Biomedical subjects

A I Caplan

Publications and source records attributed to A I Caplan.

At least 109 records · Page 6Linked to original sources

PubMed1991

Comparison of the cartilage proteoglycan core protein synthesized by chondrocytes of different ages.

Chondrocytes of different ages synthesize proteoglycans which have structural differences in both the chondroitin sulfate and keratan sulfate glycosaminoglycans. In order to ascertain whether age-dependent differences also occur in the core protein, the chick limb bud mesenchymal cell culture system was utilized to analyze newly synthesized proteoglycan core protein from undifferentiated mesenchymal cells (day 1 and 2), newly differentiated cartilage (day 4), mature cartilage (day 8), and senescent cartilage (day 16). The core protein synthesized at various times was identified by radiolabeling with [3H]leucine and [35S]sulfate immediately prior to extraction and purification. The sizes of the various core protein preparations were compared by electrophoresis on a 3% polyacrylamide gel after partial deglycosylation with chondroitinase AC and keratanase. The proteoglycans from day 4, 8, and 16 cultures each give rise to a single band of approximately 475,000 daltons. The proteoglycans from day 1 and 2 cultures also give rise to the 475,000 dalton band, but each contains several other components which produce a smear of high molecular weight material on the gel. The monomer proteoglycans were incubated with cyanogen bromide and the resultant peptides separated by electrophoresis on a 5-17.5% polyacrylamide gel. The peptide displays of core proteins synthesized on days 4, 8 and 16 are virtually identical in terms of the number and electrophoretic distribution of the core protein peptides. In contrast, proteoglycan core proteins from day 1 and day 2 cultures give rise to peptide displays which resemble those from older cultures in some respects but have distinct features as well. The absence of structural variation in the newly synthesized proteoglycan core proteins from cartilage of different ages suggests that the age-related changes in the structure of the intact proteoglycans result from differences in the glycosaminoglycan biosynthetic machinery rather than alterations in the acceptor molecule (i.e., the core protein).

Aggrecans↗

PubMed1991

Collagens of the chicken eggshell membranes.

An immunohistochemical analysis of the eggshell membranes shows the occurrence of type X collagen while type I collagen was not detected by using an appropriate monoclonal antibody with untreated shell membranes. A positive immuno-reaction for type I collagen was obtained after digestion of the shell membranes with pepsin. These observations indicate the possibility that type I collagen epitope was masked by type X collagen and that type X collagen may serve as an inhibitory boundary for biomineralization.

Acetates↗

PubMed1991

The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells.

When porous calcium phosphate ceramic is combined with marrow cells and grafted either heterotopically or orthotopically, bone forms inside the pores on the surface of the ceramic beginning at three weeks after implantation. The question remains as to whether the newly formed bone is derived from host or donor cells. To study the origin of bone cells formed in these composite grafts of marrow cells and ceramic, quail marrow cells from long bones were introduced into ceramics and the composites were implanted into subcutaneous pouches of immunologically nonreactive athymic nude mice. The ceramics were recovered at two to 84 days following surgery, fixed, decalcified, embedded, sectioned, and examined for the location of a quail-specific nucleolar marker and the binding of a specific antiserum against quail cells. Our observations indicate that ceramic-associated osteogenesis is a biphasic phenomenon: an early phase, the first three to four weeks after implantation, in which donor cells are largely responsible for the observed osteogenesis, and a second phase, eight to 12 weeks postsurgery, in which host cell actions predominate. During the second stage, the ceramic pores begin to show the formation of marrow of host origin, and the mesenchymal marrow component appears to be osteogenic because the bone formed during this late postgrafting stage contains osteocytes of host and donor origin. The second phase therefore results in chimeric bone composed of quail and mouse. These studies clearly document the donor origin of the initial bone formation and indicate that marrow contains progenitor cells capable of forming de novo bone.

Animals↗

PubMed1991

A spectrophotometric modification of a sensitive densitometric Safranin O assay for glycosaminoglycans.

Recently, an assay for quantification of glycosaminoglycans has been reported based on precipitation with Safranin O (Lammi, M. and Tammi, M. (1988) Anal. Biochem. 168, 352-357). In this procedure, the precipitate which forms when the glycosaminoglycan or proteoglycan is mixed with the Safranin O is collected with a dot-blot apparatus onto a membrane filter. The intensity of the color in the dots is measured densitometrically and is proportional to the amount of glycosaminoglycan or proteoglycan in the sample. This report describes a modification of the densitometric Safranin O assay which allows its use as a spectrophotometric assay. For this, the precipitates are solubilized in cetylpyridinium chloride and the absorbance determined for the resulting solutions. As with the densitometric method, guanidinium chloride diminishes the color intensity. However, the color is stable, even after solubilization, for at least one week. The precipitates collected from as much as 10 micrograms of material can be solubilized in as little as 100 microliters of cetylpyridinium chloride, so that increased sensitivity may be obtained if the solubilized precipitate is measured in a microcuvet. Thus, solubilization with cetylpyridinium chloride allows use of the Safranin O assay for glycosaminoglycans and proteoglycans even when a densitometer is unavailable.

Cetylpyridinium↗

PubMed1991

The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks.

When whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. As an extension of earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture, and the adherent cells were cultivated, mitotically expanded, subcultured, harvested, placed in small cubes of porous calcium phosphate ceramic, and grafted into subcutaneous sites of syngeneic rats. Primary marrow-derived, cultured mesenchymal cells introduced into ceramic showed strong osteogenic potential, with bone forming in the pore regions of ceramic as early as two weeks after in vivo implantation; cartilage was observed infrequently in pores that appeared to be avascular. Osteogenesis could be observed after the 18th subculture (over 36 population doublings) when the cells were tested in ceramic at subcutaneous sites, whereas chondrogenesis was observed with only the first and second subcultured cells in the ceramic delivery vehicle. With increasing numbers of subcultures, the initiation of osteogenesis and the apparent rate of bone formation declined, and the course of osteogenesis was delayed. Cultured, marrow-derived mesenchymal cells, even after the 21st subculture (over 40 population doublings), exhibited a positive histochemical reaction for alkaline phosphatase. However, the in vivo osteogenic potential of these cells was not correlated with their alkaline phosphatase activity. The implantation of cell pellets or the injection of cell suspensions of fresh or cultured, adherent marrow cells never produced bone or cartilage in heterotopic sites. These data indicate that porous ceramic provides an excellent delivery vehicle for cells that are capable of osteogenic expression and suggest that the composite graft of marrow-derived mesenchymal cells and porous ceramic may be useful for repair of massive bone defects. It may be possible to culture marrow mesenchymal cells as a source for reparative cells for implantation back into autogeneic sites.

Alkaline Phosphatase↗

PubMed1991

The effects of Rumalon, a glycosaminoglycan peptide complex, in a partial meniscectomy model of osteoarthritis in rabbits.

The effects of Rumalon, a glycosaminoglycan peptide complex (GP-C), were evaluated in a partial meniscectomy model of osteoarthritis (OA). When rabbits were treated with Rumalon, 0.25 ml/kg 3 times/week for 12 weeks after partial meniscectomy, the proportion of animals that developed cartilage ulceration was lower than that in untreated meniscectomized rabbits (p less than 0.05). At 6 weeks sacrifice, the same dose of Rumalon was associated with a trend toward reduction in the number of animals with ulcerations. Rumalon administered at a dose of 0.5 ml/kg for 12 weeks reduced the number of animals with ulcers, although not at the p less than 0.05 level of significance. Semiquantitative histologic evaluations revealed no differences between control and treatment groups either in the thickness of articular cartilage, density of cells present, safranin-O stain or ratio of DNA/protein. Increased proteoglycan synthesis characteristic of OA was modulated toward normal in association with Rumalon therapy. Diminished OA changes in response to Rumalon are consistent with an ameliorative response to this agent in the partial meniscectomy model in the rabbit.

Animals↗

PubMed1990

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).

Aggrecans↗

PubMed1990

Change in synthesis of sulfated glycoconjugates during muscle development, maturation and aging in embryonic to senescent CBF-1 mouse.

Previous biochemical and morphological studies have demonstrated a change in the synthetic pattern of sulfated proteoglycans during skeletal musculogenesis in the embryonic chick. These studies revealed that a transition occurs in both composition and deposition of sulfated glycoconjugates that parallels the developmental state of the tissue. The current study was undertaken to ascertain whether this transition in the embryonic chick is a conserved developmental process during musculogenesis in the mouse. Leg musculature from embryonic, newborn, juvenile, adolescent, young adult, mature adult and senescent mice, radiolabeled in vivo with [35S]sulfate, was analyzed for relative size and composition of newly synthesized sulfated macromolecules. The data reveal a transition in the synthesis of sulfated proteoglycans and glycoproteins that parallels the myogenic differentiative state of the mouse leg muscle. Embryonic mouse leg musculature synthesizes relatively large proteoglycans consisting of large chondroitin sulfate glycosaminoglycan chains. Subsequently, these major newly synthesized proteoglycans are replaced synthetically by smaller molecules composed of mixtures of dermatan sulfate, chondroitin sulfate and heparan sulfate glycosaminoglycans (newborn through 2 weeks); dermatan sulfate, heparan sulfate and chondroitin sulfate glycosaminoglycans (13 months) and heparan sulfate and dermatan sulfate glycosaminoglycans (25-26 months). The sulfated glycoproteins demonstrate a reciprocal synthetic pattern. Early in development sulfated glycoproteins form a small proportion of the newly synthesized sulfated material. With increasing developmental and maturational age, the proportion of sulfated glycoproteins increases. This continues until they become the predominant sulfated moieties synthesized by senescent mouse muscle. The results from this study thus extend observations initially made in chick to muscle development in the mouse and, therefore, suggest that the transition in synthesis of sulfated glycoconjugates is a conserved developmental process during musculogenesis.

Aging↗

PubMed1990

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a polyanhydride delivery vehicle.

Controlled release delivery vehicles for water-soluble osteogenic proteins from demineralized bovine bone matrix were constructed using polyanhydride polymers. The water-soluble proteins were isolated from a 4 M guanidine hydrochloride extract of bone matrix. The water-soluble proteins possessed Chondrogenic Stimulating Activity (CSA) when tested in stage 24 chick limb bud cell cultures, but were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice by themselves. The polyanhydride polymers alone were also incapable of inducing ectopic cartilage or bone. However, when the water-soluble proteins were incorporated into the polymeric delivery vehicle, the combination was capable of inducing cartilage and bone up to 50% of the time. These results demonstrate that it is possible to use polyanhydride polymers as controlled-release delivery vehicles for soluble bioactive factors that interact with a local cell population.

Animals↗

PubMed1990

Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation.

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.

Animals↗

PubMed1990

Chondrogenic stimulation of embryonic chick limb mesenchymal cells by factors in bovine and human dentine extracts.

Extracts of bovine or human dentine when incorporated into the medium bathing embryonic chick-limb mesenchymal cells caused the stimulation of chondrogenesis. Such extracts were prepared from demineralized bovine or human dentine by suspension in a 4 M guanidinium hydrochloride (GuHCl) solution. After stepwise dialysis down to distilled water to remove the GuHCl, the protein extracts were separated chromatographically on DEAE resin. The crude and separated extracts from both dentines produced morphological changes in the cultured mesenchymal cells similar to those reported for bovine and human adult bone extracts, as well as metachromatic staining with toluidine blue indicating that they stimulated chondrogenesis. Measurements of [35S]-SO4 incorporation and DNA content in the cultures also indicated that the extracts stimulated chondrogenesis but not cell proliferation. The similarity between silver-stained patterns on sodium dodecyl sulphate polyacrylamide gel electrophoresis of corresponding fractions of the bovine and human dentine suggests that they may contain similar or identical proteins.

Animals↗

PubMed1990

Temporal and spatial distribution of type XII collagen in high cell density culture of periosteal-derived cells.

Periosteal-derived cells of young chicks have been reported to possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes under high cell density culture conditions. In this culture, the temporal and spatial distribution of type XII collagen was immunocytochemically assessed using a monoclonal antibody. These high-density plated cells first formed a multilayer of fibroblast-like cells, in which type I and XII collagen were evenly distributed throughout the full thickness of the culture. With time, the top portion of the culture differentiated into bone tissue, while cells below this top layer differentiated into hypertrophic chondrocytes. In this transition, type XII collagen was temporally and spatially colocalized primarily with type I collagen: the top portion of bone layer was positive for both type I and XII collagens, whereas their staining intensity in the bottom portion decreased with time in culture. Using this antibody, type XII collagen was also found in developing embryonic chick tibiotarsus. These observations, taken together, suggest that type XII collagen production is a characteristic property of bone-forming cells.

Animals↗

PubMed1990

Osteogenic cell lineage analysis is facilitated by organ cultures of embryonic chick periosteum.

Monoclonal antibodies against the surface of embryonic osteogenic cells (SB-1, SB-2, SB-3, and SB-5) have been used to characterize the sequence of transitions involved in the osteogenic cell lineage. In the present study, immunohistochemical analyses of the expression of osteogenic cell surface antigens in organ cultures of folded chick periosteum were performed. Unlike traditional culture methods using isolated osteoblastic cells, periosteal explants form a mineralized bone tissue in 4 to 6 days which is virtually identical to the in vivo counterpart. Examination of fresh explants confirm that no mature osteoblastic cells were present, although a discontinuous layer of preosteoblasts was evident. As the wave of osteodifferentiation swept through the cultured tissue, antibody SB-1 reacted with the surface of a large family of cells associated with the developing bone. Antibodies SB-3 and SB-2 reacted with progressively smaller subsets of cells, namely those in successively closer physical association with the newly formed and mineralizing bone. Cells recently encased in bone matrix were stained by both SB-2 and SB-5 antibodies, while those cells deep within the matrix reacted only with antibody SB-5. Analysis of this culture model allows dissection of the discrete cellular transition steps of osteogenesis, and reveals that osteogenic precursor cells proceed through the unique lineage stages which have been documented for in vivo osteogenesis. This culture system has furthermore provided evidence which is used to refine our understanding of the osteogenic cell lineage.

Animals↗

PubMed1990

Osteochondral differentiation and the emergence of stage-specific osteogenic cell-surface molecules by bone marrow cells in diffusion chambers.

The osteochondral potential and emergence of osteogenic cell-surface molecules by avian marrow cells was evaluated in in vivo diffusion chamber cultures. The chambers were inoculated with unselected marrow cells from young chick tibiae and implanted intraperitoneally into athymic mice. At the light microscopic level, morphologic evidence of de novo bone and cartilage formation, including specific immunostaining by antibody probes, was observed in 14 out of 16 chambers incubated for 20 days or longer. In order to monitor the osteogenic differentiation of the marrow-derived cells, indirect immunofluorescence was performed with monoclonal antibodies against stage-specific cell surface antigens on cells of the embryonic osteogenic lineage. The binding of these and other specific monoclonal antibodies in the developing tissue indicates that the cell surface and extracellular matrix molecules expressed by descendants of marrow-derived mesenchymal progenitor cells are indistinguishable from their in vivo counterparts found in embryonic skeletal structures. Furthermore, the experiments reported here describe the first molecular identification of osteogenic cells by probes which are selective for stage-specific surface antigens on cells of the osteogenic lineage. Importantly, bone formation by these marrow-derived cells appears to occur through a lineage progression which is similar to that observed for embryonic tibial osteoblasts. In summary, these data support the use of diffusion chambers inoculated with avian marrow to study aspects of osteogenic and chondrogenic differentiation.

Animals↗

PubMed1990

A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine.

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.

Alkaline Phosphatase↗

PubMed1990

Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum.

Periosteal cells were enzymatically isolated from the tibiae of young chicks, introduced into cell culture, allowed to reach confluence, and subcultured. The freshly isolated or subcultured cells were loaded into diffusion chambers and implanted into the peritoneal cavity of athymic mice to test their osteo-chondrogenic potential in a contained in vivo location. Freshly isolated periosteal cells formed both bone and cartilage tissue in such test chambers, but with a relatively low incidence. In contrast, cultured periosteal cells consistently gave rise to bone and cartilage even after 10 population doublings. With further passages of cells, the osteo-chondrogenic potential diminished substantially, until complete loss of expressivity at 16 population doublings or longer. Cultured muscle fibroblasts, when loaded into diffusion chambers under identical conditions to those of cultured periosteal cells, formed neither bone nor cartilage. These observations suggest that periosteal cells of young chicks contain subsets of progenitor cells or mesenchymal stem cells which possess the potential to differentiate into osteoblasts or chondrocytes, and this potential is retained after enzymatic isolation and for several population doublings in culture.

Animals↗

PubMed1990

Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes.

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the sequence of transitions involved in the osteoblastic cell lineage. These previous data identified distinct cell stages within the osteogenic lineage, but were incomplete. To further refine and extend these observations, additional monoclonal antibodies were generated against the surface of osteogenic cells by immunizing mice with a heterogeneous population of chick embryonic bone cells. Supernatants from growing hybridoma colonies were immunohistochemically screened against frozen sections of stage 35 (day 9.5) chick tibiae. One cell line, SB-5, which secretes an antibody against the surface of osteogenic cells was successfully cloned, stabilized, and immortalized. Studies on the developmental progression of osteogenesis in the embryonic chick tibia reveal that cells within the lineage stages from Pre-Osteoblast to Secretory Osteoblast were never observed to react with antibody SB-5 at any time. By contrast, strong cell surface immunoreactivity was present on mature osteoblastic cells as they became Osteocytes. Furthermore, in cultures of osteogenic cells derived from embryonic calvaria or tibiae, cells possessing the SB-5 antigen on their surface displayed a morphology remarkably similar to that of Osteocytes found in situ. Double immunofluorescent staining of developing chick tibiae with SB-5 and SB-2, a monoclonal antibody directed against the surface of Secretory Osteoblasts, indicates that these cells proceed through an intermediate lineage step before becoming terminally differentiated Osteocytes. This transitory cell state is characterized by the simultaneous cell surface binding of antibodies SB-2 and SB-5, and is referred to as the Osteocytic Osteoblast stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.

In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratin sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories. To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached. Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alteration with lanes of the growth-promoting molecule laminin (LN). Neurite outgrowth was abundant along stripes of LN. In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border. The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing. We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present. A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG. These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth. Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycoaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition. These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.

Animals↗