Modification of fibrinogen with poly(ethylene glycol) and its effects on fibrin clot characteristics.

The suitability of existing topical fibrin glue preparations for tissue sealing or local drug delivery applications is greatly limited by their poor mechanical properties and the limited capacity of fibrinogen (Fgn) to actively bind growth factors or other therapeutic agents. Poly(ethylene glycol) (PEG) offers potential solutions to these problems by providing a mechanism for increasing the number of crosslinks between adjacent fibrin monomer molecules or for covalently crosslinking Fgn to therapeutic agents. The feasibility of this approach requires the full biological activity, or clottability, of PE glycolated Fgn. This study characterizes the clot characteristics of Fgn modified to varying degrees with monofunctional succinimidyl propionate PEG (5000 Da). The data indicate that, although thrombin clotting times are significantly altered, Fgn maintains 90% of its capacity to clot upon the addition of up to 5 PEG/Fgn. Further derivatization significantly decreases the Fgn clottability. The addition of up to 5 PEG/Fgn has little, if any, effect on the kinetics of degradation by plasmin. The results suggest that limited modification of Fgn with lysine-reactive PEG allows therapeutic enhancement of fibrin glues.

Proteoglycans at the bone-implant interface.

The widespread success of clinical implantology stems from bone's ability to form rigid, load-bearing connections to titanium and certain bioactive coatings. Adhesive biomolecules in the extracellular matrix are presumably responsible for much of the strength and stability of these junctures. Histochemical and spectroscopic analyses of retrievals have been supplemented by studies of osteoblastic cells cultured on implant materials and of the adsorption of biomolecules to titanium powder. These data have often been interpreted to suggest that proteoglycans permeate a thin, collagen-free zone at the most intimate contact points with implant surfaces. This conclusion has important implications for the development of surface modifications to enhance osseointegration. The evidence for proteoglycans at the interface, however, is somewhat less than compelling due to the lack of specificity of certain histochemical techniques and to possible sectioning artifacts. With this caveat in mind, we have devised a working model to explain certain observations of implant interfaces in light of the known physical and biological properties of bone proteoglycans. This model proposes that titanium surfaces accelerate osseointegration by causing the rapid degradation of a hyaluronan meshwork formed as part of the wound-healing response. It further suggests that the adhesive strength of the thin, collagen-free zone is provided by a bilayer of decorin proteoglycans held in tight association by their overlapping glycosaminoglycan chains.

Body weight and composition in the Sprague Dawley rat: comparison of three outbred sources.

Body weight with age and final composition for water, fat, and protein, were measured in three outbred stocks derived from the Sprague Dawley rat. Three groups of 10 individually housed male rats from three outbred colonies (Crl:CD[SD]BR, Hsd:SD[SD], and Tac:N[SD]fBR) provided with standard rodent chow and water ad libitum were weighed weekly for 56 consecutive weeks. Rats in each group were born on the same day, and each group was born within a 4-day period. Significant group effects were found for upper weight limit, carcass weight, water, and fat content (P < 0.05). The Hsd:SD(SD) and Tac:N(SD) rats grew, in proportion to their upper weight limits, at a faster rate (P < 0.01) and contained a greater percentage of body protein (P < 0.05) than did heavier, reportedly shorter-lived Crl:CD(SD)BR rats.

Effects of vitamin A on growth and differentiation of human tracheobronchial epithelial cell cultures in serum-free medium.

Differentiating epithelial cell cultures from human tracheobronchial epithelium have been propagated in serum-free medium. The major objective of this study was to examine the trophic effects of vitamin A on cell multiplication and morphology of the tracheal cell cultures. The cellular responses were analyzed in terms of growth kinetics, morphological and ultrastructural alterations and secretion of glycoconjugates. Cell cultures in control medium exhibited characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and numerous secretory vesicles in the cytoplasm. Vitamin A at 10(-6) M and 10(-7) M inhibited cell replication and enhanced the secretion of [3H]glucosamine-labeled glycoconjugates. Further, vitamin A increased the production of plasma membrane vesicles and acquisition by the cells of a highly secretory ultrastructure. This in vitro model of human epithelial cells will be important in the investigation of various aspects of growth and differentiation.

The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Isolation and characterization of the heparan sulfate proteoglycans of brain. Use of affinity chromatography on lipoprotein lipase-agarose.

Heparan sulfate proteoglycans were extracted from rat brain microsomal membranes or whole forebrain with deoxycholate and purified from accompanying chondroitin sulfate proteoglycans and membrane glycoproteins by ion-exchange chromatography, affinity chromatography on lipoprotein lipase-Sepharose, and gel filtration. The proteoglycan has a molecular size of approximately 220,000, containing glycosaminoglycan chains of Mr = 14,000-15,000. In [3H]glucosamine-labeled heparan sulfate proteoglycans, approximately 22% of the radioactivity is present in glycoprotein oligosaccharides, consisting predominantly of N-glycosidically linked tri- and tetraantennary complex oligosaccharides (60%, some of which are sulfated) and O-glycosidic oligosaccharides (33%). Small amounts of chondroitin sulfate (4-6% of the total glycosaminoglycans) copurified with the heparan sulfate proteoglycan through a variety of fractionation procedures. Incubation of [35S]sulfate-labeled microsomes with heparin or 2 M NaCl released approximately 21 and 13%, respectively, of the total heparan sulfate, as compared to the 8-9% released by buffered saline or chondroitin sulfate and the 82% which is extracted by 0.2% deoxycholate. It therefore appears that there are at least two distinct types of association of heparan sulfate proteoglycans with brain membranes.

Characterization of an amino acid fucoside of normal and SV40-transformed human embryonic lung cells.

The incorporation of radioisotopically labeled fucose into a prominent fucosylated component, i.e., fucose-labeled amino acid fucoside 4c (FL4c), of human embryonic lung cells is markedly decreased in cell lines derived from human tumors. In the current study, we have extended the above observations by examining more closely related normal and transformed human cells, e.g., Wi38 cells and SV40-transformed Wi38 cells. We have found that the level of radioisotopically labeled fucose incorporated into FL4c of the SV40-transformed human embryonic lung cells is dramatically reduced as compared to their normal counterpart cells. Additionally, we have chemically characterized FL4c. FL4c was purified from monkey tissues using a combination of gel filtration chromatography, ion-exchange chromatography, and preparative thin-layer chromatography. Analysis of this material was accomplished by amino acid analyzer and by gas-liquid chromatography; fucose, glucosamine, and aspartic acid in molar ratios of approximately 1.0:1.0:1.0 were observed. Furthermore, alpha-L-fucosidase treatment of [3H]fucose-labeled FL4c revealed that the fucose was alpha-linked and in a terminal position. Similar treatment of [3H]glucosamine-labeled FL4c yielded a component that cochromatographed with N-acetylglucosaminylasparagine. The combined results of the structural studies are consistent with the structure of FL4c being alpha-fucosyl-N-acetylglucosaminylasparagine.

Characterization of novel amino acid fucosides.

The structures of FL4b and of two other related amino acid fucosides have been determined by a combination of methylation analysis and enzymatic digestion. Additionally, the anomeric configurations of the carbohydrate moieties of FL4a, previously shown to be glucosyl(1 leads to 3)fucosyl 1 leads to threonine (Steiner, S., Via, D. P., Klinger, M., Larriba, G., Sramek, S., and Laine, R. (1978) in Glycoproteins and Glycolipids in Disease Processes (Walborg, E. F., Jr., ed) pp. 378-403, American Chemical Society, Washington, D. C.) have been determined by enzymatic digestion. The results indicate that the structures are: FL3a, fucosyl alpha 1 leads to threonine; FL3b, fucosyl alpha 1 leads to serine; FL4a, glucosyl beta 1 leads to 3 fucosyl alpha 1 leads to threonine; and FL4b, glucosyl beta 1 leads to 3 fucosyl alpha 1 leads to serine. FL4a, which appears to have the same structure as a component from human urine (Hallgren, P., Lundblad, A., and Svensson, S. (1975) J. Bil. Chem. 250, 5312-5314), and FL4b are highly unusual in that they contain fucose in a nonterminal position. The fucosyl-serine linkage found in compounds FL3b and FL4b is a novel structure.