Metabolism of glycosaminoglycans in CCl4-induced liver regeneration

The incorporation of [₃₅S]-SO₄ into glycosaminoglycans of liverin vivo and in in liver slices and into the glycosaminoglycans associated with the hepatic plasma membrane of rats at different periods after a heavy dose of CC1₄ have been studied. The incorporation of [35S]-SO4 into total glycosaminoglycans decreased to as low as 40% of the control at 24 h after the administration of CC14 and later on increased reaching a maximum on the 4th day. The amount of [35S]-SO4 incorporation into heparan sulphate was also reduced to about 40% of control at 12–24 h after the onset of injury and increased thereafter reaching a maximum on the 4th day. There was only a partial reduction in the synthesis of chondroitin sulphate in the early stage of injury and then it steadily increased reaching about 3 times the control level on 4–6 days. The [³⁵S]-SO4-incorporation into dermatan sulphate, after a slight initial decrease remained at the control levels. On the 8th day after the CCl₄-induced liver injury, the rate of [³⁵S]-SO₄-incorporation was almost equal to that in normal controls. The incorporation of [³⁵S]-SO₄ into hepatic plasma membrane glycosaminoglycans showed a similar change decreasing to about 35% of control at 24h followed by an increase, reaching normal levels on the 4th day after the administration of CC1₄. About 90% of the plasma membrane glycosaminoglycans was found to be heparan sulphate. The yield of plasma membrane from normal and CCl₄-induced regenerating liver was found to be similar and therefore the results obtained were not due to difference in the yield of the membrane preparation. The data also indicate that there was no difference in the degree of sulphation. The significance of these changes in the metabolism of sulphated glycosaminoglycans particularly plasma membrane heparan sulphate in tissue regeneration has been discussed.     

Presence of glycosaminoglycans in retinal capillary basement membrane

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [³⁵S]sulfate and [¹⁴C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase and chondroitinase ABC. Retinal microvessels also incorporated [³⁵S]- and [¹⁴C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinalmicrovessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.     

Glycosaminoglycan synthesis by glomeruli in vivo and in vitro

The distribution of glycosaminoglycans in disrupted glomerular fractions was studied using ³⁵SO₄-labeling in vivo and in vitro. The majority of ³⁵S of isolated glomerular basement membrane was found in heparan sulfate after in vivo and in vitro pulses, although the absolute proportion and the degrees of N-sulfation and N-acetylation varied with the conditions of exposure. Varying amounts of chondroitin sulfate and dermatan sulfate were found in the glomerular basement membrane fraction and larger proportions of both of these glycosaminoglycans as well as of heparan sulfate were found in various glomerular fractions. Glomerular glycosaminoglycans distribution studies must take into account the experimental conditions. Basement membrane-like components of the glomerulus such as the mesangial matrix may have varying glycosaminoglycan composition which may be found in association with glomerular basement membrane fractions.     

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Alterations in human gingival glycosaminoglycan pattern in inflammation and in phenytoin induced overgrowth

Glycosaminoglycans were extracted from normal, inflamed and phenytoin induced overgrowth of human gingival tissue by proteolysis and alcohol precipitation. Extracts were run in a Dowex-1 column and the fractions were treated with mucopolysaccharidases. Cellulose acetate electrophoresis was carried out with or without enzyme digestion for identification of individual glycosaminoglycans. Glycosaminoglycans were found to be decreased in inflammation but were observed to increase in the overgrowth. Hyaluronic acid was found to be increased in both the pathological conditions. Dermatan sulphate, chondroitin sulphate and heparan sulphate were observed to be decreased in inflammation. In overgrowth, dermatan sulphate and chondroitin sulphate were found to increase while the presence of heparan sulphate was not significant. The changes in the pattern of individual glycosaminoglycan in the two varied conditions are discussed.Abbreviations GAG glycosaminoglycan - MPS mucopolysaccharide - DS dermatan sulphate - HS heparan sulphate - CS chondroitin sulphate - HA hyaluronic acid - KS keratan sulphate     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

Structure and metabolism of rat liver heparan sulphate

Rat liver cells grown in primary cultures in the presence of [³⁵S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO₂ treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In ³H-labelled heparan sulphate, isolated after incubation of the cells with [³H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [³⁵S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [³⁵S]sulphate by rat liver cells incubated in the presence of [³⁵S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.     

The glycosaminoglycans of pig colonic wall connective tissue

Colon wall from pig, stripped of most of the mucosal layer to leave material largely composed of muscle, basement membrane, and extracellular matrix, was subjected to procedures for isolation of glycosaminoglycans. A total ethanol precipitate from a papain digest was fractionated by selective ethanol precipitation in the presence of Ca²⁺. Glycosaminoglycan fractions, freed proteolytically from a high molecular weight glycoprotein component, were further purified by Sepharose CL-6B gel-filtration or DE-52 anion-exchange chromatography. Glycosaminoglycans were identified by chemical composition, ¹³C-NMR spectroscopy and response to chondroitinase and nitrous acid degradations. The content of glycosaminoglycan in the tissue is low (0.05% dry weight) being comprised of dermatan sulphate (38%), heparin (34%), heparan sulphate (18%) and chondroitin sulphates (10%) as a percentage of total glycosaminoglycan content. Hyaluronic acid and keratan sulphate have not been detected. The composition is generally typical of a high muscle content tissue.     

Effects of hyaluronate and heparan sulphate on collagen-fibronectin interactions

Interactions of fibronectin and glycosaminoglycans and the involvement of heparan sulphate and hyaluronate in fibronectin-collagen interactions have been studied by affinity chromatography. Partially periodate-oxidized glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. The elution of fibronectin was performed by using increasing concentrations of NaCl. Of the copolymeric glycosaminoglycans, heparin and self-associating heparan sulphates display the highest affinity towards fibronectin while hyaluronic acid and chondroitin 6-sulphate do not bind fibronectin. Competitive release experiments suggest the existence of common binding sites for copolymeric glycosaminoglycans on the fibronectin backbone. Heparan sulphate favours the formation of collagen-fibronectin complexes at low molarity, while hyaluronate is ineffective at low concentrations and prevents the formation of complexes when present at concentrations > 1 mg ml^?1. It is suggested that heparan sulphate promotes the formation of complexes which bind with fibronectin thus producing steric changes that increase the affinity for collagen, while hyaluronate prevents the binding of fibronectin to collagen by a steric exclusion mechanism.     

Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1-³H]glucosamine and sodium [³⁵S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of $\text{‘O’-}\text{sulphate}$ groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of ³⁵[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.     

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Effect of magnesium deficiency on the metabolism of glycosamino-glycans in rats

Magnesium deficiency in rats has significant effect on the concentration of different glycosaminoglycans in the tissues, the nature of the change being different in different tissues. Total glycosaminoglycans, chondroitin-4-sulphate + chondroitin-6-sulphate and dermatan sulphate increased in the aorta while hyaluronic acid, heparan sulphate and heparin decreased. In the liver, total glycosaminoglycans, hyaluronic acid, chondroitin-4-sulphate + 6-sulphate and heparin decreased while total glycosamino-glycans and all the glycosaminoglycan fractions increased in the heart. In the kidney, total glycosaminoglycans showed no significant alteration, hyaluronic acid and heparin decreased while chondroitin-4-sulphate + 6-sulphate increased. Activity of biosynthetic enzymesviz. glucosamine-o-phosphate isomerase and UDPG-dehydrogenase showed decrease in the liver. The concentration of 3’-phosphoadenosine 5’-phosphosulphate, activity of sulphate activating system and sulphotransferase were also similarly altered in the liver in magnesium deficiency.     

Chondroitin sulphate at the endothelial lumen of pig aorta: Assay by radiolabelling and by X-ray microanalysis

Trypsin-releasable glycosaminoglycans from the luminal surface of intact pig aorta were measured following metabolic labelling with³⁵S]sulphate. Chondroitin sulphate was found to be present at a surface density equal to that already established for heparan sulphate (5×10¹¹ chains per cm²). This result was confirmed by X-ray microanalysis of the luminal sulphur content before and after treatment with specific glycosaminoglycan-degrading enzymes. This result implies that approximately half of the luminal surface is occupied by sulphated glycosaminoglycans.     

Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

Binding,internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Binding, internalization, and degradation of ¹²⁵I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (K_d approximately 10⁻⁸ M) and to one (K_d approximately 10⁻⁶ M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 μg/ml (approximately 10⁻⁷ M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected. J. Cell. Biochem. 64:595–604. © 1997 Wiley-Liss, Inc.