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At least 1,675 records · Page 93Linked to original sources
PubMed1993

Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.

Bacillus cereus↗
PubMed1998

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage phi29 connector particle.

The connector or portal particle from double-stranded DNA bacteriophage phi29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a = 416.86 A, b = 227.62 A, c = 236.68 A and beta = 96.3 degrees and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4(2)2(1)2 with cell dimensions a = b = 170.2 A, c = 156.9 A and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 A respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.

Bacillus Phages↗
PubMed1996

Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile.

Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associated proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel. The purified 15 kd protein was subjected to N-terminal amino acid sequencing. Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-1. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 micrograms/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control. Its isoelectric point was 6.1 without isoforms. Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein.

Amino Acid Sequence↗
PubMed2002

On the role of the crystal environment in determining protein side-chain conformations.

The role of crystal packing in determining the observed conformations of amino acid side-chains in protein crystals is investigated by (1) analysis of a database of proteins that have been crystallized in different unit cells (space group or unit cell dimensions) and (2) theoretical predictions of side-chain conformations with the crystal environment explicitly represented. Both of these approaches indicate that the crystal environment plays an important role in determining the conformations of polar side-chains on the surfaces of proteins. Inclusion of the crystal environment permits a more sensitive measurement of the achievable accuracy of side-chain prediction programs, when validating against structures obtained by X-ray crystallography. Our side-chain prediction program uses an all-atom force field and a Generalized Born model of solvation and is thus capable of modeling simple packing effects (i.e. van der Waals interactions), electrostatic effects, and desolvation, which are all important mechanisms by which the crystal environment impacts observed side-chain conformations. Our results are also relevant to the understanding of changes in side-chain conformation that may result from ligand docking and protein-protein association, insofar as the results reveal how side-chain conformations change in response to their local environment.

Computer Simulation↗
PubMed2003

Lipidic cubic phase crystal structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.35A resolution.

Well-ordered crystals of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides were grown from a lipidic cubic phase. Here, we report the type I crystal packing that results from this crystallisation medium, for which 3D crystals grow as stacked 2D crystals, and the reaction centre X-ray structure is refined to 2.35A resolution. In this crystal form, the location of the membrane bilayer could be assigned with confidence. A cardiolipin-binding site is found at the protein-protein interface within the membrane-spanning region, shedding light on the formation of crystal contacts within the membrane. A chloride-binding site was identified in the membrane-spanning region, which suggests a putative site for interaction with the light-harvesting complex I, the cytochrome bc(1) complex or PufX. Comparisons with the X-ray structures of this reaction centre deriving from detergent-based crystals are drawn, indicating that a slight compression occurs in this lipid-rich environment.

Binding Sites↗
PubMed1986

Solvation of the left-handed hexamer d(5BrC-G-5BrC-G-5 BrC-G) in crystals grown at two temperatures.

Crystals of the hexadeoxyoligomer d(5BrC-G-5BrC-G-5BrC-G) were grown at different temperatures (5 degrees C, 18 degrees C and 37 degrees C) in the absence of divalent cations. The crystals grown at 5 degrees C did not diffract X-rays, while those grown at 18 degrees C and 37 degrees C did. The oligomer adopts the left-handed ZI conformation in both crystals. The main difference resides in a more extensive hydration shell in the crystal grown at high temperature than in the crystal grown at low temperature. The high-temperature crystal displays a spine of hydration running deep in the minor groove and linking exocyclic O-2 atoms of the pyrimidine rings. In both crystalline forms, a hydrated sodium ion bound to the N-7 of a guanine ring was found. Strings of water molecules bridging phosphate anionic oxygen atoms are found along the backbone. The absolute values of the propeller-twist are also different in both structures although the values of the twist are very similar. The results point to the importance of the crystallization conditions when analysing fine structural details like solvation properties of oligomers.

Crystallization↗
PubMed2002

Cystine: a promoter of the growth and aggregation of calcium oxalate crystals in normal undiluted human urine.

PURPOSE: Many variables are known to be associated with the formation of calcium oxalate stones. We noted that on analysis a number of patients with calcium oxalate calculi also had cystine in the stones. Some but not all of these patients showed urinary cystine excretion slightly above the normal limits, resembling heterozygous carriers. This finding raised the question of whether some recurrent stone formers may be at risk for calcium oxalate calculi when they excrete cystine in above normal concentrations. MATERIALS AND METHODS: Pooled urine obtained from 3 pairs of age and sex matched controls was independently analyzed. Each urine sample was divided into spun and filtered, and ultrafiltered urine. A Multisizer II (Coulter Electronics Ltd, Beds, England) was used to measure particle number, diameter and volume. The metastable limit of each specimen was determined. Promotion activity was measured in spun and filtered, and ultrafiltered urine using 3 concentrations of cystine (80, 160 and 320 micromol./l.). Results were confirmed by measuring the incorporation of (14)C-oxalate into the crystals. Scanning electron microscopy was performed to study further the agglomerates as well as exclude cystine crystals. Each experiment was repeated 6 times. Crystalline material was collected for x-ray powder diffraction analysis. RESULTS: The urine metastable limit did not change with increasing cystine concentrations. Particle diameter increased significantly from 10.6 microm in ultrafiltered urine alone to 11.6 and 13.5 microm (p < 0.05) at 160 and 320 micromol/l. cystine, respectively. In addition, particle volume also increased proportionally in a dose response manner to cystine concentration. The dose of 320 micromol/l. cystine increased the crystal growth rate 52%. 14C-oxalate experiments confirmed these results. Scanning electron microscopy at 500x magnification revealed no cystine crystals in any experiments performed. Furthermore, x-ray powder diffraction analysis of samples revealed that experimentally determined parameters matched reference values for calcium oxalate trihydrate but not for cystine, again confirming absent cystine in the samples. CONCLUSIONS: Adding cystine to undiluted human urine resulted in the marked enhancement of calcium oxalate crystal precipitation. When considered with the finding of cystine in calcium oxalate stones in the noncystinuric population, this result implies that urinary cystine may be a risk factor for calcium oxalate calculi. Cystine was not observed in any calcium oxalate crystals, suggesting that the mechanism of crystal formation was a salting out effect.

Calcium Oxalate↗
PubMed1993

Scanning electron microscopic study of the effect of citrate and pyrophosphate on calcium oxalate crystal morphology.

The effects of citrate and pyrophosphate on the morphology of calcium oxalate monohydrate crystals formed in a supersaturated calcium oxalate solution were examined by scanning electron microscopy. Within the normal range of urinary concentrations, citrate and pyrophosphate affected the morphology of the crystals. The length/width ratio of the crystals decreased at higher concentrations of citrate and pyrophosphate. Citrate seems to have a dual mechanism of inhibition of crystal growth (binding to the crystal surfaces) and of complexation (with calcium in the solution). Pyrophosphate is believed to bind preferentially to calcium in the solid phase rather than in the solution phase, as it affected crystal morphology at very low concentrations and was detected on the crystals by X-ray microanalysis. Pyrophosphate seems to bind to the monohydrate but not to the dihydrate.

Calcium Oxalate↗
PubMed1992

The effect of two new semi-synthetic glycosaminoglycans (G871, G872) on the zeta potential of calcium oxalate crystals and on growth and agglomeration.

The effects of two new semisynthetic glycosaminoglycans (GAGs), G871 and G872, on the crystal growth and agglomeration of calcium oxalate monohydrate (COM) were studied in artificial urine in vitro. A constant composition crystallization system and a seeded crystal growth system were used to measure the rate of crystal growth and degree of agglomeration. The zeta potential on the crystal surface was measured using a Coulter DELSA 440 doppler electrophoretic light scattering analyzer. The previously reported GAG analogue, sodium pentosan polysulphate (SPP or SP54), was studied as a reference inhibitor of crystallization using the same systems. All three substances had a concentration-dependent effect on the zeta potential and on the rate of crystal growth and agglomeration of COM in artificial urine. G872 had a significantly greater effect than either G871 or SPP on all the measured parameters. It is suggested that semisynthetic GAGs, such as those reported in this paper may be of use in the prevention of the recurrence of calcium oxalate stones.

Calcium Oxalate↗
PubMed1991

Proteus mirabilis biofilm protection against struvite crystal dissolution and its implications in struvite urolithiasis.

Proteus mirabilis biofilm formation, struvite (MgNH4PO4.6H2O) crystal formation and dissolution in an artificial urine mixture were monitored using computer-enhanced microscopy (CEM) and a 1 x 3 mm. glass flow cell. Image analysis showed that P. mirabilis biofilm formation did not occur to any extent at macroenvironment flow rates greater than two mL/h (equivalent to a microenvironment flow rate of less than 5 microns./sec). Essentially, cells attached to glass surfaces, grew slowly and divided. Daughter cells were generally released directly into the medium where they could then presumably colonize other regions. Microcolonies formed by the adhesion of aggregates of cells from the medium, and over time grew into biofilms. Struvite crystallization due to urease activity and pH elevation above neutrality, was preceded by the deposition of organic matter on the glass surface, followed by the appearance of a number of tiny (one to two microns.) crystals. Crystals forming within a biofilm at low dilution rates took on a characteristic twinned or "X-shaped" appearance (crystal habit) indicative of a rapid growth rate. Those forming outside the biofilm took on a more tabular appearance reflecting their slower growth. When the macroenvironment flow rate of artificial urine (initial pH 5.8) in the glass flow cell was increased from two mL/h to four mL/h, struvite crystals not associated with biofilms dissolved within five to 10 min. Crystals entrapped within the P. mirabilis biofilm withstood flow rates up to 200 mL/h presumably due to the maintenance of an alkaline Mg-saturated microenvironment within the biofilm. These observations may suggest a mechanism by which struvite calculi can grow in spite of neutral or acidic urine pH and resist mild acidification therapy.

Bacterial Adhesion↗
PubMed1986

Modifiers of calcium oxalate crystallization found in urine. II. Studies on their mode of action in an artificial urine.

The relative potencies of various modifiers of the crystallization of calcium oxalate (CaOx) were determined under "whole urine equivalent" conditions using a batch crystallizer. The system was used to measure changes in the degree of agglomeration of CaOx crystals produced spontaneously at a level of supersaturation within the range found in the urines of recurrent CaOx stone-formers. The modifiers tested included RNA, heparin, chondroitin-4-sulphate, pyrophosphate (at pH 5, 6 and 7), Tamm-Horsfall mucoprotein and a greater than 10,000 dalton fraction of macromolecules obtained from pooled normal urine by dialysis. The effects of these urinary constituents were also measured on the zeta potential produced at the surface of CaOx crystals. In the case of pyrophosphate, there was a clear correlation between the degree of inhibition of agglomeration and the zeta potential observed on the crystal surface indicating that, for this ion, repulsive electrostatic forces dominate the tendency for CaOx crystals to agglomerate. For the other modifiers tested, the tendency to agglomerate appeared to be dependent on the balance between the positive viscous binding ("sticky") forces and the negative electrostatic forces produced by these macromolecules on the CaOx crystal surface.

Calcium Oxalate↗
PubMed1984

Ureaplasma urealyticum-induced crystallization of magnesium ammonium phosphate and calcium phosphates in synthetic urine.

Crystallization of struvite and calcium phosphates was studied in vitro as encrustations on glass rods immersed in synthetic urine, to evaluate the crystallization capacity of Ureaplasma urealyticum and compare it with that of known urease and non-urease-producing bacteria. Inoculation of the synthetic urine with Ureaplasma urealyticum resulted in alkalinization of the synthetic urine and crystallization of struvite and brushite. Inoculation with Proteus mirabilis caused a faster and more pronounced alkalinization as well as crystallization of struvite and apatite. The alkalinization and crystallization caused by Ureaplasma urealyticum and Proteus mirabilis was completely prevented by acetohydroxamic acid, a potent urease inhibitor, linking the crystallization to the urease activity of the microorganisms. When the synthetic urine was inoculated with urease-negative Escherichia coli no alkalinization and no crystallization were seen.

Calcium Phosphates↗
PubMed1983

Effects of urine pretreatment on calcium oxalate crystallization inhibition potentials.

Activated carbon and calcium oxalate solids have been used as adsorbents to remove crystallization inhibiting species from centrifuged and dialyzed normal urines. The urine supernatants, following adsorption treatments, have been examined for their calcium oxalate monohydrate crystallization inhibitory potentials by means of a constant composition potentiostatic seeded growth method. The supersaturation of the reactant solution was maintained constant during the crystallization process by the potentiometrically monitored addition of solutions containing crystal lattice ions controlled by a specific calcium ion selective electrode. The experimental results indicate that normal urine contains a number of inhibiting species of variable molecular weight and adsorbability. In addition, preliminary results indicate that substances present in normal urine interfere substantially with the adsorption of crystallization inhibiting species by calcium oxalate monohydrate crystal surfaces. Both charcoal and CaOx adsorbents have been successfully used to completely remove a synthetic, model urinary crystallization inhibitor, ethylene-diamine-tetramethylene-phosphonic acid, ENTMP, from relatively concentrated solutions in 0.15 M NaCl.

Adsorption↗
PubMed2001

Affinity binding phenomena of DNA onto apatite crystals.

The effect of DNA on the crystal growth of hydroxyapatite (HAp) and its morphology was examined. X-ray diffraction patterns of DNA-containing apatites showed typical apatitic features. However, crystal growth was greatly inhibited in the presence of DNA during synthesis; particularly, the crystallinity in the a-axis direction decreased dramatically at low concentrations of DNA. The a- and c-axis dimensions of each precipitate were almost the same, which implies that DNA molecules affect only the crystal surface. CHN analysis clearly showed the presence of these elements, which increased with an increase in DNA concentration in the solution. Scanning electron micrographs of the precipitates formed in the presence of DNA showed typical needle-like crystals, with a decreased crystal size, especially width. Infrared absorption spectroscopy of the DNA-containing apatites showed that the 1630-1700 cm(-1) absorption band due to C=C and C=N stretching increased with an increase in DNA concentration during precipitation. The ESCA spectrum of HAp(DNA 1.0) shows N 1s and C 1s peaks that are absent and weak, respectively in HAp. 31P NMR spectroscopy revealed a weak peak at the base of the 31P peak from the PO4(3-) ions in the HAp crystals. This weak 31P peak had a small positive shift from the position found in native DNA which may be due to the phosphate backbone of adsorbed DNA. The apparent solubility of the HAps increased with an increase in DNA concentration. These results suggest that there is an affinity binding between apatite crystal and DNA molecules.

Animals↗
PubMed2002

Transforming growth factor beta-3 crystals as reservoirs for slow release of active TGF-beta3.

Transforming growth factor betas (TGF-betas) play critical roles in many diseased states and injury repair processes. Exogenous delivery of TGF-beta may thus have therapeutic applications. Here, crystals of TGF-beta3 (TGF-beta3) are being evaluated as protected reservoirs for sustained local release. A sensitive Mv1Lu cell growth inhibition assay established that in vitro, active TGF-beta3 can be delivered from physically stable crystals. Non-sink release experiments revealed that crystal solubility at pH 7.4 was higher in cell culture medium (2.7+/-0.1 microg/ml) than in saline buffers (approximately 1-1.5 microg/ml, P<0.05). Addition of serum induced a five-fold delay in equilibration of soluble-crystal TGF-beta3. Semi-sink experiments cumulated in higher TGF-beta3 release than under non-sink conditions; the observed steady states correlated with crystal solubility and the frequency of buffer exchange. Release of TGF-beta3 from crystals was also strongly dependent on solubility changes as affected by pH. At neutral pH the solubilities were the lowest, and increased with both higher and lower pH. The results indicate that TGF-beta3 crystals may have promising features for local pH-triggered sustained-release applications.

Crystallization↗
PubMed1994

The effect of bile acid hydrophobicity on nucleation of several types of cholesterol crystals from model bile vesicles.

Nucleation of cholesterol crystals is thought to occur from cholesterol-phospholipid vesicles. We tested the hypothesis that bile acids are necessary for nucleation of cholesterol crystals. Model bile vesicles were prepared by KBr density ultracentrifugation of supersaturated model bile and mixed with one of the following bile acids: ursodeoxycholate, taurocholate, cholate, chenodeoxycholate or deoxycholate in final concentrations of 3, 30 and 100 mM. Vesicles were also mixed with various combinations of ursodeoxycholate and deoxycholate. Nucleation was assessed semi-quantitatively with polarizing microscopy. After 5 days, samples were again subjected to ultracentrifugation. Addition of 3 and 30 mM taurocholate, cholate, chenodeoxycholate and deoxycholate to vesicles induced nucleation. The extent of nucleation increased significantly with increasing bile acid hydrophobicity: deoxycholate > chenodeoxycholate > cholate > taurocholate (p < 0.05). At 100 mM bile acid this order was reversed (p < 0.05) because most of the cholesterol was solubilized in micelles as shown by ultracentrifugation after 5 days. Percentages of vesicular cholesterol decreased with increasing hydrophobicity: deoxycholate < chenodeoxycholate < cholate < taurocholate (p < 0.05). Ursodeoxycholate did not induce nucleation. At least seven cholesterol crystal shapes could be distinguished and all crystal types could be found after addition of various combinations of ursodeoxycholate+deoxycholate. We conclude that in this model: (a) bile acid species play an important role in the precipitation of cholesterol crystals from model bile vesicles; (b) the more hydrophobic bile acids induce more cholesterol crystal precipitation; and (c) the hydrophobicity of bile acids influences cholesterol crystal morphology.

Bile↗
PubMed1998

Effects of serpins on cholesterol crystallization in model bile.

BACKGROUND/AIMS: The serine proteinase inhibitors (serpins), alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT), are acute phase proteins synthesized by hepatocytes and excreted to some extent into bile. Their role in gallstone pathogenesis is unclear, and it was the aim of this study to determine their effect on cholesterol crystal growth rate in model bile. METHODS: Purified AAT and ACT were added to model bile at concentrations from 0.5 to 500 microg/ml. Cholesterol crystal growth was analyzed daily by polarizing microscopy and spectrophotometrically at 650 nm. Serpin inhibitory activity was measured spectrophotometrically at 405 nm, and polymerization was studied on 7.5% SDS-PAGE under non-reducing conditions, by immunoelectrophoresis and Western blotting. RESULTS: ACT added to model bile at a concentration of 0.5 microg/ml, inhibited cholesterol crystallization by 30%, had no influence at 5 microg/ml, and increased the crystallization rate 2-3 fold at concentrations of 50 and 500 microg/ml. AAT at a concentration of 0.5 microg/ml had a profound (50%) inhibitory effect on cholesterol crystal growth rate, lacked significant effect at both concentrations of 5 and 50 microg/ml, and showed stimulation of crystal growth up to 30% at a concentration of 500 microg/ml. Both serpins incubated in model bile polymerized and totally lost their inhibitory activity. CONCLUSIONS: Serpins can exhibit both inhibiting and promoting effects on the cholesterol crystallization rate in model bile, dependent on their concentrations. Since AAT and ACT are acute phase proteins, their concentrations may vary under certain pathological conditions, which may result in different actions of these serpins in gallstone formation.

Bile↗
PubMed2002

Water mediated Dickerson-Drew-type crystal of DNA dodecamer containing 2'-deoxy-5-formyluridine.

To investigate the role of divalent cations in crystal packing, a Dickerson-Drew-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2'-deoxy-5-formyluridine at X, was crystallized under several conditions with Ba(2+) ion instead of Mg(2+) ion. The crystal structure is isomorphous with the original Dickerson-type crystal containing Mg(2+) ion. In the Mg(2+)-free crystals, however, a five-membered ring of water molecules occupies the same position as the magnesium site found in the Mg(2+)-containing crystals, and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that the five-membered water molecules can take the place of the hydrated magnesium cation in crystallization. The 5-formyluracil residues form the canonical Watson-Crick pair with the opposite adenine residues.

Barium↗