Osteoblast precursor cell activity on HA surfaces of different treatments.

The clinical success of dental implants is governed by implant surfaces and bone cell responses that promote rapid osseointegration and long-term stability. The specific objective of this study was to investigate osteoblast precursor cell responses to hydroxyapatite (HA) surfaces of different treatments. Since the nature of bone cell responses in vitro is influenced by the properties of HA ceramics, this study was divided into two components: a chemical and crystallographic characterization of the HA ceramics and an in vitro cell culture study. The sintered HA samples were observed to have the highest crystallite size as compared to the as-received HA and calcined HA samples. No differences in the surface roughness and chemical composition were observed among the sintered, calcined, and as-received HA surfaces. In concurrence with the X-ray diffraction, high resolution XPS resolution of Ca 2p also indicated a higher crystallinity on sintered HA samples as compared to the calcined and as-received HA samples. As indicated by increased alkaline phosphatase-specific activity, increased cell-surface and matrix-associated protein, and 1.25 (OH2) vitamin D3-stimulated osteocalcin production, a more differentiated osteoblast-like phenotype was observed on the sintered HA surfaces compared to the as-received HA and calcined HA surfaces. An increased osteoblast-like cell activity on the sintered HA surfaces suggested that the crystallite size of HA surfaces may play an important role in governing cellular response.

Identification of N-acetylglucosamine-binding immunoglobulins in chicken egg yolk and serum distinct from the major mannose-binding immunoglobulins.

An N-acetyl-D-glucosamine (GlcNAc)-binding protein of 170 kDa has been isolated from hen serum and egg yolk. Another GlcNAc-binding protein of higher molecular mass was present only in the serum. The 170 kDa protein co-electrophoresed and co-chromatographed in gel filtration with a chicken IgG, and behaved identical to chicken IgG in double immunodiffusion with goat anti-chicken gamma chain antiserum. The sugar-binding hierarchy for the serum and yolk binding proteins, determined with bovine serum albumin neoglycoproteins, was GlcNAc greater than N-acetyl-D-galactosamine greater than glucose = galactose = L-fucose greater than mannose. This hierarchy was unlike any previously reported GlcNAc-binding proteins. The larger serum binding protein component was shown to be an IgM by double immunodiffusion with goat anti-chicken mu chain antiserum. The serum and yolk GlcNAc-binding proteins comprise a unique set of sugar-binding immunoglobulins distinct from the previously reported hen serum and yolk mannose-binding proteins (Wang et al., 1986).

Enhancement of macromolecular ligand binding by rabbit alveolar macrophages by mannose oligosaccharides and related compounds.

When rabbit alveolar macrophages were incubated with 10 mM D-mannose, binding of macromolecular ligands containing D-mannose, such as bovine serum albumin modified with mannose (Man-BSA), was enhanced more than 100%, but was inhibited at higher concentrations [C.A. Hoppe and Y. C. Lee (1982) J. Biol. Chem. 257, 12831-12834]. This phenomenon was further investigated with ovalbumin-derived glycopeptide, Asn(GlcNA2,Man5), and with a wide variety of synthetic mannose oligosaccharides. The extent of enhancement is related to the fine structure of the oligosaccharide groups, but the results are complicated by concurrent inhibition exerted by these compounds. It appears that the more inhibitory a compound is, the less capable it is of exerting the enhancement effect. Thus, small mannose derivatives such as glycosides, including clustered mannosides based on tris(hydroxymethyl)aminomethane [Y. C. Lee (1978) Carbohydr. Res. 67, 509-514], and most of the biantennary mannose oligosaccharides were found to be effective in enhancing the binding of radiolabeled Man-BSA. Triantennary oligosaccharides, on the other hand, showed only a slight enhancement effect and a much stronger inhibitory effect. The effects of ligand size, valency, as well as the fine structure on enhancement are discussed.

Preparation of phosphorylcholine derivatives of bovine serum albumin and their application to the affinity chromatography of C-reactive protein.

A simple method for the preparation of phosphorylcholine derivatives of bovine serum albumin (PC-BSA) by reductive alkylation of the amino groups of bovine serum albumin with choline phosphoryl glycoaldehyde is described. Choline phosphoryl glycoaldehyde was generated by periodate oxidation of glyceryl phosphorylcholine. PC-BSA was immobilized on SH-derivatized Toyopearl HW 65 by reacting the single SH group of PC-BSA with a bismaleimido reagent and then coupling maleimidated PC-BSA to the thiolated gel. The affinity purification of C-reactive protein (CRP), which is based on the Ca2+-dependent affinity of CRP for the phosphorylcholine residue of PC-BSA, was readily accomplished using the PC-BSA Toyopearl HW 65 column. The resulting CRP preparation from serum and pleural fluid was homogeneous as assessed by native polyacrylamide gel electrophoresis. PC-BSA derivatives were also shown to be reactive with Limulus polyphemus CRP.

Identification of the major mannose-binding proteins from chicken egg yolk and chicken serum as immunoglobulins.

Chicken egg yolk contains a mannose-binding protein that could be purified with a modification of the procedure of affinity chromatography and gel filtration used for chicken serum mannose-binding protein. The yolk protein was indistinguishable from the serum protein with respect to apparent molecular masses (169 +/- 7 kDa), subunits (74 kDa and 27 kDa, in approximately 1:1 ratio) produced after denaturation in the presence of mercaptoethanol, immunoreactivity with antibody against the chicken serum mannose-binding protein, amino acid composition, pH optimum for binding, calcium independence of binding, sugar-binding specificity, and specific-binding activity. Moreover, the chicken mannose-binding proteins cross-reacted with gamma-chain-specific antibody against chicken IgG. The binding proteins were identified as IgGs by several other criteria: identical electrophoresis pattern when subjected to reducing and nonreducing NaDodSO4/polyacrylamide gel electrophoresis, cochromatography on a Fractogel TSK HW-55(F) column, similar amino acid composition, and isolation of mannose-binding protein from purified serum IgG at a yield comparable to whole serum. These results support the notion that the mannose-binding proteins from the chicken serum and egg yolk are similar, if not identical, and are a subset of chicken IgG.

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.

Accumulation of a nondegradable mannose ligand within rabbit alveolar macrophages. Receptor reutilization is independent of ligand degradation.

Synthetic neoglycoproteins were made by reacting 5-( azidocarbonyl )pentyl 1-thio-alpha-D-mannopyranoside with poly(L-lysine) and poly(D-lysine). The 125I- Man90 -poly-D-Lys and 125I- Man104 -poly-L-Lys were tightly bound at 2 degrees C by the mannose receptor of the rabbit lung macrophage (Kd = 0.66 +/- 0.18 and 0.59 +/- 0.26 nM, respectively). Under saturating conditions in the cold, the macrophage bound 98 200 +/- 7000 and 84 200 +/- 10 500 ligand molecules per cell for the D- and L-polylysine derivatives, respectively. The cell-surface-bound ligands were dissociable by ethylenediaminetetraacetic acid and mannose at 2 degrees C. At 37 degrees C, the macrophages internalized both 125I- Man90 -poly-D-Lys and 125I- Man104 -poly-L-Lys efficiently. Although the internalized 125I- Man104 -poly-L-Lys was degraded quickly by the macrophage to small radiolabeled peptide, the internalized 125-I- Man90 -H-poly-D-Lys apparently could not be degraded or exocytosed . The amount of 125I- Man90 -poly-D-Lys which accumulated within the cell was 7-fold higher than the combined amount of surface and intracellular mannose receptors, strongly indicating reutilization of the receptors independent of degradation of the ligand.

Carbohydrate-specific adhesion of alveolar macrophages to mannose-derivatized surfaces.

The ability of rabbit alveolar macrophages to specifically recognize and adhere to surfaces derivatized with carbohydrates was examined. Otherwise inert polyacrylamide gels were derivatized with aminohexylglycosides as previously described (Guarnaccia, S. P., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14288-14292). Intact viable rabbit alveolar macrophages, isolated by lung lavage, were placed in contact with surfaces derivatized with different glycosides. Only those surfaces derivatized with alpha-D-mannose residues were capable of supporting rabbit alveolar macrophage adhesion. Adhesion was rapid, obtaining maximal levels within 10 min, and occurred readily at either 0 or 37 degrees C. The carbohydrate specificity of the cell adhesion was investigated by the use of soluble carbohydrate inhibitors. The potency of various saccharides to block the adhesion correlated with that demonstrated for blocking the uptake or binding of radiolabeled soluble glycoproteins (Shepherd, V. L., Lee, Y. C., Schlesinger, P. H., and Stahl, P. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1019-1022). Thus, the order of potency observed was: D-Man congruent to L-Fuc greater than D-GlcNAc congruent to D-Glc much greater than D-Gal congruent to D-GalNAc congruent to L-rhamnose. While soluble monosaccharides were capable of blocking adhesion when added in millimolar concentrations, polymannosylated neoglycoproteins were able to block adhesion in the nanomolar concentration range. Adhesion to the mannose-derivatized surfaces was a dynamic event even at 0 degrees C, since adhesion was less susceptible to monosaccharide inhibition at later incubation times. Surfaces derivatized with aminohexyl S-mannoside ligands were more effective in supporting adhesion than those derivatized with the corresponding O-mannosides. Soluble inhibitor studies suggest that this was due to a more favorable conformation of the S-glycoside for binding to the cell surface receptor. The results reported here demonstrate that the previously reported alveolar macrophage mannose/fucose receptor can mediate carbohydrate-specific cell adhesion.

The binding and processing of mannose-bovine serum albumin derivatives by rabbit alveolar macrophages. Effect of the sugar density.

Mammalian alveolar macrophages are known to bind, internalize, and degrade glycoconjugates containing terminal D-mannosyl residues such as bovine serum albumin modified with 2-imino-2-methoxyethyl 1-thio-alpha-D-mannopyranoside (Mann-AI-BSA) (Lee, Y. C., Stowell, C.P., and Krantz, M. J. (1976) Biochemistry 15, 3956-3963). In this report, the binding (2 degrees C) and initial uptake (37 degrees C) of Mann-AI-BSA (n = 5, 13, 24, and 43) by rabbit alveolar macrophages were examined. Man43-AI-BSA had about 400 times higher affinity (Kd = 2.0 nM) for the macrophage than the Man5-AI-BSA ligand (Kd = 820 nM) at 2 degrees C. Kinetic analysis of 125I-Man43-AI-BSA binding to macrophages at 2 degrees C yielded an association rate constant of 1.2 X 10(6) M-1 min-1 and a dissociation rate constant of 5.9 X 10(-3) min-1 (t1/2 = 117 min). The association rate constant at 37 degrees C was 2 orders of magnitude greater than at 2 degrees C. When the endocytotic process at 37 degrees C was analyzed by Michaelis-Menten-type kinetics, Man5-AI-BSA had a K uptake 20 times higher than that of Man43-AI-BSA (328 and 18 nM, respectively). The maximal uptake velocities at 37 degrees C were, however, very similar for all four Man-AI-BSA derivatives (62,600-83,000 molecules/cell/min). The rate constants for internalization and hydrolysis for 125I-Man43-AI-BSA and 125I-Man13-AI-BSA were determined using a steady state approach. The internalization rate constant for 125I-Man43-AI-BSA, 1.23 (+/- 0.20) min-1, was similar to that constant obtained for 125I-Man13-AI-BSA, 1.80 (+/- 0.67) min-1. The hydrolysis rate constants for the two ligands were also close, 7.4 (+/- 0.2) X 10(-2) and 9.2 (+/- 0.5) X 10(-2) min-1, for 125I-Man43-AI-BSA and 125I-Man13-AI-BSA, respectively.

Stimulation of mannose-binding activity in the rabbit alveolar macrophage by simple sugars.

Mammalian alveolar macrophages are known to bind glycoconjugates with terminal D-mannosyl residues (Stahl, P. D., Rodman, J. S. Miller, M. J., and Schlesinger, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1399-1403). Although macromolecules containing D-mannosyl residues, such as bovine serum albumin modified with 2-imino-2-methoxyethyl 1-thio-alpha-D-mannopyranoside (mannose-BSA) (Lee, Y. C., Stowell, C. P., and Krantz, M. J. (1976) Biochemistry 15, 3956-3963), were very potent inhibitors of 125I-mannose-BSA binding to macrophages, the monosaccharides D-mannose, L-fucose, N-acetyl-D-glucosamine, and D-glucose doubled to tripled the binding of 125I-mannose-BSA to intact rabbit lung macrophages at 2 degrees C. The monosaccharide concentration required for maximum stimulation and the extent of stimulation were dependent on the number of D-mannosyl residues attached to the BSA ligand. The lower the number of D-mannosyl residues coupled to BSA, the smaller the effect by D-mannose and the lower the D-mannose concentration at which it occurred. Equilibrium binding analysis indicated that the apparent affinity of cell surface receptor for ligand increased from Kd = 1.0 nM to Kd = 0.3 nM under conditions of maximal stimulation of 125I-mannose43-BSA binding.

Steady state and kinetic analysis of the binding of asialoorosomucoid to the isolated rabbit hepatic lectin.

The binding of 125I-asialoorosomucoid and the isolated rabbit hepatic lectin were studied at 0 degrees C. The steady state binding data were analyzed both by a direct curve-fitting procedure that utilizes nonlinear least squares regression analysis, and by conventional Scatchard plot analysis. Two classes of binding sites, present in approximately equal concentrations, were detected by both analytical procedures. The total amount of 125I-asialoorosomucoid bound was between 0.2 and 1.0 mol/mol of lectin for each class of lectin binding sites, if the molecular weight of the lectin is between 100,000 and 500,000. The estimated apparent equilibrium dissociation constants were Kapp = 0.87 nM and 1100 nM using the direct curve-fitting method and 0.63 nM and 43 nM from Scatchard plot analysis. Analysis of the forward binding reaction yielded apparent rate constants k1 = 6.8 X 10(6) M-1 and k-1 = 1.5 X 10(-2) min-1, and, therefore, Kapp = 2.1 nM. (Kapp is the apparent dissociation constant of the complex between asialoorosomucoid and hepatic lectin, K1 is the bimolecular rate constant for association of asialoorosomucoid and hepatic lectin, and K-1 is the rate constant for dissociation of the asialoorosomucoid-hepatic lectin complex.) In contrast, the reverse reaction was too slow in the presence of excess unlabeled asialoorosomucoid, and too fast in the presence of methyl beta-D-galactopyranoside to be accommodated by the simple bimolecular reaction model.