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PubMed1989

Association between cholesterol-phospholipid vesicles and cholesterol crystals in human gallbladder bile.

Rapid aggregation of cholesterol-phospholipid vesicles in gallbladder bile seems to be the first event in the production of cholesterol crystals, a prerequisite for cholesterol gallstone formation. We examined the amount of these vesicles in 33 human gallbladder biles in relation to biliary lipid composition and to the presence of cholesterol crystals. Biliary microscopy detected cholesterol crystals in all 19 biles from patients with cholesterol gallstones but in none of 14 biles from patients with pigment stones. Gel chromatography was used to separate vesicles and micelles in the native bile with an eluting buffer containing 10 mM sodium cholate to prevent disruption of micellar lipids. Cholesterol, phospholipid and bile salt concentrations were measured in every fraction collected. Bile acid, phospholipid, cholesterol and total lipid concentrations were not significantly different in samples with and without cholesterol crystals. The cholesterol saturation index (1.4 +/- 0.11 vs. 1.0 +/- 0.08) was significantly (p less than 0.01) higher in biles with crystals than without crystals. Gel filtration revealed a vesicular peak in addition to micellar fraction in 18 (23.1 +/- 3.2% of total cholesterol) of the 19 biles with crystals but only in three (15.7 +/- 2.4% of total cholesterol) of 14 biles without crystals. There was no relation between biliary lipid concentration or the cholesterol saturation index and the percentage of vesicular cholesterol in biles with or without crystals. The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals.

Bile↗
PubMed1992

Crystallization studies of glycosylated and unglycosylated human recombinant interleukin-2.

Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6(2)22 or its enantiomorph. The crystals diffract to 2.8 A and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5-5.6 in the monoclinic space group P2(1), and less frequently in the orthorhombic space group P2(1)2(1)2(1) from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3(1)21 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals.

Crystallization↗
PubMed1993

Crystallization and preliminary X-ray diffraction data of two heparin-binding fragments of human fibronectin.

Two different heparin-binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep-2A fragment, consisting of type III domains 12-14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I2(1)2(1)2(1) with a = 68.1 A, b = 88.6 A, and c = 144.9 A. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6(1)22 or P6(5)22 with a = b = 66.7 A and c = 245.7 A. The 40 kDa hep-2B fragment, consisting of type III domains 12-15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep-2A fragment with space group I222 or I2(1)2(1)2(1) and a = 67.5 A, b = 87.0 A, and c = 144.3 A. All crystal forms diffract to at least 3.5 A resolution and contain a single molecule in the asymmetric unit.

Crystallization↗
PubMed2000

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals.

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.

Apoferritins↗
PubMed1992

Human amelogenesis. I: High resolution electron microscopy study of ribbon-like crystals.

Ribbon-like crystals, from developing enamel of human fetuses, were studied by high resolution electron microscopy. These crystals were classically described as the first organized mineral formed during amelogenesis. They were characterized by a mean width-to-thickness ratio (W.T-1) of 9.5, and 40% were bent. On lattice images we noted the presence of the central dark line (CDL) associated with white spots. Both structures were found in crystals with a minimum thickness of 8-10 nm. CDLs were localized in the center of the crystals and seemed to be linked to the initial growth process, but their exact structure and function were not fully determined. We were able to study the structure of the ribbon-like crystals with a Scherzer resolution close to 0.2 nm. The good correspondence between experimental and computed images showed that their structure was related to hydroxyapatite (HA). In addition, the presence of ionic substitutions and deficiencies were also compatible with HA. In this study, about 50% of the crystals showed structural defects. Screw dislocations were the most often noted defects and were observed within crystals aligned along five different zone axes. Low- and high-angle boundaries were also detected. Low-angle boundaries, found in the center of the crystals, could thus be related to CDLs and be implicated in the nucleation step of crystal formation, whereas high-angle boundaries could result from the fusion of ribbon-like crystals. Such mechanisms could induce an acceleration of the growth in thickness of the crystal observed during the maturation stage of amelogenesis.

Amelogenesis↗
PubMed1994

Development and validation of a quantitative assay for cholesterol crystal growth in human gallbladder bile.

Crystal observation time is a rough estimate of the first microscopic appearance of cholesterol monohydrate crystals in an isotropic bile, and does not provide information on crystal growth kinetics. We have developed a method for quantitating cholesterol crystal growth in gallbladder bile. Crystals were separated from other biliary particles by ultracentrifugation on a discontinuous NaBr gradient, after bile density adjustment to d = 1.060 g/ml. More than 95% of crystals, both of native or synthetic source, floated in the density range 1.045-1.055. This density fraction was collected and crystal mass was measured by photometric turbidity, after calibration with suspensions of different-sized cholesterol crystals. The recovery of crystals added to original bile samples averaged 96.0 +/- 2.8%. Contamination with vesicles, which may potentially interfere with the turbidimetric assay, was excluded by gel-chromatography. The method was sequentially applied, until the 20th day of incubation, to biles obtained at surgery from patients with (A, n = 6) or without cholesterol gallstone (B, n = 4), and from gallstone patients pretreated for 1 week with oral ursodeoxycholic acid (C, n = 5). Crystal growth curves greatly differed, being much steeper in group A and almost flat in patients receiving ursodeoxycholic acid. The mean percent mass of biliary cholesterol in crystalline form at the 20th day was 19.2 +/- 13.5%, 1.2 +/- 0.8% and 2.7 +/- 1.1% in A, B and C, respectively (A vs. B: P = 0.014; A vs. C: P = 0.008). We conclude that the method allows a precise estimate of cholesterol crystal growth and can be usefully applied to human gallbladder biles.

Bile↗
PubMed1992

Crystal growth of ribonuclease S under microgravity.

Expecting that protein crystal growth in space would show us a new aspect of its crystallization process and might result in the improvement of crystal quality, we performed a series of growth experiments for bovine pancreatic ribonuclease S crystals under microgravity. For this purpose, the COSIMA2 system launched in September 1989 was utilized. In one of the eight crystallization chamber setups in the COSIMA2 experiment, a large "Z-form" single crystal appeared and we found that (a) the space-grown single crystal exhibited a higher fraction of significant X-ray diffraction spots than ground-grown crystals of approximately the same size and (b) the corresponding Wilson plots also tended to show a slightly lower overall temperature factor for the space-grown crystals, although the quality of the plot was not sufficient for quantitative evaluation. Thus, mainly based on the above observation (a), it is most likely that the internal order of the ribonuclease S crystal was in fact improved through the crystallization under microgravity.

Crystallization↗
PubMed2005

Control of n-alkanes crystallization by ethylene-vinyl acetate copolymers.

The crystallization of paraffins from their solution at low temperature was investigated in the presence of ethylene-vinyl acetate (EVA) copolymers that allow the control of the size of the crystals. Depending on the type of solvent and distribution of the paraffin lengths, the mechanisms of crystal formation and growth are different. Precipitation of the EVA prior to the paraffins leads to the nucleation of a large number of crystals, whereas the adsorption of EVA on the surface of the growing crystals slows down the crystal growth. EVA can act either as a nucleating agent or as a growth inhibitor. These two mechanisms were identified from the analysis of the temperature of crystallization (cloud point), the chemical composition of the crystals, and the observations of the crystal habit. The EVA was able to co-crystallize with the paraffins in crystals of an orthorhombic structure and the melting enthalpies of the crystalline paraffin did not depend significantly on their neighborhood. The energies of interaction between the different paraffinlike components are close to each other, so that minor changes of the experimental conditions may lead to dramatic effects. This is the basic rationale for the large behavioral diversity observed in these systems.

Adsorption↗
PubMed1998

Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly.

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.

Animals↗
PubMed1998

Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals. Molecular scale lattice images of these crystals were acquired in situ under growth conditions, enabling simultaneous identification of the rhombohedral LysB28ProB29 crystal form, its orientation, and its dynamic growth characteristics. The ability to obtain crystallographic parameters on multiple crystal faces with TMAFM confirmed that bovine and porcine insulins grown under these conditions crystallized into the same space group as LysB28ProB29 (R3), enabling direct comparison of crystal growth behavior and the influence of sequence variation. Real-time TMAFM revealed hexamer vacancies on the (001) terraces of LysB28ProB29, and more rounded dislocation noses and larger terrace widths for actively growing screw dislocations compared to wild-type bovine and porcine insulin crystals under identical conditions. This behavior is consistent with weaker interhexamer attachment energies for LysB28ProB29 at active growth sites. Comparison of the single crystal x-ray structures of wild-type insulins and LysB28ProB29 suggests that differences in protein conformation at the hexamer-hexamer interface and accompanying changes in interhexamer bonding are responsible for this behavior. These studies demonstrate that subtle changes in molecular conformation due to a single sequence inversion in a region critical for insulin self-association can have a significant effect on the crystallization of proteins.

Amino Acid Sequence↗
PubMed1998

Terrestrial and space-grown HAP and OCP crystals: effect of growth conditions on perfection and morphology.

This paper reports comparative characterizations of calcium phosphate crystals grown on earth and in space. At the CaCl2 and KH2PO4 + K2HPO4 solution concentrations and the pH used, only hydroxyapatite (HAP) crystals grow under terrestrial condition while both HAP and octacalcium phosphate (OCP) crystals grew during the space experiment. The space-grown OCP crystals reach 3 mm in size, the space-grown HAP crystals reach sizes up to 100 times larger than the earth-grown crystallites. It was found also that the space-grown crystallites are more perfect than the terrestrial ones, being more stable under electron beam during HRTEM examination. Spherolites of hydroxyapatite consist of small and thin HAP crystals with different orientations. Space-grown OCP crystals containing almost pure OCP phase show strong striations along the c direction due to thickness variations. Terrestrial OCP crystals grown at lowest supersaturation on earth may be almost as large as the space-grown ones, possess a regular habit and are homogeneous in thickness. However, they always contain substantial regions of HAP structure. Also, in these crystals electron irradiation induces phase transformation from crystalline to amorphous (disordered) state during transmission electron microscopy observations. In the space-grown crystals, such transformation needs longer radiation time. We believe that the differences described above come from much lower supersaturation and different pH for crystals nucleating and growing in space compared to those formed on earth.

Calcium Phosphates↗
PubMed1995

Multiple crystal forms of endoglucanase CelD: signal peptide residues modulate lattice formation.

The crystal structure of Clostridium thermocellum endoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a = 99.3 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 A resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C.

Amino Acid Sequence↗
PubMedDate not supplied

Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1.

Lamellar single crystals of four random copolymers of (R)-3-hydroxybutyrate with different hydroxyalkanoates: poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyvalerate) (P(3HB-co-8%-3HV)), poly(3-hydroxybutyrate-co-10 mol%-4-hydroxybutyrate) (P(3HB-co-10%-4HB)), poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyhexanoate) (P(3HB-co-8%-3HH)) and poly(3-hydroxybutyrate-co-10 mol%-6-hydroxyhexanoate) (P(3HB-co-10%-6HH)), were grown from dilute solutions of chloroform and ethanol. All single crystals have lath-shaped morphology and the second monomer units seem to be excluded from the P(3HB) crystal, on the basis of the electron diffraction diagrams. The enzymatic degradation of P(3HB-co-8%-3HH) and P(3HB-co-10%-6HH) single crystals was investigated with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Adsorption of an extracellular PHB depolymerase, examined using an immuno-gold labelling technique, demonstrated a homogeneous distribution of enzyme molecules with a low concentration on the crystal surfaces. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield narrow cracks along their long axes and the small crystal fragments. Lamellar thicknesses of single crystals and molecular weights of copolymer chains remained unchanged during the enzymatic hydrolysis. The above results support the hypothesis that the hydrophobic adsorption of the enzyme contributes to increase the mobility of molecular chains of single crystals and generate the disordered chain-packing regions. The active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends with endo-exo enzymatic hydrolysis behaviour, termed processive degradation.

Adsorption↗
PubMed2001

In situ observation of crystal growth for poly[(S)-lactide] by temperature-controlled atomic force microscopy.

The crystallization behavior and crystalline morphologies of poly[(S)-lactide] (P[(S)-LA]) in thin films crystallized isothermally at over 160 degrees C were characterized by transmission electron microscopy and atomic force microscopy (AFM). The dendritic crystal and hexagonal crystal were formed in thin film with thicknesses below 30 nm or over 50 nm, respectively. The crystal structures of dendritic and hexagonal crystals were identical, suggesting that the crystalline morphology of P[(S)-LA] is strongly dependent upon the film thickness. In situ observation of the crystal growth in the P[(S)-LA] thin film at 165 degrees C from the melt was carried out by using temperature-controlled AFM equipped with a heating stage. The initial stage of crystallization and development of lamellae were successfully observed during isothermal crystallization at 165 degrees C. The first forming crystal showed the edge-on orientation, and grew to S-shaped edge-on lamellae. Dendritic flat-on crystals were developed from the S-shaped edge-on lamellae. The growth rates of flat-on and edge-on lamellae were almost identical.

Biocompatible Materials↗
PubMedDate not supplied

Enzymatic degradation processes of lamellar crystals in thin films for poly[(R)-3-hydroxybutyric acid] and its copolymers revealed by real-time atomic force microscopy.

Enzymatic degradation processes of flat-on lamellar crystals in melt-crystallized thin films of poly[(R)-3-hydroxybutyric acid] (P(3HB)) and its copolymers were characterized by real-time atomic force microscopy (AFM) in a phosphate buffer solution containing PHB depolymerase from Ralstonia pickettii T1. Fiberlike crystals with regular intervals were generated along the crystallographic a axis at the end of lamellar crystals during the enzymatic degradation. The morphologies and sizes of the fiberlike crystals were markedly dependent on the compositions of comonomer units in the polyesters. Length, width, interval, and thickness of the fiberlike crystals after the enzymatic degradation for 2 h were measured by AFM, and the dimensions were related to the solid-state structures of P(3HB) and its copolymers. The width and thickness decreased at the tip of fiberlike crystals, indicating that the enzymatic degradation of crystals takes place not only along the a axis but also along the b and c axes. These results from AFM measurement were compared with the data on crystal size by wide-angle X-ray diffraction, and on lamellar thickness and long period by small-angle X-ray scattering. In addition, the enzymatic erosion rate of flat-on lamellar crystals along the a axis was measured from real-time AFM height images. A schematic glacier model for the enzymatic degradation of flat-on lamellar crystals of P(3HB) by PHB depolymerase has been proposed on the basis of the AFM observations.

Biophysical Phenomena↗
PubMed1997

Effects of sorbed water on the crystallization of indomethacin from the amorphous state.

The water sorption isotherm and the crystallization rates for amorphous indomethacin were determined at 30 degrees C as a function of relative humidity (RH), along with the effects of water content on the glass transition temperature (T(g)). Below 43% RH, only the stable gamma crystal form appears, whereas at higher RH, only the metastable alpha crystal form appears. The tendency for the alpha crystals to form at higher RH is consistent with the Ostwald step rule. The crystallization rate of the alpha form continuously increased with increasing RH due to increasing molecular mobility. The crystallization mechanism of the gamma form changed from surface-initiated to bulk-initiated crystallization at 21% RH, and although crystallization rates of the gamma form increased with increasing RH in both cases, they were higher when crystallization was surface-initiated. The complex crystallization behavior of the gamma form is explained by the higher water content and molecular mobility of the surface relative to the bulk and the general effect of water on alpha or gamma crystal form selection as described by the Ostwald step rule.

Absorption↗
PubMed1982

Inhibitory activity of human urine on calcium oxalate crystal growth: effects of sodium urate and uric acid.

1. Freshly voided urine from healthy subjects was pooled and ultrafiltered (10,000 daltons). The ultrafiltrable and the macromolecular portions were preincubated with calcium oxalate, uric acid and sodium urate crystals and the effects on the inhibitory activity of calcium oxalate crystal growth assessed by monitoring the disappearance of [14C]oxalate from the solution. 2. The inhibitory activity of the ultrafiltrate was higher than that of the urinary macromolecular fraction. Both uninhibited and inhibited crystal growth processes followed second-order kinetics. 3. Calcium oxalate crystals adsorbed almost all the urinary macromolecular inhibitors whereas sodium urate or uric acid crystals adsorbed only 10-20%. 4. In the presence of a metastable solution of calcium oxalate, the incubation of the urinary macromolecular fraction with sodium urate crystals caused a pronounced reduction in the inhibitory activity. A similar effect was seen with uric acid crystals, but to a lesser degree. 5. We conclude that the effect of sodium urate or uric acid crystals alone on naturally occurring urinary macromolecular inhibitors of calcium oxalate crystal growth is weak, but that in the presence of metastable calcium oxalate this is greatly enhanced. A substantial adsorption of the inhibitors on to the crystals is suggested, possibly secondary to epitaxial growth of calcium oxalate on the surface of the urate crystals.

Calcium Oxalate↗
PubMed2000

Sialic acid and crystal binding.

BACKGROUND: We studied the role of cell surface sialic acid in the adherence of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells. METHODS: Studies were performed with undifferentiated (crystal-binding) cells in subconfluent cultures and maturated (noncrystal-binding) cells in confluent cultures. Lectins were used to study the emergence and abundance of oligosaccharides at the cell surface during epithelial development. The effect of neuraminidase treatment on crystal binding was studied with [14C]COM crystals, and the enzyme-induced release of cell surface-associated sialic acid molecules was monitored by labeling the cells metabolically with [3H]glucosamine. RESULTS: Binding studies with lectins derived from Maackia Amurensis II (MALII) and Sambucus Nigra (SNA) demonstrated that the cells expressed terminal sialic acids attached to penultimate galactose through alpha 2,3 and alpha 2,6 bonds at different stages of epithelial development. Neuraminidase treatment strongly reduced the affinity of the cell surface for COM crystals in subconfluent cultures. Nevertheless, neuraminidase cleaved more sialic acids from cells in confluent cultures than from those in subconfluent cultures. Peanut agglutinin (PNA), which binds only to sialylated terminal galactose units, adhered to developing but not to maturated cells, unless the latter were pretreated with neuraminidase. Both results indicate that the surface of maturated MDCK cells is more heavily sialylated than that of undifferentiated cells. Free sialic acid molecules showed little or no affinity for COM crystals and did not affect the adherence of the crystals to undifferentiated cells. CONCLUSIONS: There are at least two models that may explain these results. First, sialic acids are presented at the surface of immature cells in an orientation that specifically matches crystal surface characteristics favoring crystal-cell interactions. Second, sialic acid molecules are not directly associated with the crystals, but may be involved in the exposure of another crystal binding molecule at the cell surface.

Animals↗