Prospective study of the significance of contact sensitization caused by metal implants

Cell nuclei of mouse hepatoma contain various proteoglycans (PG) which include heparan sulfate proteoglycan (HS-PG), dermatan sulfate proteoglycan (DS-PG), and chondroitin sulfate proteoglycan (CS AC-PG). The latter is not found in cell nuclei of normal mouse liver. Heparan sulfate (HS) and dermatan sulfate (DS) are the main constituents of carbohydrate chains of nuclear proteoglycans of tumor and normal cells, respectively. Changes in the composition of nuclear PG during malignant transformation are discussed considering the concept of their possible involvement in the regulation of cell mitotic activity.     

Knee osteoarthritis after meniscectomy: prevalence of radiographic changes after twenty-one years, compared with matched controls

Aggrecan, a large aggregating proteoglycan, is one of the major structural components of cartilage. Its core protein contains three glubular domains and two glycosaminoglycan-attachment domains. These domains play various roles to maintain cartilage structure and function. An N-terminal globular domain binds hyaluronan and link protein to form huge aggregates. The chondroitin sulfate (CS) chains attach to the CS domain and provide a hydrated, viscous gel that absorbs compressive load. Two autosomal recessive chondrodysplasias, cartilage matrix deficiency (cmd) in mice and nanomelia in chicken are both caused by aggrecan gene mutations. Cmd homozygotes die shortly after birth, while the heterozygotes are born normal. However, cmd heterozygotes develop late onset of spinal disorder, which suggests aggrecan as a candidate gene predisposing individuals to spinal problems. Nanomelia is a useful model to elucidate intracellular trafficking of proteoglycans. Further studies on aggrecan will lead to prophylaxis and treatment of joint destructive diseases such as osteoarthrosis and to elucidation of cartilage development, which is essential for skeletal formation.     

Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries

Although a large percentage of the volume of human restenotic arterial lesions is occupied by extracellular matrix (ECM), the composition and organization of this ECM are not well characterized. In this study, restenotic segments taken from 30 human peripheral arteries by directional atherectomy at times ranging from 13 days to 36 months after angioplasty were analyzed for specific patterns of ECM composition and organization by light and electron microscopic histochemistry and immunohistochemistry. Histochemical analysis revealed the presence of distinct zones, enriched either in proteoglycans or fibrillar collagen. Most sections contained these regions juxtaposed to each other. The frequency of these two distinct ECMs did not change as a function of time after angioplasty. The collagen-rich zone usually contained elongated smooth muscle cells spaced close together except in regions resembling fibrous plaques. The proteoglycan-rich ECM contained both elongated and stellate-shaped smooth muscle cells randomly arranged and separated by wide distances. This region resembled the loose-connective-tissue-containing myxoid region typical of restenotic lesions. Immunohistochemical analysis of these regions revealed that the proteoglycan-containing zone stained intensely for versican, a large interstitial chondroitin sulfate proteoglycan, whereas the collagen-containing areas were mostly negative for versican but positive for type I collagen. The versican-positive regions also immunostained for biglycan, a small leucine-rich dermatan sulfate proteoglycan, and sparsely for elastin. However, both of these ECM molecules were present in the versican-negative type I collagen-positive regions of the lesions. These results suggest that the development of restenotic lesions involves localized deposits of specific ECM molecules that may play a role in the asymmetric renarrowing of this tissue after angioplasty.     

Isolation and characterization of the proteoglycans synthesized by adult human pulp fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human adult dental pulps have been isolated and characterized on the basis of their glycosaminoglycan content, molecular size and charge. The proteoglycans were identified by their labelling with [35S] sulphate and susceptibility to digestion by papain. The sulphated glycosaminoglycans associated with the proteoglycans were identified following specific enzymatic and chemical degradations as chondroitin sulphate, dermatan sulphate and heparan sulphate. Dermatan sulphate and chondroitin sulphate and heparan sulphate were the principal glycosaminoglycans associated with the cell layers. The proteoglycans could be fractionated on the basis of their charge and size into a number of heterogeneous pools. The principal proteoglycans isolated were small and contained either chondroitin sulphate or dermatan sulphate and most likely correspond to decorin and biglycan. Other molecules with features similar to versican and syndecan were also identified.     

Overproduction of Campylobacter ferritin in Escherichia coli and induction of paracrystalline inclusion by ferrous compound

Levels of the proteoglycans hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate in brains were measured. The samples were from 25 brains obtained at autopsy from patients who had senile dementia of Alzheimer's type (SDAT) (60 to 97 years old) and 10 brains from non-demented elderly subjects (75 to 101 years old). Proteoglycan levels in the brains of patients with SDAT were twice as high in the hippocampus and four times higher in the superior frontal gyrus than the levels in brains of non-demented elderly subjects. The content of heparan sulfate proteoglycan was greatest nine times higher in the hippocampus and seven times higher in the superior frontal gyrus. By immuno-electronmicroscopic examination, heparan sulfate proteoglycan was found in the basement membranes of capillaries, and in a portion of the cores of the senile plaques.     

Human follicular fluid proteoglycans in relation to in vitro fertilization

OBJECTIVE: To determine whether the concentrations of proteoglycans and hyaluronan in human follicular fluid (FF) are associated with follicular volume, oocyte fertilization, and ET during IVF. DESIGN: The FF from individual follicles were collected. Enzyme-linked immunosorbent assay methods for quantification of a larger chondroitin sulfate proteoglycan and a smaller composite heparan-chondroitin sulfate proteoglycan were established. Hyaluronan and E2 were measured by RIA techniques. PATIENT(S): Sixteen infertile women participating in the IVF program. MAIN OUTCOME MEASURE(S): Concentrations of the proteoglycans, follicular volume, fertilization, and ET rates. RESULT(S): The follicles contained high concentrations of proteoglycans with an average of 0.8 mg/mL of FF, and approximately 70% consisted of the larger chondroitin sulfate proteoglycan, and 30% of the heparan-chondroitin sulfate proteoglycan. A negative correlation was found between the follicular volume, the chondroitin sulfate proteoglycan (r = -0.43), and hyaluronan (r = -0.56). The percentage of embryos developed in culture was significantly higher in follicles larger than 2 mL. A significant and 35% lower concentration of the chondroitin sulfate proteoglycan was found in larger follicles from which subsequent ET was observed. THe heparan-chondroitin sulfate proteoglycan and hyaluronan were both unrelated to fertilization and ET in vitro. CONCLUSION(S): Lower concentrations of chondroitin sulfate proteoglycan were associated with higher follicular volumes and greater fertilization and ET rates. These associations could merely reflect the maturation of the follicle or a role of the chondroitin sulfate proteoglycan in the fertilization process.     

Five familial hypercholesterolemic kindreds in Japan with novel mutations of the LDL receptor gene

BACKGROUND AND OBJECTIVES: Proteoglycans of the extracellular matrix are vital to the growth and evolution of malignant neoplasms. The present study determined the composition of proteoglycans isolated from paired specimens of normal breast and adenocarcinoma of the breast harvested from each patient (n = 8). The proteoglycans were then tested for their ability to stimulate endothelial cell proliferation. METHODS: Proteoglycans were isolated by extraction with 4 M guanidine hydrochloride and purified by CsCl density-gradient centrifugation. The proteoglycans were characterized and tested for their ability to simulate endothelial cell proliferation. RESULTS: In each case, the total proteoglycan content of the tumor was significantly greater than that of the corresponding normal tissue. The proteoglycans isolated from the carcinoma contained 32.2% (13.7/42.5) more chondroitin sulfate, 18.5% (5.6/30.2) less dermatan sulfate, and 29.6% (8.1/27.3) less heparan sulfate than did the proteoglycans of normal breast tissue. Proteoglycans from normal tissue did not stimulate endothelial cell proliferation, whereas those from malignant tissue stimulated proliferation by 1.3- to 1.5-fold. CONCLUSIONS: These results indicate that malignant breast tissue exhibits both qualitative and quantitative changes in proteoglycan composition, which, in turn, may stimulate endothelial cell proliferation.     

Critical roles of glycosaminoglycan side chains of cartilage proteoglycan (aggrecan) in antigen recognition and presentation

Systemic immunization of BALB/c mice with proteoglycan (aggrecan) from fetal human cartilage induces progressive polyarthritis, an experimental disease similar to human rheumatoid arthritis. The development of the disease in this genetically susceptible murine strain is based on cross-reactive immune responses between the immunizing fetal human and mouse self-proteoglycans. One of the cross-reactive and arthritogenic T cell epitopes (92GR/QVRVNSA/IY) is localized in the G1 domain of human/murine proteoglycan. Susceptible BALB/c mice, however, develop arthritis only if both the chondroitin sulfate (CS) and keratan sulfate (KS) side chains of the arthritogenic human proteoglycans are removed. The function of these two glycosaminoglycan side chains is opposite. The presence of a KS side chain in adult proteoglycan inhibits the recognition of arthritogenic T cell epitopes, prevents the development of T cell response, and protects animals from autoimmune arthritis. In contrast, the depletion of the CS side chain generates clusters of CS stubs and provokes a strong B cell response. These carbohydrate-specific B cells are the most important proteoglycan APC. Taken together, proteoglycan-induced progressive polyarthritis is dictated by three major components: genetic background of the BALB/c strain, highly specific T cell response to epitope(s) masked by a KS chain in aging tissue, and the presence of proteoglycan (CS stub)-specific B cells required for sufficient Ag presentation.     

Proteoglycans are present in the transverse tubule system of skeletal muscle

The transverse tubule system (T-tubule, T-system) of skeletal muscle is a membranous network that penetrates the interior of myofibers. The T-system is continuous with the sarcolemma and therefore provides a path for membrane excitation to reach internal myofibrils. In this study we demonstrate that T-tubules in elasmobranch fish, frog, and rat skeletal muscle contain a matrix of chondroitin sulfate proteoglycans. We used anti-T1, a mouse monoclonal antibody that recognizes a rare chondroitin sulfate epitope, for immunolocalization and biochemical studies. First, we find that T1 immunoreactivity colocalizes with a T-tubule marker, the dihydropyridine receptor alpha 2 subunit, in both frog and fish muscle. Secondly, the distribution of T1 immunoreactivity exactly matches the different distribution of T-tubules in rat and frog muscle. In rat muscle, two bands of T1 immunoreactivity are detected per sarcomere, a distribution that corresponds to the T-tubules located at the two A-I junctions of each sarcomere. In frog muscle, we detect one band of T1 immunoreactivity per sarcomere that corresponds to the one T-tubule per sarcomere located at the Z line. Lastly, we have isolated and biochemically characterized T1 antigenicity from fish skeletal muscle. Like extracellular matrix proteoglycans of cartilage, T1 antigenicity requires denaturing conditions to be solubilized. In fish muscle, two chondroitin sulfate proteoglycans bear T1: a heavily glycosylated proteoglycan with a molecular mass of about 1000 kDa, and a smaller proteoglycan that has a mobility on SDS-PAGE like a protein of molecular mass 280 kDa. We propose that proteoglycans function as structural components in the T-system. The proteoglycans may form a matrix, like the one formed by the cartilage proteoglycans they resemble, that can withstand the cytosolic osmotic pressures present in muscle cells and therefore may prevent the T-tubule from collapsing. We present a quantitative argument in support of this hypothesis.     

Polarized infrared spectra of crystalline glycosaminoglycans

Polarized infrared spectra have been recorded for oriented, crystalline specimens of hyaluronates, chondroitin 4-sulfate and 6-sulfate, dermatan sulfate, and a cartilage proteoglycan, having different known chain conformations as determined by X-ray diffraction. The dichroism data for the vibrational modes of the amide and carboxyl groups have been interpreted with respect to the particular molecular structures.     

Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.     

Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell

The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

The microfibrillar proteins fibulin-1 and fibulin-2 were previously identified as prominent components of the endocardial cushion tissue (ECT) during heart development and shown to persist in adult valves and septa. Immunogold staining has now been used to compare their localization in embryonic (days 9-11) and adult mouse heart with that of fibronectin and the chondroitin sulphate proteoglycan versican. All four proteins were deposited in the ECT, which consists of a hyaluronan-rich, mainly unstructured matrix, but were barely detectable in myocardial basement membranes or within endocardial cells. Digestion with hyaluronate lyase selectively released the fibulins and versican but not fibronectin from the ECT. Yet neither of the two fibulins bound to hyaluronan in solid-phase assays, in contrast to versican. In the adult heart valve, all four proteins could be detected close to cross-striated collagen fibrils or microfibrils, but only versican was lost upon exposure to hyaluronate lyase. The data indicate that fibulins are associated with the hyaluronan-matrix of ECT through a bridge of versican, but that this association changes upon valve development to another supramolecular, presumably microfibrillar organization based on fibronectin and/or fibrillins.     

Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.     

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.     

Dermatan sulfate released after injury is a potent promoter of fibroblast growth factor-2 function

Proteoglycans have been shown in vitro to bind multiple components of the cellular microenvironment that function during wound healing. To study the composition and function of these molecules when derived from an in vivo source, soluble proteoglycans released into human wound fluid were characterized and evaluated for influence on fibroblast growth factor-2 activity. Immunoblot analysis of wound fluid revealed the presence of syndecan-1, syndecan-4, glypican, decorin, perlecan, and versican. Sulfated glycosaminoglycan concentrations ranged from 15 to 65 microgram/ml, and treatment with chondroitinase B showed that a large proportion of the glycosaminoglycan was dermatan sulfate. The total glycosaminoglycan mixture present in wound fluid supported the ability of fibroblast growth factor-2 to signal cell proliferation. Dermatan sulfate, and not heparan sulfate, was the major contributor to this activity, and dermatan sulfate bound FGF-2 with Kd = 2.48 microM. These data demonstrate that proteoglycans released during wound repair are functionally active and provide the first evidence that dermatan sulfate is a potent mediator of fibroblast growth factor-2 responsiveness.