Trypsin Extracts Research Articles

Extraction of trypsin from bovine pancreas by applying polyethyleneglycol/sodium citrate aqueous two-phase systems

The goal of this work was to determine the optimal conditions for separating trypsin (TRP) from alpha-chymotrypsin (ChTRP) and to apply them for trypsin purification from bovine pancreas by liquid–liquid extraction with polyethyleneglycol/sodium citrate (PEG/NaCit) aqueous two-phase systems. Partitioning behaviours of TRP and ChTRP are demonstrated to be very sensitive to variables such as PEG molecular weight, pH and tie line length. Aqueous two-phase systems (ATPSs) formed by PEG of MW 3350 and NaCit pH 5.20 showed the best separation capability. The addition of NaCl up to a final concentration of 7% (w/w) and the decrease of top/bottom volume ratio to 0.1 led to the recovery of 60% of pancreatic TRP in a concentrated form in the top phase with a 3-fold purification. Biomass presence up to 25% (w/w) of the total system mass did not affect significantly yield and purification parameters.

Hyaluronan Facilitates Transforming Growth Factor-β1-mediated Fibroblast Proliferation

This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-beta(1) (TGF-beta(1))-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-beta(1), with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-beta(1)-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-beta(1) signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-beta(1), which is associated with differences in HA generation. TGF-beta(1) regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response.

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning tunneling microscope(STM) was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium (AOT)/ iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.Imol·L −1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca 2+, Na +, K + which were in the range of 0.2–1.0mol·L −1, and influence of concentration of Ca 2+ is greater than that of Na +, and K + which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsim were: AOT reversed micelles 0.05mol·L −1, trypsin concentration in crude pancreatin solutin 3mg.ml −1, pH 5.2–5.3, ratio (by volume) of extraction phase to strip-extraction phase 1:1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginine ethyl ester (BAEE) U-mg −1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L −1 ceryl-trimethyl-ammonium bromide (CTAB), total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg −1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Combined sensitive analytical methods for cholecystokinin levels and tryptic activity in individual fish larvae

A protocol for combined analysis of cholecystokinin (CCK) levels based on radioimmunoassay (RIA) and fluorescence tryptic enzyme activity (FTA) was developed in order to accomplish a sensitive analysis of individual bodies and gut segments of fish larvae. Methanol was used for CCK extraction. The gut of herring larvae contained 8.9±1.2 fmol CCK/mg dry weight and in the post-larval Atlantic halibut the CCK levels varied significantly ( p<0.05) from 20.9±15.6 to 101.8±56.7 fmol/mg dry weight for separated intestinal and pyloric segments, respectively. Acid solution, 0.02 mol/l HCl–CaCl 2 (pH 1.8), and alkaline solution, 0.1 mol/l Tris–0.02 mol/l CaCl 2 (pH 8.0) were tested to prepare crude trypsin extracts from Coregonids and Atlantic halibut larvae. The tryptic activity of crude extracts prepared with acid solution was enhanced by a factor of 3.19±0.52 compared to the tryptic activity of crude extracts prepared with alkaline solution. The larval trypsins (from yolk sac larvae) were stable in methanol, preserving 88% of its starting activity after 6 days of storage. Based on the results, the method of extraction using methanol and acid solution (pH 1.8) was suitable for the combined analysis for CCK levels and FTA in gut segments or single larvae. The potential application of these analytical tools may allow a better understanding of the individual variability of gut functionality, nutritional condition and the feeding activity of developing fish based on their content of CCK and tryptic activity.

Comparison of enzymatic extraction procedures for use with directly coupled high performance liquid chromatography-inductively coupled plasma mass spectrometry for the speciation of arsenic in baby foods

A method has been developed for the speciation of arsenic in a variety of baby foods using directly coupled high performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICP-MS). Ion exchange HPLC separation using gradient elution and sodium sulphate as the mobile phase resulted in good resolution and a fast separation of the following species: arsenobetaine, monomethylarsonic acid, dimethylarsinic acid and arsenate. The arsenicals were extracted by two enzymatic digestion procedures, based on the action of trypsin or pancreatin. The results from the enzyme digested samples were compared with those obtained for “total” arsenic by microwave-oven digestion. The reliability of the procedure was also checked by analysing the total reducible arsenic species by hydride generation-AAS and by analysing two certified reference materials. The enzymatic digestion procedures were also evaluated on pure standards to ensure that no species alteration occurred during the extraction process. Trypsin was found to work well although the pancreatin extraction had a deleterious effect on the chromatography, with the dimethylarsinic acid peak co-eluting with the arsenobetaine. Pancreatin was therefore concluded to be unsuitable for the speciation process, although the total amount of arsenic extracted was comparable to the trypsin extraction. Values for the total arsenic content in the baby foods were found to range between 0.25 and 4.7 μg g −1. Arsenobetaine was the only arsenic species detected in most samples, but an unidentified species was detected in one sample.

Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes.

A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PAGE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectrofocusing values (pI 5.8-6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP-HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT-PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino-acid sequence is most similar to Xenopus oocyte-specific GV lamin B3, but differs from somatic lamins (A, B1 or B2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV-type lamin (the homolog of Xenopus lamin B3).

Optimizing the Determiniaton of Trypsin Inhibitor Activity in Chickpeas by Response Surface Methodology

Abstract Response surface methodology (RSM) was used to optimize determination of trypsin inhibitor activity (TIA) in chickpea. TIA was determined by measuring the hydrolysis rate of N-α-benzoyl-DL-arginine-p- nitroanilide hydrochloride (BAPNA) in the presence of both pig pancreatic trypsin (EC 3.4.21.4) and chickpea extract containing trypsin inhibitor. Three critical parameters in TIA determination—buffer extraction pH (pH), stirring extraction time (Set), and development color time (Dct)—were optimized. Highest TIA values were obtained when pH = 11, Set = 150 min, and Dct = 10.2 min.

Molecular Diversity Among the Trypsin Resistant Surface Proteins of Group B Streptococc

The alpha (alpha) component of the c protein and R proteins are trypsin resistant, but antigenically distinct, proteins on the cell surface of some but not all strains of group B streptococci (GBS). These two classes of proteins, along with the group and type polysaccharide, can be used to characterize strains of GBS. Four species of R protein (R1 through R4) have been described. We studied trypsin extracts from numerous strains of GBS by immunodiffusion in agarose and polyacrylamide gel electrophoresis/Western blot. Sera monospecific for alpha, R1 and R4 were used to immunoprecipitate/blot the proteins. The molecular weight of the blotted proteins was determined. Although by immunodiffusion the proteins within a class were identical to each other, great heterogeneity in size and blotting pattern was found within each class. Variation was independent of the polysaccharide serotype. Multiple molecular weight species were seen for alpha, R1 and R4 proteins. For a given strain, the various forms of alpha or R1 appeared to form a multiple size ladder; those of R4 were fewer and closer in size. The highest form of alpha ranged from 85 to 170 kDa, with 45 kDa being the highest form for some rare GBS strains. For R4 the predominant and highest form varied from 84 to 197 kDa, whereas some strains with R1 had the highest form over 200 kDa. Our results indicated that despite similarities, there is great diversity among the alpha, R1 and R4 trypsin resistant proteins of GBS.

Polypeptides associated with tufts of cell-surface fibrils in an oral Streptococcus.

Cells of the oral bacterium Streptococcus oralis CN3410 produce lateral tufts of cell-surface fibrils of two lengths. Treatment of cells with trypsin resulted in loss of the tufts and release of longer fibrils intact. SDS-PAGE analysis of trypsin extracts containing fibrils revealed two groups of high molecular mass polypeptides which were denoted group A (molecular mass 227-246 kDa) and group B (molecular mass 175-208 kDa). Antibodies were raised to these two groups of trypsin-extracted polypeptides (TEPs) and to purified fibrils, and the reactivities of the three different antisera were found to be similar both on nitrocellulose blots of cell-surface polypeptides and in ELISA with whole cells. Similar patterns of TEPs were obtained from cells of a spontaneously derived mutant strain, KP34V, which lacked the short fibril components of tufts. Cells of strain KP34V had similar cell-surface hydrophobicity to strain CN3410 cells, and adhered to the same extent to parotid salivary pellicle or human buccal epithelial cells (BECs) as the wild-type cells. Trypsin treatment of strain CN3410 cells abolished their surface hydrophobicity and ability to adhere to BECs, but did not affect streptococcal cell binding to experimental salivary pellicle. Antibodies to TEPs or fibrils had no effect on cell adhesion to BECs or salivary pellicle. The results imply that the short fibril components of tufts are not involved in the cell adhesion properties tested. It is suggested that the TEPs are components of long fibrils, but they are not determinants of streptococcal cell adhesion to pellicle or to epithelial cells.

Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. (c) 1995 John Wiley & Sons, Inc.

Recovery and Characteristics of Carotenoprotein from Lobster (Homarus americanus) Waste

Carontenoproteins were recovered from lobster (Homarus americanus) waste with the aid of bovine trypsin and semi-purified trypsin extracts from Atlantic cod offal. The use of the enzyme preparations resulted in more than a two-fold increase in the yield of the product as compared with the extraction process carried out without enzymes. The use of EDTA to dematerialize the raw material resulted in a greater removal of ash and chitinous material form the products than the use of HCI or hear treatments (i.e., 50o C for 30 min). The procedures that combined EDTA treatment and (NH4)2SO4 precipitation; (iii) heat treatment and (NH4) 2SO4 precipitation; or (iv) heat treatment and precipitation with 1 M HCI. SDS-polyacrylamide gel electrophoresis of lobster carotenoprotein resolved the latter into two major bands with molecular weights of 43, 900 and 89, 500. The high contents of protein, fat and essential amino acid associated with the H. americanus carotenoproteins suggest that the products have potential for u...

Characterization of chicken lung angiotensin I-converting enzyme.

Angiotensin I(AI)-converting enzyme (ACE) (EC 3.4.15.1) was solubilized from the membrane fraction of chicken lung using trypsin and nonidet P40 extraction, and then purified to homogeneity by captopril affinity chromatography. Comparison of trypsin-extracted and detergent-solubilized membrane-bound converting enzyme by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane-binding sequence contributed to a large extent to the size and charge of the enzyme. Both forms of the enzyme were glycoproteins but they differed in the glucidic content; 4.5% by weight of the enzyme in the trypsin-extracted ACE and 15% by weight of the enzyme in the detergent-solubilized ACE. In both cases hexoses were the most abundant residues. Both forms of the enzyme were found to contain 1 g-atom zinc/mol enzyme. The purified enzymes did not only split Hip-His-Leu but also AI and bradykinin. The Michaelis constant (Km) and maximum velocity (Vmax) values of the trypsin-extracted ACE for Hip-His-Leu were 52 x 10(-5) mol/l and 15.36 nmol/min respectively, and for AI they were 7.8 x 10(-5) mol/l and 0.45 nmol/min respectively. The Km and Vmax values of the detergent-solubilized ACE for Hip-His-Leu were 32 x 10(-5) mol/l and 11.75 nmol/min respectively, and for AI they were 6.5 x 10(-5) mol/l and 0.97 nmol/min.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple assay for determining keratin and collagen degradation in vitro

A simple assay measuring degradation of human epidermal keratin and bovine tendon collagen is presented. Insoluble protein substrate (30 mg) was incubated with 1 ml buffer and enzyme sample for 1 h at 37 degrees C, following addition of 1 ml distilled water and removal of the remaining substrate by filtration/centrifugation. The protein content was determined in the filtrate/supernatant by the Lowry method. Keratin was prepared as follows: Freeze-drying, homogenization in a mortar or beetling mill, extraction in 0.9% (w/v) NaCl followed by water and 100% ethanol, drying at 37 degrees C. The assay was tested with pig pepsin, bovine trypsin, and crude extract of fish stomach, demonstrating that these preparations are effective in degrading human epidermal keratin.

Developmentally-related changes in surface membrane glycopeptides of murine haemopoietic cells.

We have carried out a comparative study of mature murine granulocytes with two immature haemopoietic cell lines (multipotential cells, FDCP-Mix, and granulocyte progenitor cells, FDCP-2) with respect to the structure and composition of their surface membrane glycopeptides. The glycopeptides were labelled biosynthetically by incubation of the cells for 1-3 days with [3H]glucosamine. Cell-associated glycopeptides were released by treatment with trypsin and the trypsin extract was exhaustively digested with Pronase to remove most residual peptide. Radiolabelled materials were fractionated by chromatography on lectin affinity columns connected in the series: lentil lectin (LCA), concanavalin A (Con A) and wheat germ agglutinin (WGA). Lectin-binding glycopeptides were eluted with appropriate competing sugars and further analysed by gel filtration, base/borohydride elimination and susceptibility to degradation by glycosidases including endo-beta-galactosidase. Abundant quantities of N-linked polylactosamine-type glycopeptides, which bound only to the WGA columns, were identified on mature granulocytes but the molecules were highly-branched (i.e. resistant to endo-beta-galactosidase). In contrast, there seemed to be very little branching in the polylactosamine chains from FDCP-2 cells, whilst corresponding carbohydrates from multipotential FDCP-Mix cells gave evidence for both linear and branched domains in the same, large complex glycans. O-Linked tetrasaccharides of general structure: NeuAc-Gal-(NeuAc)-GalNAc were found in clusters on WGA-binding glycopeptides from all cell types, these components being especially prominent on mature granulocytes. FDCP-2 cells were distinguished by the presence of monosialylated and non-sialylated counterparts of the foregoing tetrasaccharides. The relative amount of LCA-binding glycopeptides was low on FDCP-Mix cells by comparison with FDCP-2 cells and mature granulocytes. Our findings therefore demonstrate that notable differences in gross composition and molecular fine structure of surface membrane glycopeptides are detectable in haemopoietic cells at different stages of development. The relationship of these differences to the biological properties of cell surfaces remains to be established.

Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities.

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.

Does trypsin extract soluble antigen meet the criteria of immunogenicity without allergenicity?

We were unable to confirm the finding of Crowle that TESA possesses the attribute of immunogenicity and absence of allergenicity which are desired in a non-living antituberculosis vaccine. This disagreement could be a function of the particular lot of TESA examined and/or differences in the animal test systems used for the assay of protective potency. The relative contribution of these two possibilities could be determined by the simultaneous assay of a potent TESA product in different laboratories using different animal models.

A phosphatidylethanolamine-containing complex on human B cells that mediates rosette formation with mouse erythrocytes.

The maturation-associated human B cell rosette receptor (MER) for mouse erythrocytes has been solubilized from B cells by mild trypsinization. It specifically agglutinates mouse red cells. Material with hemagglutinating activity partitioned into the lipid-soluble phase of a Folch partition of the trypsin extract was sensitive to phospholipase C and alkali, and on two-dimensional thin layer chromatography, it co-migrated principally with phosphatidylethanolamine (PE). Phosphatidylcholine, the major lipid present, was inactive. The relationship of phospholipid structure to hemagglutinating activity has been described. PE in the crude trypsin extract was associated with unidentified glycoprotein and albumin. Material containing hemagglutinating lipid bound to a wheat germ lectin-Sepharose column and was released by N-acetylglucosamine, indicating that the PE was complexed with glycoprotein. When the crude trypsin extract or eluate from the lectin column was extracted with aqueous phenol, hemagglutinin in the aqueous phase no longer bound to wheat germ lectin-Sepharose; however, albumin was greatly enriched, indicating that some of the PE exists in a complex with albumin. The molar ratio of PE to albumin was approximately 200:1. After delipidation, this albumin (in molar excess) inhibited hemagglutination by PE in the same way as a recently described subclass of serum albumin. Studies with phospholipase-treated B cells were also consistent with PE being the MER. We conclude that MER is PE, existing in a complex containing glycoprotein and a subclass of albumin. The capacity to form rosettes can be transferred to nonrosetting Raji B cells by the complex, but not pure PE, indicating that the proteins may be involved in orienting PE correctly for it to function as the MER.

Changes in the synthesis, distribution and sulphation of glycosaminoglycans of cultured human skin fibroblasts upon ascorbate feeding.

The effect of ascorbic acid on the synthesis, distribution and sulphation of glycosaminoglycans by human skin fibroblasts has been examined. Medium was supplemented with ascorbate over several days, and cultures incubated with [3H]glucosamine and Na2(35)SO4 for 48 h, followed by analysis of the glycosaminoglycans in the medium, in collagenase and trypsin extracts, and in cell fractions. Ascorbate feeding resulted in a reduction in hyaluronate synthesis, which was the main 3H-labelled component and was distributed mainly in the medium fractions. Sulphated glycosaminoglycans showed a reduction in incorporation of 3H label, but increased sulphation following ascorbate feeding. In control cultures 53% of 3H-labelled sulphated glycosaminoglycans and 63% of 35S-labelled glycosaminoglycans were present in the medium fraction, while in ascorbate-fed cultures, 41% of 3H label and 38% 35S label were incorporated into medium-sulphated glycosaminoglycans. Ascorbate also caused an increase in cell density and in collagen production and deposition.

Synthesis of glycosaminoglycans by cloned bovine endothelial cells cultured on collagen gels.

Sulphated glycosaminoglycans have been analysed in cloned bovine aortic endothelial cells cultured on collagen gels after incubation with [3H]glucosamine and Na2(35)SO4. Radioactive products were analysed in the culture medium, in sequential collagenase and trypsin extracts of the cell monolayer and the associated extracellular matrix, and in the remaining viable cells. Heparan sulphate and chondroitin sulphate were found in each compartment: the heparan sulphate had a low degree of sulphation (approximately 0.4 N-sulphate and 0.2 O-sulphate groups per disaccharide unit on average). In the nitrous acid scission products of heparan sulphate, O-sulphated substituents were confined to disaccharide and tetrasaccharide fragments, indicating that local regions of the chain (which might be susceptible to excission by the platelet endoglycosidase) are highly sulphated. Only minor structural differences in heparan sulphate were observed between the various compartments. In contrast the chondroitin sulphate found in the collagenase extract had a higher iduronic acid content than corresponding material in the trypsin extract and the culture medium, indicating that collagenase and trypsin may extract glycosaminoglycans from different regions of the extracellular and pericellular matrix.

Role of the cellular matrix in haemopoiesis. I. Synthesis of glycosaminoglycans by mouse bone marrow cell cultures.

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.