Delta Di-4S Research Articles

Changes in glycosaminoglycan characteristics during progression of a human gingival carcinoma xenograft line in nude mice.

We investigated changes in the glycosaminoglycans (GAGs) during progression of a human gingival carcinoma xenograft line, GK -1, in nude mice. The GAGs extracted from cancers 3, 5, 7, 10 and 15 weeks after transplantation consisted of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS) as major components, and dermatan sulfate (DS) as a trace component for all cancers. HPLC analysis revealed that the HA content per defatted tissue dry weight increased in the cancers 5 weeks after transplantation compared to those of 3 weeks (p < 0.05), while CS for cancers at 10 weeks decreased compared with 7 weeks (p < 0.05). However, HS showed no significant change. Both the CS and DS contained primarily 4-sulfated disaccharide units. Immunohistochemical staining with antibody 2-B-6 for the PGs having delta DI-4S produced by chondroitinase ABC digestion showed that CS is located in the tissue surrounding the cancer nests and mass. These results indicate that the location of accumulation of CS, which primarily contains 4-sulfated disaccharide units, plays an important role in cancer progression.

High-performance liquid chromatography analysis of chondroitin sulfate isomers in human periodontium.

We investigated the chondroitin sulfate in human periodontal samples (gingiva, periodontal ligament, cementum and alveolar bone) collected for orthodontic reasons. Glycosaminoglycans (GAGs) were extracted from the periodontium by enzyme digestion, and unsaturated disaccharide isomers of chondroitin sulfate were obtained by chondroitinase ACII and hyaluronidase digestion. The isomers were analyzed by high-performance liquid chromatography. Chondroitin sulfate was found in all four types of periodontal tissue; its unsaturated disaccharide isomers consisted in delta Di-0S, delta Di-6S, delta Di-4S, delta Di-diSE and delta Di-triS. These four types of periodontal tissue showed different molar ratios of the unsaturated disaccharides. The ratio of delta Di-4S to delta Di-6S was greater in the calcified than in the uncalcified tissue.

Short-end injection capillary electrophoresis for quantification of plasma chondroitin sulfate isomer disaccharides

We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Delta di-0S and Delta di-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.

Isolation and identification of chondroitin sulfates from the mud snail.

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.

Intra-articular levels of prostaglandin E2, hyaluronic acid, and chondroitin-4 and -6 sulfates in the temporomandibular joint synovial fluid of patients with internal derangement.

This study was conducted to measure the intra-articular levels of prostaglandin E2 (PGE2), hyaluronic acid, and chondroitin-4 and -6 sulfate in patients with temporomandibular joint (TMJ) internal derangement involving a closed lock, and to see if these levels correlate with the clinical or arthroscopic findings. Fifteen female patients (16 joints) with a mean age of 36.7 years were diagnosed as having a closed lock by clinical examination and diagnostic MR imaging. The patient's subjective pain was assessed by a visual analog scale (VAS) and a pain questionnaire (pain score), and the interincisal opening was measured. TMJ aspirates were obtained by washing of the joint with saline containing vitamin B12 as a marker for calibration of data. The samples were assayed for PGE2 with a radioimmunoassay, and the concentrations of unsaturated disaccaride isomers of hyaluronic acid (delta di-HA), chondroitin-4 sulfate (delta di-4S), and chondroitin-6 sulfate (delta di-6S) were measured by high-performance liquid chromatography. Immediately after collection of the synovial aspirates, diagnostic arthroscopy was performed on all but three joints to evaluate the severity of synovitis and cartilage degeneration. The degree of arthroscopic pathology was scored quantitatively. Intra-articular levels of PGE2, delta di-HA(HA), delta di-4S(C4S), and delta di-6S(C6S) were compared with patient's age, mouth opening, VAS rating, pain scores, and arthroscopic scores for synovitis and cartilage degeneration. The PGE2 level did not correlate with the clinical or arthroscopic parameters. HA had a weak correlation with mouth opening (0.54). C4S and C6S were correlated with arthroscopic scores of TMJ degeneration (0.97, 0.89) and with age (0.75, 0.62). The ratio of C4S and C6S to HA was also correlated with the arthroscopic indices of degeneration (0.93, 0.8) and PGE2 level (0.74, 0.69), but not with age. The PGE2 level in the TMJ synovial fluid does not specifically reflect the intensity of pain or synovitis, but the detection of high concentrations of C4S and C6S, compared with the amount of HA, is a possible marker of proteoglycan degradation in the TMJ.

Disaccharide Analysis of Skin Glycosaminoglycan in Localized Scleroderma

Altered metabolism of skin glycosaminoglycan has rarely been investigated in localized scleroderma in contrast to systemic sclerosis. The aim of this investigation is to elucidate the change in skin glycosaminoglycan of localized scleroderma. We analyzed 5 skin samples of localized scleroderma and 10 site-matched control skin samples using high performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling. The involved skin constantly showed an increased amount of delta Di-4S(DS), the main disaccharide unit of dermatan sulfate, a decreased amount of delta Di-HA, the main disaccharide unit of hyaluronic acid, and an elevated ratio of delta Di-4S(DS)/delta Di-HA as compared with the uninvolved skin or the site matched control skin. These results correlate well with those findings in systemic sclerosis, indicating that the alteration in skin GAG may be closely related to the fibrotic process.

High-performance liquid chromatography analysis of chondroitin sulphate isomers in human whole saliva in a variety of clinical conditions.

Tests have been carried out to assess the level of unsaturated disaccharide isomers obtained from chondroitin sulphate in whole saliva, which contains chondroitin sulphate derived from gingival crevicular fluid (GCF). Whole saliva was collected from periodontally diseased subjects (PDS), clinically healthy subjects (CHS) and edentulous subjects (ES). Glycosaminoglycans (GAG) were liberated by digestion with Pronase E, and precipitated with cetylpyridinium chloride and ethanol. The unsaturated disaccharides obtained by chondroitinase ACII digestion of the liberated GAG were analysed by high-performance liquid chromatography. The unsaturated disaccharides included delta Di-0S, delta Di-6S and delta Di-4S. Analysis of data indicated that delta Di-0S, delta Di-6S and delta Di-4S were found in all PDS samples. The amount (ng ml-1 collected whole saliva) of delta Di-0S, delta Di-6S and delta Di-4S (P < 0.01) indicated significant differences between CHS and PDS whole saliva samples. The quantities of delta Di-0S and delta Di-4S (P < 0.01) indicated significant differences between PDS and ES whole saliva. The amount of delta Di-0S (P < .05) and delta Di-6S (P < 0.01) also indicated significant differences between CHS and ES whole saliva. These results indicate that chondroitin sulphate in PDS and CHS whole saliva is representative of that previously reported in gingival crevicular fluid and so provides a useful and alternative means of assessing the role of GAG as indicators of periodontal disease.

Age changes in the rat temporomandibular joint articular disc: a biochemical study on glycosaminoglycan content.

The temporomandibular joint (TMJ) articular discs were removed from female Sprague-Dawley rats 3, 5, 10, 32, 90 and 130 weeks of age. Glycosaminoglycans (GAGs) were extracted from the discs by heat treatment, alkali treatment and digestion with Pronase E, and purified by precipitation with cetylpyridinium chloride and ethanol. The concentration of total GAG was highest in the 3 week extracts and tended to decrease with age. Dermatan sulphate was the predominant GAG detected in all age groups along with chondroitin sulphate, hyaluronic acid and heparan sulphate. The disaccharides obtained from chondroitin sulphate were delta Di-4S, delta Di-6S and delta Di-0S, with delta Di-4S being the predominant isomer followed by delta Di-6S for all ages of all the GAG examined. The concentration of chondroitin sulphate showed a decrease with age. Quantitative changes of GAG with age may be related to functional changes in TMJ discs.

Disaccharide analysis of chondroitin sulfate in peri-implant sulcus fluid from dental implants.

We collected peri-implant sulcus fluid by capillary tubes from sites around titanium osseointegrated implants and determined the chondroitin sulfate released into the peri-implant sulcus fluid by high-performance liquid chromatography. Chondroitin sulfate was found in all peri-implant sulcus fluid samples, and its content was similar to that in gingival crevicular fluid obtained around natural teeth. The predominant unsaturated disaccharide isomer was delta Di-0S, followed by delta Di-4S. Delta Di-6S was present in trace amounts. The amount of delta Di-0S was greater in peri-implant sulcus fluid than in gingival crevicular fluid. Assaying chondroitin sulfate disaccharides in peri-implant sulcus fluid may be an effective method of monitoring the peri-implant condition of dental implants.

Structure of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates.

The structure of chondroitin sulfate on aggrecan isolated from the rib and proximal tibial growth plates of bovine fetuses was investigated, and the previously reported increase in the hydrodynamic size of chondroitin sulfate chains between the reserve and hypertrophic zones of the rib was confirmed in the tibial growth plate. Superose 6 gel chromatography, calibrated for chondroitin sulfate chain length by monosaccharide analysis, showed that the average molecular mass of chondroitin sulfate in the reserve and maturing zones of both growth plates was 21,600 and 30,400, respectively. Determination by capillary zone electrophoresis of the disaccharide composition of chains following chondroitinase digestion showed that delta Di-0S, delta Di-4S, and delta Di-6S together accounted for more than 98% of the disaccharides in the digests from all zones of both growth plates; delta disulfated and delta trisulfated disaccharides were not detected. Furthermore, this analysis revealed a gradient in chondroitin sulfate composition from the reserve to the hypertrophic zone, characterized by a marked increase in the content of delta Di-6S (from about 32% to about 52%) and a marked decrease in the content of delta Di-4S (from about 53% to about 35%). Moreover, this altered pattern of sulfation was detected on chains of all sizes in the hypertrophic zone, suggesting that a proportion of the reserve zone aggrecan might be removed and replaced with aggrecan rich in chondroitin-6-sulfate synthesized during the proliferative and maturation stages of the resident chondrocytes. These data are discussed in relation to the biosynthetic mechanisms that control chondroitin sulfate chain length and sulfation on aggrecan and their modification during chondrocyte proliferation, maturation, and hypertrophy in the growth plate.

Modulation by transforming growth factor-beta 1 and interleukin-1 beta of proteoglycan release and chondrodisaccharide composition in porcine articular cartilage.

The effects of IL-1 beta and TGF-beta 1 on proteoglycan release and modifications in porcine articular cartilage explants are described. Proteoglycan release in culture medium was markedly increased when cartilage was treated with IL-1 beta (5 to 100 ng/mL). Addition of IL-1 beta alone or in combination with TGF-beta 1 to cartilage culture stimulated the production and release of PGE2. Indomethacin (10(-6) M) did not modify the IL-1 beta effect on proteoglycan release. Both the spontaneous and IL-1 beta-induced proteoglycan release were downregulated by TGF-beta 1 (50 ng/mL). Basal and stimulated loss of proteoglycans was reduced by the addition of cycloheximide (10(-6) M) and by metalloprotease inhibitors. GAGs of conditioned medium and extracellular matrix proteoglycans were digested to about 90% by chondroitin ABC lyase, suggesting that CS is the major GAG present. Structural analysis of disaccharides, by CZE, revealed a different pattern of oxysulfation when cartilage explants were treated either with IL-1 beta or TGF-beta 1. Indeed, analysis of GAGs, released into the medium from TGF-beta 1-treated cartilage, showed a reduction in the level of 4-S-disaccharide (delta Di4S and an increase in desulfated disaccharides delta Di0S). In the extracellular matrix, IL-1 beta and TGF-beta 1 induced a more complex rearrangement of the GAGs, in that the levels of delta Di0S and delta Di4S were increased whereas those of delta Di6S were decreased. Altogether, these results suggest that TGF-beta 1 does not only counteract the effect of IL-1 beta on proteoglycan release, but modifies the structure of GAGs, probably by interfering with the activity or the synthesis of sulfotranspherases involved in GAG biosynthesis.

Immunohistochemical characterization of extracellular matrix components of granulosa cell tumor of ovary.

In order to clarify the characteristics of granulosa cell tumors of the ovary, extracellular matrix components were investigated by immunohistochemical techniques. Twenty-three granulosa cell tumors (GCT; eight juvenile and 15 adult type) were studied in comparison with non-neoplastic granulosa cells of human ovaries. In all 23 cases of GCT, chondroitin 6-sulfate proteoglycan revealed with antibody 3B3 was characteristically observed in the extracellular matrix in the solid nest, as well as in microfollicles. In the juvenile cases, the extracellular matrix also contained large proteoglycan (PG) revealed with antibody 2B1. Macrofollicles as well as microfollicles contained PG chondroitin 6-sulfate side chains with a significant amount of chondroitin 4-sulfate. By biochemical analysis using high pressure liquid chromatography, it was also found that disaccharide composition of glycosaminoglycan fractions extracted from granulosa cell tumor tissues consisted mainly of 2-acetamide-2-deoxyl-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S). The characteristic feature of granulosa cell tumors is the accumulation of chondroitin sulfate PG, especially chondroitin 6-sulfate PG, which may be synthesized by the tumor cells themselves. Immunohistochemical characterization of the extracellular matrix components (collagen, laminin, heparan sulfate PG, chondroitin 4-sulfate PG) was also studied in relation to chondroitin 6-sulfate PG localization.

Sulphation of proteochondroitin and 4-methylumbelliferyl beta-D-xyloside-chondroitin formed by mouse mastocytoma cells cultured in sulphate-deficient medium.

Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.

Quantitation of chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic joint fluid.

To investigate the correlation between joint disease and the composition of chondroitin sulfate in the joint fluid, unsaturated disaccharide isomers of chondroitin 4-sulfate (delta di-4S) and chondroitin 6-sulfate (delta di-6S) were measured in joint fluids obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA), or traumatic arthritis (TA). These pathologic joint fluids were digested with chondroitinase ABC, and the delta di-4S and delta di-6S produced were determined by high performance liquid chromatography combined with fluorometry. Total content of delta di-4S plus delta di-6S was 71.8 +/- 30.0 nmoles/ml (mean +/- SD) in OA, 55.4 +/- 29.3 nmoles/ml in RA, and 211 +/- 149 nmoles/ml in TA joint fluids. The ratio of delta di-6S to delta di-4S was 3.81 +/- 0.992 in OA, 1.13 +/- 0.527 in RA, and 5.75 +/- 2.46 in TA joint fluids. Differences between groups were statistically significant. These results strongly suggest that the levels of chondroitin sulfate isomers and the delta di-6S: delta di-4S ratio in joint fluid reflect the proteoglycan metabolism of joint tissues, particularly of articular cartilage; hence, they could be used to diagnose joint diseases and to predict articular cartilage destruction from such joint diseases.

Analysis of chondroitin sulfate isomers in the periodontium of the monkey using high-performance liquid chromatography.

Glycosaminoglycan (GAG) was extracted from monkey periodontium, consisting of gingiva, periodontal ligament, alveolar bone and cementum, and from dental pulp and dentin by digestion with Pronase E. Unsaturated disaccharide isomers formed by chondroitinase AC digestion from chondroitin sulfate were labeled with dansylhydrazine and analyzed by high-performance liquid chromatography. These tissues showed different molar ratios of the unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lowest in the dental pulp, followed by the gingiva, periodontal ligament, dentin, alveolar bone, and cementum, in that order. It was greater in the calcified than in the uncalcified tissues.

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.

Continuous Release of Secretory Granule Proteoglycans From a Cell Strain Derived From the Bone Marrow of a Patient With Diffuse Cutaneous Mastocytosis

A human cell strain (designated HBM-M) that was derived from the bone marrow of a child with diffuse cutaneous mastocytosis was previously found to possess features that suggested it belonged in the mast cell/monocyte lineage. HBM-M cells synthesized approximately 150-Kd Pronase-resistant proteoglycans that were recognized by an antihuman secretory granule proteoglycan peptide core antibody. These cells also contained in relatively high abundance the same sized mRNA transcript that encodes the peptide core of proteoglycans that are normally localized to secretory granules of hematopoietic cells. However, unlike most other hematopoietic cells, HBM-M cells continuously released their newly synthesized 35S-labeled proteoglycans rather than retaining them in an intracellular storage compartment. Chondroitinase ABC, nitrous acid, and heparinase degraded approximately 76%, 17%, and 7%, respectively, of the HBM-M cell-derived 35S-labeled proteoglycans. As assessed by high performance liquid chromatography, 91% of the unsaturated 35S-labeled disaccharides generated by treatment with chondroitinase ABC were delta Di-4S. The remaining chondroitin sulfate 35S-labeled disaccharides appeared to be primarily a complex mixture of disulfated disaccharides. The 35S-labeled glycosaminoglycans that were not degraded by chondroitinase ABC migrated in two-dimensional cellulose acetate electrophoresis as if they were heparan sulfate or under-sulfated heparin. Thus, although the HBM-M cell-derived proteoglycans had some of the features of proteoglycans produced by normal human mast cells, the heparin-like and chondroitin sulfate glycosaminoglycans bound to the HBM-M cell proteoglycans were considerably less sulfated. Because the only human cell types that have so far been shown to synthesize proteoglycans that have heparin-like glycosaminoglycans bound to a protease-resistant peptide core are mast cells and basophilic leukocytes from patients with myelogenous leukemia, it is possible that the HBM-M cell is a mast cell progenitor cell.

Continuous release of secretory granule proteoglycans from a cell strain derived from the bone marrow of a patient with diffuse cutaneous mastocytosis

A human cell strain (designated HBM-M) that was derived from the bone marrow of a child with diffuse cutaneous mastocytosis was previously found to possess features that suggested it belonged in the mast cell/monocyte lineage. HBM-M cells synthesized approximately 150-Kd Pronase-resistant proteoglycans that were recognized by an antihuman secretory granule proteoglycan peptide core antibody. These cells also contained in relatively high abundance the same sized mRNA transcript that encodes the peptide core of proteoglycans that are normally localized to secretory granules of hematopoietic cells. However, unlike most other hematopoietic cells, HBM-M cells continuously released their newly synthesized 35S-labeled proteoglycans rather than retaining them in an intracellular storage compartment. Chondroitinase ABC, nitrous acid, and heparinase degraded approximately 76%, 17%, and 7%, respectively, of the HBM-M cell-derived 35S-labeled proteoglycans. As assessed by high performance liquid chromatography, 91% of the unsaturated 35S-labeled disaccharides generated by treatment with chondroitinase ABC were delta Di-4S. The remaining chondroitin sulfate 35S-labeled disaccharides appeared to be primarily a complex mixture of disulfated disaccharides. The 35S-labeled glycosaminoglycans that were not degraded by chondroitinase ABC migrated in two-dimensional cellulose acetate electrophoresis as if they were heparan sulfate or under-sulfated heparin. Thus, although the HBM-M cell-derived proteoglycans had some of the features of proteoglycans produced by normal human mast cells, the heparin-like and chondroitin sulfate glycosaminoglycans bound to the HBM-M cell proteoglycans were considerably less sulfated. Because the only human cell types that have so far been shown to synthesize proteoglycans that have heparin-like glycosaminoglycans bound to a protease-resistant peptide core are mast cells and basophilic leukocytes from patients with myelogenous leukemia, it is possible that the HBM-M cell is a mast cell progenitor cell.

Disaccharide analysis of the skin glycosaminoglycans in systemic sclerosis.

The disaccharides constituting chondroitinase-digestible glycosaminoglycan (GAG) in the skin lesions of patients with systemic sclerosis were determined using high-performance liquid chromatography (HPLC). In scleroderma there was an increase in the amount of delta Di-4S(DS), the main disaccharide unit of dermatan sulphate, and a decrease in delta Di-HA, the disaccharide unit of hyaluronic acid, as compared with normal skin from a similar site. The distribution pattern of the main disaccharides constituting chondroitin sulphate and dermatan sulphate in scleroderma differed from that in scars or scleredema.

Glycosaminoglycans in the lamina propria and submucosal layer of the monkey palatal mucosa.

Glycosaminoglycans (GAGs) in monkey palatal lamina propria plus both fatty and glandular zones of palatal submucosa were compared. Chemical analysis revealed that GAG contents of the lamina propria and glandular zone were higher than that of the fatty zone. The four GAGs identified by electrophoretic analysis were hyaluronic acid, dermatan sulfate, chondroitin sulfate and heparan sulfate. Each mucosal layer contained all four GAG components. The predominant GAG in both the lamina propria and glandular zone was dermatan sulfate followed by hyaluronic acid. The reverse situation (predominant hyaluronic acid, less prominent dermatan sulfate) was noted in the fatty zone of the submucosa. The three tissue regions showed different molar ratios of unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lower in the lamina propria than in either the fatty or glandular submucosal zones.