Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease.

BACKGROUND: The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties. METHODS: Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts' adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL. RESULTS: ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin ß3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption. CONCLUSIONS: ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis. GENERAL SIGNIFICANCE: The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.

Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide.

The polysaccharides of cranical cartilage were isolated by ethanol precipitation after papain digestion and beta-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39 200 on weight basis (Mw) and 31 400 on number basis (Mn).

Extraction and fractionation of proteoglycans from squid skin.

The extractability of squid skin proteoglycans with solutions of varying concentrations of guanidine-HCl, urea and SDS was studied; 4 M guanidine-HCl, being the best extractant, removed 95% of the tissue proteoglycans (glycosaminoglycan uronic acid). The proteoglycans in the 4 M guanidine-HCl extract were fractionated by repeated ion exchange and gel chromatography on Sepharose CL-4B to give three main populations, all being present in about equal proportions. Two populations (Kd 0.34 and 0.56) contained only chondroitin (proteochondroitin) and the other (Kd 0.50) only oversulphated chondroitin sulphate (oversulphated proteochondroitin sulphate). Two minor populations, one containing chondroitin and chondroitin sulphate and the other chondroitin sulphate and oversulphated chondroitin sulphate, were also identified.

Effects of glycosaminoglycans on proliferation of epithelial and fibroblast human malignant mesothelioma cells: a structure-function relationship.

Proteoglycans interact with other effective macromolecules regulating a variety of cellular events via their glycosaminoglycan (GAG) chains. The effects of all known glycosaminoglycans (GAGs) produced by normal cells and tissues on the proliferation of two human malignant mesothelioma cell lines, one with fibroblast-like morphology and the other with epithelial differentiation - both able to produce hyaluronan (HA), galactosaminoglycans (GalAGs) and heparan sulphate (HS) containing proteoglycans - have been studied. Cell proliferation was assessed by measuring [3H]thymidine incorporation and cell number. GalAGs, i.e. chondroitin sulphates (CSs) and dermatan sulphate (DS), strongly stimulate the proliferation of fibroblast-like cells in a dose-dependent manner (170-250% at 100 microg/ml), independently of their sulphation pattern. In epithelial cells, however, only DS stimulates cell proliferation. The effects of CSs on proliferation of epithelial cells are not depended on their sulphation pattern. Thus, CSs either with -[GlcA-GalNAc-(-6-O-SO(3)-)]- or -[GlcA-GalNAc-(-4-O-SO(3)-]- as the commonest unit, had no significant effect. L-Iduronic acid (IdoA)-rich heparin and fast-moving HS (fm-HS), a HS fraction with a heparin-like structure, had significant antiproliferative effects on mesothelioma cells of both types (30-70% at 1.0 microg/ml and 85-90% at 100 microg/ml, respectively). GlcA-rich HS, however, had no significant effects. HA inhibits only the proliferation of fibroblast-like cells by 25% at 50 and 100 microg/ml. Keratan sulphate suppresses cell proliferation (10-30%) in both cell lines. In the view of these findings, a structure-function relationship of GAGs on cell proliferation of the two human malignant mesothelioma cell lines is discussed. Other factors, such as chain conformation and geometry, as well as interactions of growth factors with GAGs, possibly involved in the regulation of cell proliferation, are also discussed.

Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin.

A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (Mr) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (Mr 33 kDa) bound on a protein core of Mr 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain.

Isolation, biochemical and immunological characterisation of two sea urchin glycoproteins bearing sulphated poly(sialic acid) polysaccharides rich in N-glycolyl neuraminic acid.

Two different sialoproteins were isolated from the sea urchin shell by guanidine hydrochloride extraction in the presence of Triton X-100. The sialoproteins (SP I and SP II) were purified on DEAE-Sephacel and Sepharose CL-6B and separated from each other by density gradient centrifugation. The ratio between recovered SP I and SP II was 1:4.5 and their M(r)s 650 and 600 kDa, respectively. They were degraded by neuraminidase, endoglycosidase F and peptide N-glycosidase F resulting in fragments of similar relative molecular mass (M(r)s). Although their protein cores have approximately the same relative molecular mass of 500 kDa, they differ markedly in their contents of aspartic acid/asparagine, glycine, leucine and phenylalanine, as well as in the primary amino acid sequence of their N-terminal peptides. Carbohydrate analyses showed that the sialic acid content was higher in SP I (11.4% of dry tissue weight) than in the more prominent SP II (5.3%). Two types of carbohydrates, O-glycosidically-linked polysaccharides and N-glycosidically-linked oligosaccharides are present in both sialoproteins. SP I contains 10-11 polysaccharide chains whereas SP II contains 5-6. The polysaccharides are linked to protein cores via galactosamine, have approximately the same M(r) of 12 kDa and contain 32-33 N-glycolyl neuraminic acid, 10-11 glucosamine, 6-7 sulphate and 6-8 neutral monosaccharide residues. Sialic acid residues are organized in a poly(sialic acid) unit which is present in the non-reducing terminal of the polysaccharides and degraded by neuraminidase. Hexosamines, sulphates and neutral monosaccharides are all constituents of the sialic acid free region of the chain near the reducing end. Two oligosaccharide populations were isolated from SP I, one major (70% of the total oligosaccharides) with M(r) of approximately 3 kDa and the other with M(r) of 1.5 kDa. In SP II, however, only a 3-kDa oligosaccharide population was present. The oligosaccharides from both sialoproteins are N-glycosidically linked to asparagine via the glucosamine and contain mannose, glucosamine, galactosamine and sialic acids. Antibodies against SP II were raised in rabbits and it was shown that the antigenicity of SP II was lost on either neuraminidase or trypsin digestion, indicating that both the poly(sialic acid) units of the polysaccharide and the protein core are antigenically active. As expected, SP II showed considerable cross-reactivity with SP I due to the common poly(sialic acid) structure. There were no significant reactivities of SP II and SP I with antibodies to bovine bone sialoprotein and osteopontin. The biological role of the two sea urchin sialoproteins as developmentally regulated products of the tissue remains to be elucidated.

Human abdominal aortic aneurysm is closely associated with compositional and specific structural modifications at the glycosaminoglycan level.

Human abdominal aortic aneurysm (AAA) is a commonly occuring disease of blood vessels and is related to alterations in extracellular matrix molecules. In this study we report on the type and fine structural characterization of glycosaminoglycans (GAGs) present in AAA as compared with those present in normal abdominal aorta. Hyaluronan (HA), the galactosaminoglycans-chondroitin sulfate (CS) and dermatan sulfate (DS) with average molecular size (Mr) of 35-kDa-as well as heparan sulfate (HS) with Mr of 40-kDa were identified in both tissues. No significant intrabatch differences in total GAG content were identified in normal and aneurysmal aortas. Comparing, however, tissue composition and structure of GAGs between AAAs and normal aortas, significant differences (P < or = 0.001) were found. The overall GAG content in AAAs was approx. 60% lower than the normal ones. A 90% decrease in HS content, and 65 and 73% in CS and HA, respectively, were also recorded. In contrast, only a slight decrease in the amount of DS was noted (8%). Structural alterations in disaccharide composition of GAGs correspond mainly to significant decreases (P < or = 0.001) of HS-derived N-sulfated disaccharides, CS-derived 6-sulfated disaccharide and DS-derived disulfated disaccharides. These results demonstrate that the development of AAA is related to dramatic quantitative and structural modifications at the GAG level and this may well be attributed to the destruction of arterial wall architecture and further significant functional inadequacies of the tissue.

Oxytocin and lysine-vasopressin with N5,N5-dialkylglutamine in the 4 position: effect of introducing sterically hindered groups into the hydrophilic cluster of neurohypophyseal hormones.

The synthesis and pharmacological potencies of oxytocin and lysine-vasopressin analogues are reported in which the N5-amide of their glutaminyl residues are dialkylated. These analogues have been studied as an ongoing exploration of the biological effects on the natural hormones of substituting individually one of the amino acid residues, which has a hydrophilic side chain and which are thought to be part of the hydrophilic surface of the hormones. [4-(N5,N5-Dimethylglutamine)]oxytocin (17), [4-(N5,N5-di-n-propylglutamine)]oxytocin (18), and [4-(N5,N5-dimethylglutamine)] lysine-vasopressin (19) were synthesized by clasical solution techniques. Potencies in the in vitro rat uterotonic, avian vasodepressor, rat pressor, and rat antidiuretic assays were determined and are as follows, respectively: for compound 17 3.01 +/- 0.14 units/mg, 4.55 +/- 0.03 units/mg, tachyphylaxis and tachyphylaxis; for compound 18 less than 0.1, less than 0.1, less than 0.05, and less thad 1.88 +/- 0.04 units/mg. The potencies in all cases are significantly less than those of the parent hormone. The results are discussed in terms of the proposed biologically active conformations of the hormones at the uterotonic receptor and at the antidiuretic receptor.

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate.

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 micrograms of delta-disaccharides. As little as 0.8-5 ng of delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (> 90%) to delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/- 6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled cepolymerized heparin fragment of M(r) 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.

An enzyme immunoassay to determine the levels of specific antibodies toward bacterial surface antigens in human immunoglobulin preparations and blood serum.

Human polyvalent intravenous immunoglobulin (IVIG) preparations are used as a complementary aid to the proper antimicrobial treatment of severely septic patients in intensive care units (ICUs) and/or as a prophylactic agent to immunocompromised hosts, particularly prone to bacterial infections. There is skepticism about the usefulness of IVIGs since it is not known whether their administration ensures the enhancement of humoral immune responses by providing a sufficient amount of specific antibodies towards the specified bacterial pathogen to be treated. In this report, a simple and reproducible enzyme-linked immunosorbent assay for determining the content of specific antibodies against bacterial surface antigens in commercially available IVIG preparations is described. The method is also easily applied to determine the amount of bacterial antibodies in blood serum. The levels of specific antibodies toward gram positive and negative pathogenic isolates often encountered in ICUs were estimated in two IVIG (Sandoglobulin and Gamimmune) preparations. Significant differences regarding the content of antibodies to certain clinically bacterial isolates were identified not only between the two IVIG preparations tested, but also among various lots from each IVIG preparation. No significant variation (P < or = 0.001) among the bottles derived from the same lot was determined in both preparations. The variation in the levels of specific antibodies in IVIG preparations may be attributed to differences between the donor pools as well as the manufacturing procedure. Application of the method to patients with primary immune deficiencies showed that infusion of highly reactive IVIG preparations enhanced significantly their humoral response toward various pathogens. The results of this study suggest that the content determination of pathogen-specific antibodies in IVIG preparations before administration may be of great importance for treating bacterial infections.

Isolation and analysis of a novel acidic polysaccharide from the case of squid pen.

A new non-sulphated acidic polysaccharide with an average molecular mass of 55 kDa was isolated from squid pen case after papain digestion and beta-elimination. This polysaccharide contains mainly L-iduronic acid, D-glucuronic acid, D-galactosamine, D-glucosamine and significant amounts of neutral sugars as glucose, galactose and fucose. The polysaccharide was not degraded to the relative disaccharides by chondroitinases ABC, AC and B, hyaluronidase and keratanase or by treatment with heparinases, suggesting a structure different from those of known glycosaminoglycans. The polysaccharide cannot form self aggregates.

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues. Structure-activity studies.

Analogues of [Orn6]-SP6-11 have been synthesized in which the methionyl residue is replaced by glutamine gamma-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by CONHCH3, CON(CH3)2, CONHPh and CONCH3Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH3)2]11 and the Glu(NHCH3)11 analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH3)11 analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH3Ph)11 analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)11 analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine gamma-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met11 side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.

Effects of mitogens on synthesis and distribution of heparan and chondroitin sulphates by human leukemic B, T cells and monocytes studied by high-performance liquid chromatography and radiochemical detection.

Human leukemic cell lines, Jurkat (T-cell leukemia), Daudi (Burkitt's lymphoma, B-cell leukemia) and THP-1 (acute monocytic leukemia) synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. CS is the major secreted GAG in all cell lines, as well as the major cell-retarded glycosaminoglycan (GAG) in Jurkat and Daudi, whereas HS is the major GAG in the cell membrane of THP-1. The effects of mitogenic substances on both synthesis and distribution of GAGs in Jurkat, Daudi and THP-1, independently of their effect on cell proliferation, were studied. The secretion of CS and HS from Jurkat was significantly suppressed by using 12-O-tetradecanoylphorbol 13-acetate (TPA), phytohaemagglutinin (PHA) and anti-CD3 monoclonal antibody (OKT3). These mitogens had different effect on the synthesis of cell-associated GAG by Jurkat, depending on the mitogen type. Addition of TPA or lipopolysaccharide (LPS) in Daudi's culture medium resulted in increased synthesis of HS, while no effect on CS synthesis was noticed. Furthermore, in the presence of LPS, THP-1 produce slightly lower amounts of CS, whereas this mitogen significantly suppresses the HS synthesis in both culture medium and cell membrane. The obtained data clearly demonstrate that the various mitogenic substances participate in the regulation of GAG synthesis. The effects are dependent on the type of mitogen and the cell line.

Application of carboxylic-phosphinic mixed anhydrides in the fragment peptide synthesis of protected analogues of substance P.

Mixed carboxylic-phosphinic anhydrides derived from peptide acids and 1-oxo-1-chlorophospholane have been applied in the synthesis of the protected [Leu11]-SP by the fragment coupling strategy. The yields from fragment couplings were ca. 75%, the products were of high purity while the conditions of formation and coupling of the corresponding mixed phosphinic anhydrides, for optimum yields, have been evaluated.

Analysis of N-acetyl and N-glycolylneuraminic acid in rat serum and tissues with Walker 256 carcinoma by high-performance liquid chromatography.

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

Application of carboxylic-phospholanic mixed anhydrides to fragment coupling in peptide synthesis.

The application of 1-oxo-1-chlorophospholane as a novel reagent for the in situ activation of peptide fragments for use in peptide bond forming reactions, either in liquid or solid phase, has been examined. 24.1 MHz 31P NMR spectroscopy has been employed to follow the formation, stability and reactivity of the intermediate phospholanic-carboxylic mixed anhydride.

Isolation and chemical study of the glycosaminoglycans from squid cornea.

1. Oversulphated chondroitin sulphate (ca 93% of tissue glycosaminoglycans) with average molecular weight 72,500, chondroitin sulphate (5%) and small amounts of lowsulphated chondroitin sulphate were isolated from squid cornea. 2. The sulphation pattern of oversulphated chondroitin sulphate was delta di-4S (52%), delta di-diSD (28%), delta di-6S (9%) and delta di-OSCS (11%) and that of chondroitin sulphate 49, 1, 20 and 30% respectively. 3. All glycosaminoglycans contained neutral monosaccharides, glucose being the predominant neutral monosaccharide in oversulphated chondroitin sulphate and chondroitin sulphate and fucose in low-sulphated chondroitin sulphate. 4. Although L-iduronic acid was not detected, the digestion of oversulphated chondroitin sulphate with chondroitinases ABC and AC gave unexpected results.

Screening of N-acylneuraminic acids in serum and tissue specimens of mouse C57BI with Lewis' lung cancer by high-performance liquid chromatography.

Serum and tissue specimens from healthy C57BI mice and from mice with Lewis' lung cancer after metastasis were analyzed for N-acetyl- and N-glycolylneuraminic acid by high-performance liquid chromatography. Both neuraminic acids were present, while N-glycolylneuraminic acid was the predominant sialic acid in all tissues. Samples from mice with metastatic cancer showed a significant increase (67-200%) of total sialic acids mainly as a result of increased N-glycolylneuraminic acid synthesis. These results suggest that cancer metastasis in various tissues is closely associated with increased synthesis of the predominant neuraminic acid and may help to understand the underlying mechanisms of tumor development.

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection.

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.

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.