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At least 1,261 records · Page 70Linked to original sources
PubMed2002

Calcium oxalate monohydrate crystal binding substance produced from Madin-Darby canine kidney cells.

BACKGROUND: The interaction between kidney urothelium and crystals is a critical event in the growth of renal calculi. When studying calcium oxalate monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells in culture, we observed that crystals also attached to areas on the coverslips devoid of cells. This phenomenon could be the result of substances produced by the cells that adhere to the glass and subsequently bind COM crystals. We investigated the characteristics of this COM binding substance. METHODS: Media was collected from cultures of MDCK cells (conditioned media) and proteins were separated by high performance liquid chromatography. The molecular weights and purity of isolated proteins were determined by polyacrylamide gel electrophoresis. The conditioned media and each separated fraction were applied to glass and to MDCK cells and COM-binding ability determined using 14C-labeled crystals. The binding of radio-labelled calcium oxalate dihydrate, brushite, uric acid, and apatite to coverslips were also studied. RESULTS: Fourteen times more COM bound to coverslips incubated with conditioned media than those with control media. The molecular weight of the protein bound to the glass was determined to be 200 kDa. The COM crystals binding to this protein was 1.5 micro g/ng. Other crystals bound to a lesser extent. The incubation of cells with this protein inhibited COM binding by 39%. CONCLUSION: The MDCK cells produce a 200-kDa protein that has a high binding affinity for COM crystals. This protein binds to glass and is responsible for crystal binding to areas devoid of cells. This protein also has an inhibitory effect on COM binding to MDCK cells in culture.

Animals↗
PubMed2005

Kinetics of two-step nucleation of crystals.

When the nucleation of a stable crystalline phase directly in a supersaturated old phase is greatly retarded, the crystal nuclei might nucleate within faster-forming particles of an intermediate phase. Here we present a theoretical investigation of the kinetics of this two-step nucleation of crystals and derive general expressions for the time dependence of the number of crystals nucleated within the particles of the intermediate phase. The results reveal that crystal nucleation can be strongly delayed by the slow growth of the particles and/or by the slow nucleation of the crystals in them. Furthermore, the linear part of the time dependence of the number of nucleated crystals is determined by the formation rate of the intermediate particles. This is in contrast with the one-step nucleation of crystals when this linear part is determined by the rate of crystal nucleation directly in the old phase. Criteria are proposed for distinction between the one- and two-step nucleation mechanisms, based on the supersaturation dependence of the delay time for nucleation. The application of the theoretical approach to the analysis of experimental data on the nucleation of crystals and other ordered aggregates of protein and other soluble materials is discussed.

Journal Article↗
PubMed2006

Confined crystallization of cylindrical diblock copolymers studied by dynamic Monte Carlo simulations.

We report dynamic Monte Carlo simulations of polymer crystallization confined in the cylindrical microdomains of diblock copolymers. The microdomains were prepared via spontaneous microphase separation from homogeneous melt, and the major component was then frozen in a vitreous amorphous state to make a hard confinement to the crystallization of the minor component. We found that during the isothermal crystallization at high temperatures, crystal orientations are dominantly perpendicular to the cylinder axis at the early stage of crystal nucleation and remain to the final state; while if the block junctions are broken before crystallization, crystal orientations are dominantly parallel at the early stage of crystal nucleation, and eventually other orientations take the place of parallel preferences. Analysis of bond orientations in the heterogeneous melts demonstrates the microscopic origin of oriented crystal nucleation.

Journal Article↗
PubMed2005

NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline.

In the Joint Center for Structural Genomics, one-dimensional (1D) 1H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D 1H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1D 1H NMR spectra, the proteins are classified into four groups, "A" to "D." A-type proteins are candidates for structure determination by NMR or crystallography; "B"-type are earmarked for crystallography; "C" indicates folded globular proteins with broadened line shapes; and "D" are nonglobular, "unfolded" polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D 1H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C- and D-type proteins.

Animals↗
PubMed1982

Oriented properties of the chlorophylls: Electronic absorption spectroscopy of orthorhombic pyrochlorophyllide a-apomyoglobin single crystals.

The orientations of the transition dipole moments in chlorophyll (Chl) are among the most useful spectroscopic properties for determining macromolecular architecture in photosynthetic complexes; however, the relationships between these orientations and the Chl molecular geometry are unknown. In order to solve this problem, we have prepared single crystals of the synthetic 1:1 complex between pyrochlorophyllide a and apomyoglobin. The protein crystallizes readily in the orthorhombic (B) form, space group P2(1)2(1)2(1), and the unit cell dimensions are determined to be within 0.5% of those for native MetMb crystals of the same type. These green crystals are highly dichroic, and the strong absorption along the crystallographic a axis in the Q(y) band is red-shifted by about 9 nm, relative to the corresponding feature in a solution of the protein. Although the crystal structure for native Mb in this space group has not been determined, the direction cosines of the heme normal relative to the crystal axes have been measured. By using these values, an appropriate trigonometric analysis, and the measured polarized single-crystal spectra, the orientation of the Chl transition dipole moment for the Q(y) transition can be specified relative to the crystal axes. With the completion of the protein crystal structure, this result will lead directly to the orientations of the optical transition dipole moments relative to the molecular geometry. The effects of vibronic coupling and the protein environment on the absorption properties of Chl are discussed in detail.

Journal Article↗
PubMed1998

Molecular mechanism of basic calcium phosphate crystal-induced activation of human fibroblasts. Role of nuclear factor kappab, activator protein 1, and protein kinase c.

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor kappaB and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts. In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications.

3T3 Cells↗
PubMed2002

COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells.

Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 microg/cm(2)) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. We also show that specific inhibition of p38 MAPK by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) or by overexpression of a dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells.

Animals↗
PubMed1990

Crystallization and preliminary crystallographic study of bacterial alpha-amylases.

Crystallization of bacterial alpha-amylases has been achieved by the hanging-drop vapor diffusion method. The crystals of Bacillus licheniformis and B. licheniformis 584 amylases are isomorphous to each other. The crystals of B. licheniformis amylase belong to the tetragonal system, space group P4(2)2(1)2 with cell dimensions of a = 119.3 and c = 85.4 A. The asymmetric unit contains one molecule of amylase, with a volume per molecular mass, Vm, of 2.75 A3/Da. The crystals of B. licheniformis and B. licheniformis 584 amylases diffract beyond 2.5 A resolution and are suitable for X-ray diffraction analysis. The crystals of B. amyloliquefaciens amylase are orthorhombic, and have space group C222(1), with cell dimensions of a = 154, b = 298, and c = 90 A. The asymmetric unit contains three to five molecules. In the crystallization of B. licheniformis and B. licheniformis 584 amylases, the addition of EDTA was indispensable to obtain large single crystals, while it had an adverse effect on the crystallization of B. amyloliquefaciens amylase, producing a large amount of small crystals.

Bacillus↗
PubMed2000

Electron microscopy of satellite tobacco mosaic virus crystals: metal-coated, negatively stained and stereo pairs.

Highly purified virions of satellite tobacco mosaic virus (STMV) were found to crystallize at relatively low concentrations (300-500 microg ml(-1)) in pure water. Small crystals of these preparations were examined in the transmission electron microscope after either being rotary shadowcast with metal or negatively stained with 4% uranyl acetate. Stereo views were also obtained of both types of preparations. Stereo pairs of metal-coated crystals provided good three-dimensional images. When stereo pairs of negatively stained crystals were printed from second negatives, they provided striking images although the three-dimensional aspect was not so pronounced. Images of both types of preparations were compared with a computer-generated model of the virus. This model was based on data obtained in earlier X-ray diffraction crystallographic studies. Measurements of crystal axes on the EM images were somewhat lower than those of the computer model. It is assumed the reason for this is the dehydration of crystals during preparation for electron microscopy. The EM images did verify the type of crystal lattice determined in the X-ray diffraction studies. Conversely, knowing the exact unit cell parameters and the distribution of virions in the crystal from X-ray diffraction data aids in the further interpretation of electron micrographs of virus crystals.

Crystallography↗
PubMed2006

Basic calcium phosphate crystals: pathways to joint degeneration.

PURPOSE OF REVIEW: Basic calcium phosphate crystals have long been associated with rheumatic syndromes. Although an understanding of the molecular mechanisms involved in generating these pathological effects has been significantly advanced in recent years, it is still incomplete. RECENT FINDINGS: Basic calcium phosphate crystals have been shown to increase prostaglandin E(2) production in human fibroblasts, mediated by the induction of both cyclooxygenases 1 and 2. Basic calcium phosphate crystals have also been found to upregulate IL-1beta in fibroblasts and chondrocytes. The upregulation of inducible nitric oxide synthase and stimulation of nitric oxide production in chondrocytes by octacalcium phosphate crystals has been demonstrated. The involvement of protein kinase C isoforms in basic calcium phosphate crystal-mediated matrix metalloproteinase 1 and 3 expression in human fibroblasts has been clarified. Two pathways are involved: protein kinase Calpha mediates the calcium-dependent pathway, whereas protein kinase Cmu activates the extracellular-regulated kinase pathway in a calcium-independent cascade. In addition, basic calcium phosphate crystals activate the transcription factor Egr-1, an effect that may contribute to the mitogenic effect of these crystals on fibroblasts. SUMMARY: Recent findings have emphasized the potential for basic calcium phosphate crystals to stimulate the production of a variety of inflammatory mediators such as prostaglandin E(2), nitric oxide, IL-1beta and matrix metalloproteinases, and have helped to elucidate the mechanisms of these effects. Such advances are essential for the ongoing search for effective therapies for basic calcium phosphate crystal-associated diseases.

Calcinosis↗
PubMed1999

Crystallization by settling in suspensions of hard spheres.

We examine crystallization by settling for two different model hard-sphere suspensions. Sedimentation velocities, internal shock velocities, and crystal growth velocities are measured. Dynamic light scattering experiments measure volume fraction profiles in fluid phases, while Bragg scattering experiments determine volume fraction profiles in crystal phases. Centrifugation experiments determine the particle Péclet number above which samples will not crystallize. The sedimentation velocities, as a function of volume fraction, agree with other "hard-sphere" data. Remarkably, the value of the reduced crystal growth velocity (approximately 0.075) obtains for two orders of magnitude of the particle Péclet number. Kynch theory provides an adequate description of the data in the fluid phase, but is less adequate for volume fraction profiles in the crystal phase. The crystals in the dense sediment are compressed more along a vertical axis relative to the horizontal axis. Predicted Wilson-Frenkel crystal growth velocities, calculated using known hard-sphere equations of state and a short-time self-diffusion constant, rationalize the measured crystal growth velocities.

Journal Article↗
PubMed2002

Acoustic crystal thermodynamic integration method.

The acoustic crystal thermodynamic integration method is a generalization of the Einstein crystal method developed by Frenkel and Ladd. The name is derived from the acoustic branches of the phonon spectrum of the reference system. The method is designed to calculate the Helmholtz free energy of classical solid phases using Monte Carlo or molecular dynamics simulations. It has several advantages over the Einstein crystal method. For large systems, the Einstein crystal method suffers from very long correlation times near the zero coupling limit because the reference system breaks the overall translational symmetry of model systems. The acoustic crystal method does not break translational symmetry, so correlation times for the acoustic crystal are small. This makes the acoustic crystal method superior to the Einstein crystal method for large system sizes. Also the acoustic crystal method does not artificially introduce long-range order in low-dimensional systems.

Journal Article↗
PubMed2000

Increased resolution data from a large unit cell crystal collected at a third-generation synchrotron X-ray source.

A third-generation synchrotron source was used to collect data from crystals with a very large unit cell. There was an increase in the effective resolution of the data from 5 to 3.5 A. Data were collected on crystals of HK97 mature empty capsids, space group P2(1), with unit-cell parameters a = 580, b = 625, c = 790 A, beta = 90.0 degrees. Like other crystals with very large unit-cell dimensions, the intensity falls off rapidly as a function of resolution, with a precipitous drop beginning at 9 A resolution. Synchrotron data from these crystals were previously observed at the CHESS F1 beamline to about 3.5 A resolution, but the intensities could not be accurately measured beyond 5 A. In experiments conducted at the Advanced Photon Source (APS) beamline 14-BM-C, data from identical crystals could be processed to a resolution of 3.5 A. The lifetime of the crystals in the beam was increased from one exposure per crystal volume to between four and eight exposures per crystal volume. More than 500 images were collected in two trips, allowing the extension of the resolution of the data set and the structure determination to 3.5 A resolution. Factors in the increased resolution may include X-ray flux, beamline geometry and low background scatter. These results suggest that other crystals with large unit cells and pronounced intensity falloff with resolution may benefit from the use of this or similar beamlines.

Capsid↗
PubMed2004

Expression, purification and crystallization of an extended-spectrum beta-lactamase from Klebsiella oxytoca.

OXY-1a is an extended-spectrum beta-lactamase from the conditional pathogenic bacterium Klebsiella oxytoca. OXY-1a is responsible for the antibiotic resistance of this pathogen. A soluble form of OXY-1a with a His tag at its C-terminus was overexpressed in Escherichia coli. The recombinant protein was purified and crystallized at room temperature using PEG 4000 as the main precipitant. Two crystal forms were obtained from the same growth conditions. One was orthorhombic, with crystals that diffracted to better than 1.9 A, while the other was tetragonal, with crystals that only diffracted to about 3.0 A. Complete data sets were collected from both crystal forms. The orthorhombic crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.54, b = 73.43, c = 84.56 A, while the tetragonal crystal has unit-cell parameters a = b = 73.72, c = 96.81 A. The asymmetric unit of the orthorhombic crystal is estimated to contain one OXY-1a molecule, giving a crystal volume per protein weight (V(M)) of 2.25 A(3) Da(-1) and a solvent content of 45%.

Crystallography, X-Ray↗
PubMed2004

Spectacular improvement of X-ray diffraction through fast desiccation of protein crystals.

Succeeding in getting a protein to crystallize is not always the final hurdle in the determination of its three-dimensional structure. A relatively frequent and particularly vexing situation is the production of macroscopically well formed crystals that exhibit no suitable diffraction pattern. In this paper, three independent cases (i.e. proteins and crystallization conditions) are reported of spectacular diffraction-pattern improvement through a simple crystal-handling procedure that was discovered serendipitously. The procedure basically consists of removing a non-diffracting frozen crystal from the X-ray beam, plunging it into a soaking solution made of the original crystallization solution supplemented with a traditional cryoprotectant and then letting it dry in the evaporating sitting drop for some time (15 min to several hours). The treated crystals are then remounted and exhibit a huge improvement in their diffraction intensity and resolution. In all three cases presented here, the crystal quality shifted from unusable to perfectly suitable for structure determination. In addition to being a 'last resort' procedure for experimentalists struggling with non-diffracting crystals, this puzzling effect constitutes one more challenging problem for theoretical protein crystallographers.

Candida albicans↗
PubMed2005

Structure determination of GGA-GAE and gamma1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic.

Crystallization of protein-protein complexes is an important step in the studies of biological functions of proteins. However, weak and transient, even though specific, interactions often present difficulties in crystallization of protein complexes due to the heterogeneity of the sample mixture. For example, the gamma1-ear domain of the AP-1 complex and the GAE domain of GGA1, responsible for the interaction with accessory proteins involved in vesicular transport, are known to interact with target proteins with affinities of the order of 1-100 microM. Such low affinities have hampered crystallization trials of the complexes. To overcome this problem, the gamma1-ear and GAE domains were first co-crystallized with excess amounts of the peptides. Co-crystals of both domains were obtained and the complex structures were determined at 2.5-2.9 A resolution. Based on the crystal packing of gamma1-ear and the cognate peptide, gamma1-ear fused with a peptide tag at the N-terminus was prepared. The peptide-tagged gamma1-ear readily crystallized and the crystal diffracted far better, 1.9-2.2 A resolution, compared with the co-crystallized complex, giving significantly more details without affecting the overall complex structure.

ADP-Ribosylation Factors↗
PubMed1994

Extension of the diffraction resolution of crystals.

The diffraction resolution of crystals of the guanine nucleotide-exchange factor complex, EF-Tu-Ts, has been extended from 5.0 to 2.5 A by lowering the solvent content in the crystals as well as the temperature of data collection. The common form of EF-Tu-Ts crystal belongs to space group P2(1)2(1)2(1) with a = 81.1, b = 109.9, c = 207.5 A and has a solvent content of 61%. The crystals diffract to a resolution of 5.0 A at 293 K and 4.0 A at 273 K. When cryoprotective agents are slowly diffused into the crystals, the cell constants shrink to a = 74.4, b = 109.9, c = 198.7 A and the solvent content falls to 55%. After the cryoprotective agent has been added, the crystals diffract to 2.7 A resolution at 293 or 273 K and 2.5 A at 250 K. X-ray diffraction data, collected before and after the transformation of individual EF-Tu-Ts crystals, demonstrate that a large percentage of the improvement in diffraction resolution is due solely to the addition of cryoprotective agents. The transfer procedures for the successful introduction of cryoprotective agents into EF-Tu-Ts crystals as well as the general applicability to other crystal systems will be discussed.

Journal Article↗
PubMed1996

Crystallization of apocrustacyanin on the International Microgravity Laboratory (IML-2) mission.

Rod-shaped crystals of apocrustacyanin C1 have been grown under microgravity on the International Microgravity Laboratory (IML-2) NASA space shuttle mission using the vapour-diffusion set-up of the Advanced Protein Crystallization Facility (APCF). The crystals obtained under microgravity are compared with crystals grown simultaneously in ground control experiments in identical APCF reactors, and with those obtained in the laboratory. The degree of reproducibility of the results in microgravity was also tested. Statistically, the microgravity-grown crystals are larger and of better X-ray diffraction quality than those grown in the ground controls but inferior to the best crystals grown in sitting drops, in the laboratory. Diffracting crystals, the best to 2.3 A, were produced in seven out of the eight reactors in microgravity, whereas the eight ground control reactors yielded only one poorly formed crystal suitable for diffraction studies, which also diffracted to 2.3 A. The crystals belong to the space group P2(1)2(1)2(1) with two subunits per asymmetric unit.

Journal Article↗