Incorporation Of Radioactive Sulfate Research Articles

Ruthenium(II)- and copper(I)-catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan priming activity

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and β-1,4-galactosyltransferase 7 (β4GalT7).

Matrix Disruptions, Growth, and Degradation of Cartilage with Impaired Sulfation

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

Human cationic trypsinogen is sulfated on Tyr154

The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-β activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.

4:27 Transduction of TGF-B1 inhibits proteoglycan synthesis in intervertebral disc cells in a three-dimensional culture

Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and a subsequent decrease in the disc's hydrostatic capacity. Growth factors, such as TGF-B1, administered in recombinant form or synthesized endogenously by means of gene therapy, stimulate proteoglycan synthesis in intervertebral disc cells in monolayer cultures. However, these cultures do not accurately reflect the native environment of intervertebral disc cells, and therefore, conclusions regarding TGF-B1's in vivo potential are difficult to extrapolate. The purpose of this study was to evaluate the effect of adenoviral-mediated delivery of TGF-B1 on proteoglycan synthesis in human intervertebral disc cells cultured in a three-dimensional system.Methods used: Nucleus pulposus cells were isolated from human intervertebral discs by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with Ad-TGF-B1 at either 50, 75 or 100 multiplicity of infection (MOI). After 48 hours of incubation, cells were trypsinized and incorporated into a three-dimensional "pellet" culture system. After an additional 48 hours, proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count.of findings: Cultures treated with Ad-TGF-B1 demonstrated a progressive decrease in active proteoglycan synthesis with an increasing MOI. For an MOI of 50, proteoglycan synthesis was 53.4% of control; for an MOI of 75, 24.8% of control and for a MOI of 100, 11.2% of control (p<.05). Additionally, a dose-dependent response was shown to be significant with increasing viral concentrations (p<.001).Relationship between findings and existing knowledge: The inhibition of proteoglycan synthesis by TGF-B1 observed in this study contrasts with the stimulatory effect previously reported in monolayer cultures. The concentrations employed in our work were 80% less than those previously demonstrated to be stimulatory, yet proteoglycan synthesis in the three-dimensional culture was uniformly diminished in a dose-dependent manner (Table 1)Table 1Inhibitory effect of TGF-B1 on proteoglycan synthesis50 MOI75 MOI100 MOITGF-B153.4%24.8%11.2%.Overall significance of findings: Although exogenous administration of the growth factor TGF-B1 has been shown to favorably modify proteoglycan synthesis in human intervertebral disc cells in vitro, its brief half life limits its potential role in the treatment of chronic disorders, such as degenerative disc disease. Experiments with adenoviral-mediated delivery of TGF-B1 in monolayer cultures demonstrate a successful increase in proteoglycan synthesis as well. However, this experimental system does not reflect the in vivo environment, and as a result, conclusions regarding the clinical potential of the gene delivery of TGF-B1 drawn from these experiments may be misleading. As the potential for practical application of gene therapy becomes increasingly feasible, experimental models that accurately simulate the in vivo environment acquire greater significance. The inhibitory response to TGF-B1 seen in our study may result from the increased cell-to-cell interactions within the three-dimensional pellet culture, which in turn may produce higher, and potentially toxic, TGF-B1 concentrations in the microcellular environment. Further experimentation is warranted to clarify whether TGF-B1 possesses therapeutic potential for the future treatment of intervertebral disc disease.Disclosures: No disclosures.Conflict of interest: No conflicts.

4:21 Gene delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 increases proteoglycan synthesis in human intervertebral disc cells

Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and subsequent disc dehydration. Previous studies demonstrate increased matrix metalloproteinases in degenerated discs compared with controls and propose an intimate role of these catabolic proteins in the degenerative process. Gene therapy has been investigated as a method to induce endogenous synthesis of therapeutic growth factors to stimulate proteoglycan synthesis. Although many anabolic growth factors increase matrix synthesis, the use of catabolic inhibitors has yet to be explored. The objective of this study was to evaluate the effect of adenoviral-mediated delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 (TIMP–1), as well as the anabolic potential of bone morphogenic protein–2 (BMP–2), on proteoglycan synthesis.Methods used: Human nucleus pulposus cells were isolated by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with either Ad-TIMP-1 or Ad-BMP-2 at 50, 75, 100 or 150 multiplicity of infection (MOI). After 48 hours of incubation, cells were incorporated into a three-dimensional “pellet” culture system. After an additional 48 hours of incubation, active proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count.of findings: Both Ad-TIMP-1 and Ad-BMP-2 increase proteoglycan synthesis compared with controls (p<.05). Cultures treated with Ad-BMP-2 demonstrated a progressive increase in synthesis with increasing viral MOI (190%, 212%, 310% and 466%). Cells treated with Ad-TIMP-1 exhibited a maximum response at a MOI of 100 (65%, 289%, 378% and 249%).Relationship between findings and existing knowledge: Although anabolic factors have previously been shown to increase proteoglycan synthesis in cultured disc cells, this is the first report demonstrating a similar upregulating capacity with the use of catabolic inhibitors.Overall significance of findings: This study demonstrates that both catabolic inhibitors (TIMP–1) and anabolic growth factors (BMP-2) are successful at increasing measured proteoglycan synthesis in cultured human intervertebral disc cells. The optimal regulation of matrix synthesis may be achievable with gene therapy using a combination of both anabolic factors and catabolic inhibitors. Further studies are warranted to assess the potential of combination therapy with these growth factors to upregulate proteoglycan synthesis, as well as to determine if such combinations can minimize the total vector load required to elicit a beneficial, and potentially therapeutic, response (Table 1)Table 1Stimulatory effect on proteoglycan synthesis50 MOI75 MOI100 MOI150 MOITIMP–165%289%378%249%BMP–2190%212%310%466%.Disclosures: No disclosures.Conflict of interest: No conflict.

Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rab11-GTPases.

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.

Storing live embryonic and adult human cartilage grafts for transplantation using a joint simulating device

Objectives: Cartilage transplantation as a means to replace damaged articular surfaces is of interest. A major obstacle is the long-term preservation of cartilage grafts. The commonly used technique of freezing the grafts inevitably leads to cellular death. The current study compares the technique to an innovative approach using a pulsed-pressure perfusion system termed a joint simulating device (JSD), intended to simulate intra-articular mechanical forces. Methods: Human articular cartilage explants were harvested from both embryonic epiphyseal tissue and femoral heads of elderly women (over 70 years of age) undergoing a partial joint replacement (hemi-arthroplasty) and were divided in two groups: half of the samples were incubated in the JSD while the remaining half were grown in static culture within tissue culture plates. After 10 days all samples were evaluated for: (a) cell vitality as assessed by image analysis and XTT assay; (b) biosynthetic activity as expressed by radioactive sulfate incorporation into glycosaminoglycans (GAG’s); and (c) proteoglycan content as assessed by alcian blue staining intensity. Results: A 10-fold increase in sulfate incorporation in samples held in the JSD compared to the static culture group was observed in embryonic cartilage. In adult cartilage culture in the JSD elevated sulfate incorporation by threefold as compared to static culture. Central necrosis was observed in specimens grown in the static culture plates, while it did not occur in the samples held in the JSD. Cell vitality as assessed by XTT assay was significantly better in the JSD group as compared to static culture. The difference was more pronounced in the embryonic specimens as compared to adult cartilage. The specimens cultured within the JSD retained proteoglycans significantly better than those cultured in static culture. Conclusions: Maintenance of cartilage specimens in a JSD was highly effective in keeping the vitality of cartilage explants in vitro over a 10-day period. A possible future application may be a long-term preservation of chondral grafts, without freezing. Aviodance of freezing of cartilage grafts, might prevent the cartilage degeneration often observed in frozen osteochondral grafts.

Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle

Mdx mice uniquely recover from degenerative dystrophic lesions through an intense myoproliferative response. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix is essential for normal myogenesis. Proteoglycans are abundant components of the extracellular matrix. The synthesis of proteoglycans in mdx mice during skeletal muscle regeneration was evaluated. Incorporation of radioactive sulfate demonstrated a significant increase in the synthesis of several types of proteoglycans in mdx animals compared to age-matched controls. The size and charge of proteoglycans synthesized by the mdx mice remained unchanged. In particular, one of the up-regulated proteoglycans, the small chondroitin/dermatan sulfate proteoglycan decorin which is known to bind and to sequester transforming growth factor-beta, was investigated. Immunocytolocalization and in situ hybridization studies showed that decorin mainly accumulated in the endomysium, i.e. around individual skeletal muscle fibers from M. tibialis anterior and diaphragm.

Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus.

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69-80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915-934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949-966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

Antioxidant effects of hyaluronan and its α-methyl-prednisolone derivative in chondrocyteand cartilage cultures

Objective: To compare hyaluronan and its α-methyl prednisolone derivative(HYC-141) with respect to their potential to directly cause tissue damage and to protect tissues from attack by reactive oxygen species. Methods: Cartilage samples and chondrocytes were isolated from 15-day chickembryos and were exposed to enzyme-based and activated inflammatory cell-free radical generating systems in the presence of varying concentrations of α-methyl prednisolone, hyaluronan, and HYC-141. Tissue and cell vitality was measured by determining the incorporation of radioactive sulphate into newly synthesized glycosaminoglycans. Results: Only α-methyl prednisolone caused a significant decrease in biosyntheticactivity. All the tested substances were capable, to some extent, of protecting tissues and cells from damage by reactive oxygen species; HYC-141 demonstrated the greatest protective effect. Conclusion: These data suggest that HYC-141 may possess certain advantages over the individual component molecules in the local treatment of arthropathies.

Effects of parathyroid hormone‐related peptide on chick growth plate chondrocytes

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH) have similar biological effects in vitro that are mediated through the PTH receptor. PTH receptors have been demonstrated in the zone of provisional calcification and the hypertrophic zone of the cartilaginous growth plate. The current study examined the biological effects of PTHrP on chick growth plate chondrocytes. Chondrocytes were exposed to varying doses of PTHrP for 24 h, and the incorporation of radioactive thymidine into DNA was used as an index of proliferation. A dose-dependent stimulation of proliferation was seen, with a maximal 27-fold increase at 50 nM PTHrP. A dose-dependent stimulation of cAMP was seen, with a maximal effect at a dose of 50 nM. Proteoglycan synthesis, measured by incorporation of radioactive sulfate, was stimulated, with a maximal effect of 65% at 1 nM. Collagen synthesis and alkaline phosphatase activity from both cellular and matrix vesicle sources decreased in a dose-dependent fashion, with a maximal inhibition of approximately 50% of the control value. The physiologic significance of the PTH and PTHrP-responsiveness of growth plate chondrocytes is uncertain at the present time. It is possible that PTH or PTHrP, or both, act as a systemic, developmental modulator of cellular proliferation and differentiation in the growth plate.

Proteoglycans and endothelial barrier function: effect of linoleic acid exposure to porcine pulmonary artery endothelial cells

Certain fatty acids induce changes in endothelial barrier function which may be mediated by alterations in normal proteoglycan synthesis/metabolism. To test this hypothesis, pulmonary artery derived endothelial cells were treated with media supplemented with linoleic acid (18:2), and/or a known proteoglycan synthesis inhibitor, β- d-xyloside. Independent exposure to 1 MM β- d-xyloside or 90 μM 18:2 increased albumin transfer, i.e., decreased barrier function, when compared with control cultures. 18:2 and β- d-xyloside increased albumin transfer additively, suggesting that the mechanisms by which 18:2 and β- d-xyloside alter the proteoglycan metabolism are different. Compared with the control group, treatment with 18:2 inhibited proteoglycan synthesis, decreased anionic properties of heparan sulfate proteoglycans in the cell monolayers and caused the release of a unique chondroitin sulfate proteoglycan into the culture media. Treatment with β- d-xyloside caused an increased incorporation of radioactive sulfate into glycosaminoglycans but inhibited proteoglycan synthesis. These results suggest that the fatty acid- and β- d-xyloside-induced impairment in endothelial barrier function may involve changes in the synthesis, release and physicochemical properties of proteoglycans.

A high molecular weight proteoglycan is differentially expressed during development of the mollusc Concholepas concholepas (Mollusca; Gastropoda; Muricidae).

Incorporation of radioactive sulfate to hatched veliger larvae of the gastropod muricid Concholepas concholepas indicated that over 87% of the sulfated macromolecules were found in the detergent insoluble fraction, rich in extracellular matrix (ECM) components. The sulfated material was solubilized with guanidine salt followed by urea dialysis and fractionated by DEAE-Sephacel chromatography. Three sulfated compounds eluting at 0.7, 1.1, and 3.0 M NaCl, called peaks I, II, and III, respectively, were obtained. The sulfated compound present in peak I was degraded by pronase or sodium alkaline treatment to a small sulfated resistant material, suggesting the presence of a proteoglycan (PG). Filtration analysis on Sephacryl S-500 and SDS-PAGE of the intact PG indicates that it has a high molecular weight (360,000 to over 1 x 10(6)). Monoclonal antibodies (mAb) against this PG were produced. The specificity of one mAb, the 6H2, was demonstrated by size chromatography and ELISA analysis. The epitope recognized by this mAb seems to be present in the core protein of the PG. Both the extent of sulfation and the presence of different sulfated species of PGs were evaluated during the development of this mollusc. A twelvefold increase in the incorporation of sulfate to PGs per milligram of protein was found in veliger larvae compared to blastula-glastula stages. This change correlated well with the differential expression of the sulfated PG present in peak I. Biochemical and immunological analysis indicate that high levels of this PG are found in veliger and trocophore larvae in comparison with blastula-gastrula and early juveniles.(ABSTRACT TRUNCATED AT 250 WORDS)

Decorin, a chondroitin/dermatan sulfate proteoglycan is under neural control in rat skeletal muscle.

Proteoglycans (PGs) are abundant components of the extracellular matrices (ECM) of skeletal muscle. We have previously found that the synthesis of skeletal muscle PGs present at the ECM increase after denervation. The experiments reported here were undertaken to identify which PG(s) increase after denervation of rat leg muscles. Incorporation of radioactive sulfate demonstrated the presence of a chondroitin/dermatan sulfate PG of 70-90 kDa in the skeletal muscle ECM, which increased after denervation. The PG has a core protein of 39-45 kDa after treatment with chondroitinase ABC. Antibodies against rat decorin, a chondroitin/dermatan sulfate PG synthesized by various cell types, specifically immunoprecipitated this PG from a mixture of PGs. Immunocytolocalization of this PG indicated that the chondroitin/dermatan sulfate PG accumulates at the perimysium of skeletal muscle after denervation. Finally, Northern blot analysis indicated an increase of muscle transcripts for decorin after denervation. The data reported here suggest that a chondroitin/dermatan sulfate PG present at the skeletal muscle ECM, very similar if not identical to decorin, increases after denervation.

Suppression of proteoglycan synthesis in chondrocyte cultures derived from canine intervertebral disc.

Chondrocytes were dissociated enzymatically from canine nucleus pulposus. After attaining monolayer growth, the cells were subpassaged and incubated with sodium salicylate, indomethacin, or sodium meclofenamate (10(-2) M to 3 x 10(-6) M). When incubated with nonsteroidal anti-inflammatory drug concentrations mimicking therapeutic plasma levels, proteoglycan synthesis was significantly suppressed. After 24 hours of incubation, there was a 15% decrease in radioactive sulfate (35SO4) incorporation for salicylate (10(-3) M), a 29% decrease for indomethacin (3 x 10(-6) M), and a 75% decrease for sodium meclofenamate (10(-5) M). This inhibition was both dose- and time-dependent. Despite the effect of these nonsteroidal anti-inflammatory drugs on quantitative proteoglycan synthesis, there was no demonstrable effect of these drugs on the size of proteoglycan monomers or the degree to which proteoglycan aggregate formation occurred.

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor β1 and β2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor β (TGF-β) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor β isoforms (TGF- β 1 and TGF- β 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGFβ was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-β isoforms.

Physico-chemical and immunological characteristics of pituitary prolactin from water buffaloes (Bubalus bubalis)

Prolactin (PRL) was purified from freshly frozen pituitary glands of water buffaloes (Bubalus bubalis) by a combination of existing procedures of Ellis and Jiang and Wilhelmi involving serial extraction of different pituitary proteins. The partially purified preparation was further fractionated on DEAE-Sephadex followed by Sephadex G-100 chromatography. This was finally purified on HPLC. This preparation was found to be homogeneous by SDS-PAGE and HPLC and had a single N-terminus amino acid (Threonine). The molecular size was estimated to be 24K +/- 0.5 by SDS-PAGE and approximately 25K by GPC-HPLC. The buffalo PRL gave a dose dependent inhibition curve in a rat liver based radio receptor assay with a potency of 30-35 I.U./mg and also in a partial homologous RIA using 125I-buffalo PRL and rabbit anti-oPRL serum giving a potency of 30 I.U./mg. Metabolic labelling studies using 35SO4(2-) with buffalo pituitary minces showed the incorporation of radioactive sulfate into immunoprecipitable PRL-like material. Physico-chemical characterization of the site of the linkage between sulfate and PRL revealed the presence of Tyr-O-SO4 in bu-PRL. A high affinity monoclonal antibody (MAB) with Ka of 10(10) L/M, belonging to IgG1 isotype, and capable of cross reacting with ovine and bovine PRL was generated. This MAB was conformation specific as reduced and carboxymethylated PRL did not react with it. A homologous RIA system using this MAB has been standardised.

Connective Tissue Metabolism in Bone and Cartilage of Adjuvant Arthritic Rat

The metabolism of connective tissue matrix components such as glycosaminoglycans and glycoproteins was investigated in normal as well as pathological tissues of bone and cartilage associated with adjuvant arthritis using rat as animal model of the disease. The inflammatory process of adjuvant arthritis was induced in rats with the inoculation of Freund's adjuvant containing heat killed Mycobacterium tuberculosis suspended in paraffin oil. The changes in the metabolism of matrix components in bone and cartilage were examined using radioactive isotopic labeling measurements during the acute as well as chronic phases of arthritic disease. The glycosaminoglycans were fractionated into sulfated and non-sulfated glycosaminoglycans by chemical and enzymic modifications. The biosynthesis of sulfated glycosaminoglycans was evaluated using radioactive labeled (35S)-sulfate. Alterations were demonstrated in the metabolism of connective tissue in the bone and cartilage tissues of arthritic rat. The results obtained showed an increased incorporation of radioactive sulfate in specimens of bone and cartilage during the process of adjuvant arthritis. The contents of sulfated as well as non-sulfated glycosaminoglycans were found to be increased in both the tissues of arthritic rat. Similarly, the amount of total glycosaminoglycans was also found to be increased significantly in the diseased tissues. In addition, various components of tissue glycoproteins such as fucose, sialic acid and total hexose were found to be elevated in insoluble fractions of bone and cartilage during the diseased state. The effects of experimentally induced adjuvant arthritis on the connective tissue were discussed in the light of changes taking place in the metabolism of glycosaminoglycans and glycoproteins in bone and cartilage of arthritic rat.

Adaptive changes in sulfoglycolipids of kidney cell lines by culture in anisosmotic media

(1) The effects of osmolarity environments on renal glycolipid composition were examined using established renal cell lines. The profile of glycosphingolipids of Madin-Darby canine kidney cells (MDCK) in culture with anisosmotic media showed that a hyposmotic medium reduced the concentration of GalCer I 3-sulfate and LacCer II 3-sulfate. (2) The concentrations of sulfoglycolipids were increased by maintaining the culture in a hyperosmotic media prepared by the addition of various sodium salts to the control isosmotic medium, while the contents of most of the neutral glycolipids were reduced. The hyperosmotic medium supplemented with nonelectrolytes, mannitol, sucrose or urea, also increased the concentration of sulfoglycolipids. (3) Both sulfoglycolipids were increased linearly with gradual increases of sodium chloride in the medium. Hyperosmolarity produced by the addition of a nonelectrolyte, mannitol, also increased the levels of sulfoglycolipids. In both series of media, the most prominent accumulation was observed in LacCer II 3-sulfate. (4) The incorporation of radioactive sulfate into sulfoglycolipids was elevated in cells adapted to high NaCl or mannitol. The increase of the label was observed not only in MDCK but also in three other established cell lines of renal tubular origin, JTC-12, LLC-PK 1 and MDBK. (5) It was established, using the culture system of homogeneous cell lines, that the mechanism of increasing the amount of sulfoglycolipids is independent of the integral regulatory mechanism of animals and resides in the renal epithelial cell itself. These results suggest that by culture in hyperosmotic media, the elevated level of intracellular cations stimulated the activity of GalCer and LacCer sulfotransferase, inducing the increased expression of sulfoglycolipids.