values were 60% and 80%, respectively. In the presence of dU, an appearance of crystals was recorded at volume reductions of less than 20%. In solutions with an initial pH of 5.80 and 6.45, the crystal number was greater with dU than without; such a difference was not recorded at pH 7.28. In samples containing dU, the mean crystal volume (MCV) varied very little when the sample volume was reduced. The same was found in solutions without dU when the initial pH was 5.80 and 7.28, whereas the MCV was greater in the samples with pH 6.45. Scanning electron microscopy of solutions reduced to 30-40% of the original volume showed that calcium phosphate had formed in solutions with a starting pH of 7.28 and 6.45. In solutions with pH 5.80 calcium oxalate crystals were observed with calcium phosphate.

Calcium Oxalate↗

Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells.

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3)(1)2(1)2 with cell constants of a=b=105.6 angstrom, c=506.9 angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil. Thus in the region of 10-5.5 angstrom resolut along the c*-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 A along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 angstrom. Data analysis is under way on a data set from a PtCl4 derivative.

Amino Acid Sequence↗

Inhibition of nucleation and crystal growth of calcium carbonate by human lithostathine.

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 mumol/L, could be involved in such a control. Lithostathine in concentrations greater than 1.5 mumol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO3 crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO3 crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (Kd) = 0.9 x 10(-6) mol/L. The glycosylated amino-terminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO3 crystal growth with a Kd = 3.0 x 10(-6) mol/L, similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

Adsorption↗

Macrofiber structure and the dynamics of sickle cell hemoglobin crystallization.

Fibers of deoxyhemoglobin S undergo spontaneous crystallization by a mechanism involving a variety of intermediate structures. These intermediate structures, in common with the fiber and crystal, consist of Wishner-Love double strands of hemoglobin S molecules arranged in different configurations. The structure of one of the key intermediates linking the fiber and crystal, called a macrofiber, has been studied by a variety of analytical procedures. The results of the analysis indicate that the intermediates involved in the fiber to crystal transition have many common structural features. Fourier analysis of electron micrographs of macrofibers confirms that they are composed of Wishner-Love double strands of hemoglobin molecules. Electron micrographs of macrofiber cross-sections reveal that the arrangement of the double strands in macrofibers resembles that seen in micrographs of the a axis projection of the crystal. This orientation provides an end-on view of the double strands which appear as paired dumb-bell-like masses. The structural detail becomes progressively less distinct towards the edge of the particle due to twisting of the double strands about the particle axis. Serial sections of macrofibers confirm that these particles do indeed rotate about their axes. The twist of the particle is right handed and its average pitch is 10,000 A. The effect of rotation on the appearance of macrofiber cross-sections 300 to 400 A thick can be simulated by a 15 degrees rotation of an a axis crystal projection. The relative polarity of the double strands in macrofibers and crystals can be determined easily by direct inspection of the micrographs. In both macrofibers and crystals they are in an anti-parallel array. On the basis of these observations we conclude that crystallization of macrofibers involves untwisting and alignment of the double strands.

Crystallization↗

Calculations on crystal packing of a flexible molecule, Leu-enkephalin.

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.

Chemical Phenomena↗

Optimization of glass-ceramic crystallization based on DTA exotherm analysis.

OBJECTIVES: Crystallization of glass-ceramics is traditionally achieved through a two-stage heat treatment consisting of an isothermal nucleation stage followed by an isothermal growth stage. A method for determining a more efficient heat treatment schedule for a glass-ceramic material using a thermal analysis technique is proposed. The goal of an optimized heat treatment schedule is the production of a glass-ceramic with a desired microstructure (number of crystals per volume) in the shortest amount of time. METHODS: The proposed method involves differential thermal analysis (DTA) to measure glass crystallization exotherm characteristics which are correlated with the population density of growing crystals, and therefore, to the effectiveness of any prior heat treatment. Traditional thermal processing parameters were investigated and optimized. A method for generating a more efficient heat treatment schedule composed of a series of increasing heating rates was also demonstrated. RESULTS: The thermal analysis method measured a significant effect upon the number density of crystals generated as a function of several experimental variables. Micrographs from samples crystallized with a more time-efficient heat treatment schedule were shown to have equivalent crystal number densities compared to those crystallized with a more time consuming, traditional schedule. SIGNIFICANCE: This work demonstrated that a rapid thermal analysis method was capable of measuring the relative effectiveness of heat treatment schedules to generate crystalline populations. A novel heat treatment schedule was developed based on progressive adjustment of processing heating rates to generate the maximum crystal population in the shortest amount of time.

Ceramics↗

HREM study of irradiation damage in human dental enamel crystals.

Several phenomena have been observed during the examination of human dental enamel crystals (mainly constituted by hydroxyapatite (OHAP] by high-resolution electron microscopy (HREM) at 300 and 400 keV: orientation-dependent damage in the form of mass loss from voids or uniform destruction of crystal structure, beam-induced diffusion creating outgrowths at the crystal surfaces, recrystallization of the bulk crystal and crystallization of the inorganic components of the matrix surrounding the crystals. These beam-induced crystals have the CaO structure. The phenomena observed are most likely due to various electron-crystal interaction mechanisms (ballistic knock-on damage, electronic excitations, temperature rise, etc.). In this paper, the contribution of the ballistic process to the phenomena observed is discussed. The quantitative description of the knock-on collisions rests on the McKinley-Feshbach cross-section formula. The minimum ion displacement energies which appear in this expression have been estimated on the basis of the electrostatic ion binding energies, and the covalent bond energies if required. It is shown that hydroxyl, calcium and oxygen ions can effectively be displaced by the incident 300 and 400 keV electrons. Thus, the formation of CaO crystals by the combination of calcium and oxygen ions diffusing from their initial sites inside the OHAP lattice can tentatively be explained.

Adult↗

Crystallization of HLA-DR antigens.

The class II major histocompatibility antigens HLA-DR1, DR2, DR3, DR4, DR7 and DR8 were purified by immunoaffinity chromatography from homozygous human B lymphoblastoid cell lines. The purified, detergent-soluble molecules were cleaved with the protease papain to remove the hydrophobic transmembrane regions and cytoplasmic tails. Crystals were obtained for each of the papain-solubilized fragments. DR1 crystallized under a variety of different conditions, resulting in two different orthorhombic crystal forms, one of which diffracts as far as 3.5A. Crystals of DR2, DR3, DR4 and DR8 have the same unit cell dimensions as the DR1 crystals, and crystals of DR3 and DR4 have the same diffracting power as the DR1 crystals. The best DR7 crystals obtained thus far are hexagonal and diffract to only about 8A. Crystals of similar hexagonal form have also been observed for most of the other DR subsets.

Alleles↗

Mechanism of formation of multilayered 2D crystals of the enzyme IIC-mannitol transporter.

We have recently reported the crystallization by reconstitution into lipid bilayer structures of Enzyme IIC(mtl), the transmembrane C-domain of the mannitol transporter from E. coli. The projected structure was determined to a resolution of 0.5 nm [J. Mol. Biol. 287 5 (1999) 845]. However, further investigation proved that these crystals were multilamellar stacks instead of 2D crystals, and therefore were unsuitable for three-dimensional structural analysis by electron crystallography. Understanding the crystallogenesis of these crystals could reveal the mechanism of formation of multilayers. In the present study, cryo-electron microscopy (cryo-EM) and turbidimetry are used to study the successive steps of reconstitution of Enzyme IIC(mtl) into phospholipid-containing structures and its crystallization under different conditions. Our experimental approach enabled us to distinguish the separate steps of reconstitution and crystallization. The salt concentration especially influenced the nature of the vesicles, either half open unilamellar or aggregated multilamellar, formed during reconstitution of Enzyme IIC(mtl). The presence of DOPE and DOPC and the temperature influenced the type of lipid structures that were formed during the crystallization phase of Enzyme IIC(mtl). Cryo-EM showed that protein crystallization is closely associated with the formation of isotropic lipid (cubic) phases. We believe that DOPE is responsible for the formation of these lipid cubic phases, and that crystallization is driven by exclusion of protein from these phases and its concentration into the lamellar phases. This mechanism is inextricably associated with the formation of multilayers.

Cholic Acids↗

Basic calcium phosphate crystals stimulate the endocytotic activity of cells--inhibition by anti-calcification agents.

Pathological calcifications are associated with many medical conditions including diabetes, breast cancer, and crystals-associated osteoarthritis. The deposition of calcium-containing crystals on cells induces detrimental cellular effects and speeds up the progression of associated diseases. We carried out the present study to test the hypotheses that calcium-containing crystals may stimulate the influx of other molecules existing in the extracellular fluid disturbing normal molecular signaling and that anti-calcification agent will inhibit such endocytotic process. We found that basic calcium phosphate (BCP) crystals greatly stimulated the endocytotic activity of cells by rendering the cells more permeable and that the anti-calcification agent phosphocitrate and several others inhibited the crystals-mediated endocytosis. This is the first study reporting that the endocytotic activity of cells is affected by BCP crystals and that such endocytotic activity can be inhibited by anti-calcification agents. Since calcium-containing crystals are associated with many human diseases and in many circumstances are associated with apoptotic bodies, extracellular and matrix vesicles where DNA fragments, small peptides, and minerals are released into extracellular space, the findings reported here are important for our understanding of the complex biological effects and the potential pathological role of calcium-containing crystals in crystals-associated diseases, and for the development of disease modifying agents as well.

Breast Neoplasms↗

Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins.

Dihedral torsion angles evaluated for the phospholipid molecules resolved in the X-ray structures of transmembrane proteins in crystals are compared with those of phospholipids in bilayer crystals, and with the phospholipid conformations in fluid membranes. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid headgroup conformations in protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phospho-diester, that is characteristic of phospholipid bilayer membranes. This suggests that the lipids that are resolved in crystals of membrane proteins are not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from energetically disallowed skew conformations. This indicates a configurational heterogeneity in the lipids at a single binding site: eclipsed conformations occur also in some glycerol backbone torsion angles and C-C torsion angles in the lipid headgroups. Stereochemical violations in the protein-bound lipids are evidenced by one-third of the ester carboxyl groups in non-planar configurations, and certain of the carboxyls in the cis configuration. Some of the lipid structures in protein crystals have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in structures of the phytanyl chains associated with bacteriorhodopsin crystals are in the incorrect S-configuration.

Binding Sites↗

Retardation of crystallization-induced destabilization of PMF-in-water emulsion with emulsifier additives.

An oil-in-water (O/W) emulsion, in which the oil phase is semi-solid fat, is easily destabilized when stored below the crystallization temperature of the oil phase. Such destabilization, characterized by loss of fluidity at chilled temperature and oil-water separation after re-heating, is caused by inter-droplet bridging of fat crystals protruding out of the emulsion droplets. In the present study, we found that the simultaneous use of additives of highly hydrophobic sucrose oligoester (SOE; P-170) and highly hydrophilic SOE (P-1670) containing palmitic acid moiety remarkably retarded the crystallization-induced destabilization of the O/W emulsion that contains palm-mid-fraction (PMF) as the oil phase. Without the additives, destabilization occurred when the emulsion was cooled from 60 to 0 degrees C and kept at 0 degrees C for 1 day. Microscopic observation revealed that destabilization was caused by coalescence of the oil droplets, which was triggered by the growth of needle-shaped PMF crystals protruding out of the emulsion membranes. However, the addition of P-170 to PMF increased the crystallization temperature of PMF and at the same time retarded the destabilization. Furthermore, the simultaneous addition of P-170 and P-1670 retarded the crystallization-induced destabilization even more. Optical observation, DSC, and synchrotron radiation X-ray diffraction measurements indicated that the P-170 additive enhanced interfacial heterogeneous crystallization to form tiny PMF crystals in the droplets, and that the P-1670 additive retarded morphological change of the PMF crystals into long needle shapes in association with polymorphic transformation from alpha to beta'.

Crystallization↗

Hydrogen bonding interactions between adsorbed polymer molecules and crystal surface of acetaminophen.

The objective of this work was to investigate whether or not the hydrogen bonding interaction between polymer and crystal surface can be detected by the etching pattern changes in the presence of polymers. The (010) face of acetaminophen single crystal was used as a model solid surface. The etching patterns on the (010) face of acetaminophen crystal by water are in the directions of a- and c-axes, which are the same as the directions of the dominant attachment energies on the (010) face. In the presence of polymer, the hydrogen bonding interactions between adsorbed polymer and crystal surface can affect surface diffusion of acetaminophen molecules and change the etching patterns in the direction of a-axis, i.e., the direction of one hydrogen bond chain. Studies with 2-hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC) and poly(vinyl alcohol) (PVA) showed that polymers, which can form hydrogen bonds with acetaminophen crystal surface, can change etching patterns in the direction of a-axis. Study with Dextran suggested that if a polymer cannot form hydrogen bonds with crystal surface due to steric repulsion, it will not change the etching pattern in the direction of a-axis. Studies with poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) further confirmed that only if a polymer can form hydrogen bonds with acetaminophen on crystal surface, the etching patterns in the direction of a-axis will be affected. The study results suggest that in the presence of polymers, the etching pattern change in the direction of hydrogen bond chain, the a-axis of acetaminophen crystals, can be used to indicate the existence of the hydrogen bonding interactions between adsorbed polymers and acetaminophen crystal surface.

Acetaminophen↗

Preliminary analysis of two and three dimensional crystals of vault ribonucleoprotein particles.

Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults.

Animals↗

Use of detergents in two-dimensional crystallization of membrane proteins.

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.

Crystallization↗

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

Crystallization↗