The glycosaminoglycans of estrogen-induced medullary bone in Japanese quail

Glycosaminoglycans were isolated from the femurs of estrogen-treated male Japanese quail. During the 72 h after the injection of estrogen the incorporation of a 1-h pulse of H₂³⁵SO₄ into keratan sulfate increased more than 100-fold in a pattern corresponding to the production of the induced medullary bone. The rate of incorporation into chondroitin 4-sulfate, the only other glycosaminoglycan detected, remained constant throughout the same time period. The rate of incorporation of the 1-h pulse of sulfate into chondroitin 4-sulfate and keratan sulfate was the same at 48 h of estrogen treatment. When birds (48 h estrogen) were allowed to live 6 h after the injection of the isotope, chondroitin 4-sulfate accumulated 5-fold over that found for similar animals labeled for only 1 h. Keratan sulfate, into which the isotope was incorporated at the same rate as the chondroitin sulfate in this experiment, did not accumulate much more in 6 h of labeling than in 1 h of labeling. This suggests that the keratan sulfate turns over more rapidly than the chondroitin 4-sulfate in this tissue. Autoradiography showed that the chondroitin 4-sulfate was associated mainly with the marrow cells near the cortical bone and the keratan sulfate with the newly synthesized medullary bone. These results suggest that keratan sulfate is a specific marker for this secondary bone matrix.     

In vitro synthesis of proteoglycans associated with medullary bone in Japanese quail

A short-term incubation system was used to study proteoglycan synthesis during the early stages of medullary bone formation in estrogen-treated male Japanese quail. The proteoglycans were separated by chromatography on a DEAE Bio-Gel A column eluted with a 400-ml 0-1 M NaCl gradient. The profile from uninjected control birds showed a single peak, whereas profiles from estrogen-treated birds showed development of another peak. Incorporation of [35S]sulfate into the estrogen-induced proteoglycan increased most dramatically between 25 and 37 h after hormone treatment. The estrogen-induced proteoglycan has a Kav = 0.65 on Sepharose CL-4B, an average buoyant density of 1.50 g/ml, and contains keratan sulfate as its constituent glycosaminoglycan. The second proteoglycan has a Kav = 0.52 on Sepharose CL-4B, an average buoyant density of greater than or equal to 1.7 g/ml, and has chondroitin sulfate as it major glycosaminoglycan. It may also contain some heparin or heparan sulfate. The results support the usefulness of the incubation system for studying the dynamics of bone matrix production.     

Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II

A keratan sulfate proteoglycan was isolated under denaturing conditions from the mineral compartment of rabbit cortical bone. This small proteoglycan (Kd = 0.39 on Superose 6, Mr approximately 20,000 on sodium dodecyl sulfate gels) contained small keratan sulfate chains that were distinctly bimodal in size. The keratanase and endo-beta-galactosidase digestible glycosaminoglycan chains were O-linked to a core protein of Mr approximately 80,000. This core protein had several properties in common with the bone sialoprotein II molecule of bovine and human bone including: a closely spaced doublet band on sodium dodecyl sulfate electrophoresis gels; a high staining intensity with Stains All that was greatly diminished by neuraminidase; a significant amount of small O-linked oligosaccharides; and an amino-terminal amino acid sequence that was nearly identical to human bone sialoprotein II. (In contrast, bone sialoprotein II in human, bovine, and rat bone does not appear to have any keratan sulfate chains.) Antiserum made against the keratan sulfate proteoglycan reacted with its core protein on electrotransfers from sodium dodecyl sulfate-polyacrylamide gels.     

Keratan sulfate proteoglycan isolated from the estrogen-induced medullary bone in Japanese quail

1. Two proteoglycans isolated from the femurs of quail actively producing medullary bone were separated using DEAE Bio-Gel A. 2. The first to elute in the gradient was a keratan sulfate proteoglycan with an average buoyant density of 1.53 g/ml and a Kav = 0.57 on Sepharose CL-4B. 3. The second proteoglycan to elute contained chondroitin 4-sulfate. 4. Apparently only the keratan sulfate proteoglycan is associated with the new medullary bone matrix.     

Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.     

Estrogens and the Hypothalamus: Nuclear Receptor and RNA Polymerase Activation

Abstract

To further the understanding of estrogen action in the central nervous system, we have developed a procedure for quantitation of nuclear estrogen receptor (R_nE) in adult rat brain. Crude chromatin is separated from soluble and membranous fractions by centrifugation of brain homogenate through 1.2 M sucrose pads. Incubation of the pellet with [³H] estradiol at elevated temperature and precipitation of receptor-steroid complexes with protamine sulfate allows measurement of R_nE. The yield of DNA by this procedure is 70–80% and the amount of R_nE measured is linear with respect to the amount of DNA added. Using this procedure, we have measured hypothalamic R_nE in the ovariectomized adult rat as a function of time after injection of 5 ug estradiol and found a peak R_nE accumulation of about 30 fmol/hypothalamus at 1 h. By 12 h, the levels of R_nE return to control (uninjected) values.

We have also measured the time course of estradiol induced activation of hypothalamic endogenous nuclear RNA polymerases I and of II, suggested to be initial steps in estrogen action. RNA polymerase I activity was unaffected by estrogen treatment, while RNA polymerase II activity showed a transient activation with a time course approximating that of R_nE accumulation. For early times after estrogen treatment (10–90 min), the amount of R_nE present in the hypothalamus is correlated with the extent of RNA polymerase II activation. The absence of sustained activation of RNA polymerases I or II in the hypothalamus is consistent with the lack of long term retention of R_nE in nuclei of this, tissue and the lack of estrogen induced hypertrophy and hyperplasia.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

Further purification and characterization of newly synthesized anionic glycoconjugates secreted by cultured UMR-106 cells: evidence that the major anionic glycoconjugate secreted by these cells is similar to bone sialoprotein II.

Following incubation of UMR-106 cells for 48 h in the presence of [3H]glucosamine and [35S]sulfate, the newly synthesized anionic glycoconjugates were isolated from the culture medium by cetylpyridinium chloride/ethanol precipitation and further separated by DEAE-Sephacel chromatography into two radiolabelled fractions, a major component, UM I, and a minor component, UM II. UM I appeared to be homogeneous as shown by Sepharose CL-4B chromatography under dissociative conditions, and SDS-polyacrylamide gel electrophoresis. It showed a molecular mass of approximately 93 kDa on 4-15% gels. UM I was partially degraded by brief treatment with trypsin, releasing a small, terminal peptide that contained 47.6% of 35S but no 3H. Treatment of UM I with neuraminidase and 0.1 N H2SO4 (1 h at 80 degrees C), respectively, released 27% 3H and 38.4% 3H plus 41% 35S, suggesting the presence of a significant number of sialic acid residues, as shown by Sephadex G-50 chromatography of the digests. Amino acid analysis showed that the UM I glycoconjugate was rich in acidic amino acids (12.6% aspartic acid and 21.2% glutamic acid residues) and its N-terminal sequence was Phe-Ser-Met-Lys-Asn-Phe-, which is identical to the published N-terminal amino acid sequence of rat bone sialoprotein II. Keratanase treatment of UM I released 26% of the incorporated radioactivity, suggesting the presence of keratan sulfate chains. UM II contained a chondroitinase ABC-sensitive proteoglycan.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

Relationship of sulfation to ongoing chondroitin polymerization during biosynthesis of chondroitin 4-sulfate by microsomal preparations from cultured mouse mastocytoma cells

A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.     

Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans

Proteoglycans from bovine tracheal cartilage were digested with trypsin and chymotrypsin by procedures similar to those described by Mathews (Biochem. J.125, 37 (1971)). Chondroitin sulfate-peptide fragments in the digest were precipitated with cetylpyridinium chloride and subsequently fractionated on a preparative Sepharose 6B column. The fragments, which emerged from the column as a broad peak, were divided into five fractions. Rechromatography of these fractions on an analytical Sepharose 6B column indicated that they had K_av values from 0.17 (fraction 1) to 0.62 (fraction 5). The weight average molecular weight values obtained by meniscus depletion equilibrium centrifugation were 193,000, 126,000, 80,000, 46,000, and 23,000 for fractions 1 to 5, respectively. Values for the molecular weights and for the limiting viscosity numbers, [η], of the fractions were used to determine estimates for α of 0.40–0.46 and for K of 0.43–0.88 in the equation [η] = K·M_v^α. These values for α are consistent with a branched structure for the chondroitin sulfate fractions. Papain digests of each of the fractions were chromatographed on Sephadex G-200. The observed distributions of the monomer chains released by this protease were almost the same for each sample, which indicates that the individual chondroitin sulfate chains in all of the original fractions had nearly the same average molecular weights. The data in sum indicate that peptide fragments which contain from 1 to 8 polysaccharide chains are released when the proteoglycans are digested with trypsin-chymotrypsin.Analytical data indicated that all fractions contained 3–11% of their polysaccharide as keratan sulfate. This indicates either that about 50% of the keratan sulfate chains in the original proteoglycan molecules are located in close proximity to the chondroitin sulfate chains or that some peptides contain large numbers of keratan sulfate chains. Proteoglycan preparations which differed by a factor of about 6 in their ratio of chondroitin sulfate to protein yielded very similar elution patterns on Sepharose 6B after trypsin-chymotrypsin digestion.     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.     

Growth modulation and proteoglycan turnover in cultured mesangial cells

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with ³⁵SO₄^2? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (K_d of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (K_d = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with K_d values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (K_d = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular ³⁵S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [³H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G₁/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc.     

Degradation of pre-messenger RNA in bovine thyroid slices; effect of thyrotropin

Summary Bovine thyroid RNA labeled by incubation of slices in the presence of ³²P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of ³²P-labeled pre-mRNA and a proportional increase of ³²P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.Abbreviations SDS sodium dodecyl sulfate - TIPNS triisopropylnaphthalene disulfonic acid, sodium salt - TSH thyrotropin-stimulating hormone     

Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase

We have previously found that the purified chondroitin 6-sulfotransferase(C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate(PAPS) to position 6 of N-acetylgalactosamine in chondroitin,catalyzed the sulfation of keratan sulfate, and that both theC6ST activity and the keratan sulfate sulfotransferase (KSST)activity were expressed in COS-7 cells when C6ST cDNA was transfected.In this report we describe some properties of the KSST activitycontained in the purified C6ST, and characterize the sulfatedproducts formed from keratan sulfate and partially desulfatedkeratan sulfate. Optimal pH, requirement for cationic activators,and K_m value for PAPS of the KSST activity were very similarto those of the C6ST activity. ³⁵S-Labeled glycosaminoglycansformed from keratan sulfate and partially desulfated keratansulfate were N-deacetylated by treatment with hydrazine/hydrazinesulfate and then cleaved with HNO₂ at pH 4, and the resultingproducts were reduced with NaB³H₄. Analysis of the degradationproducts with paper chromatography and high performance liquidchromatography provided evidence that C6ST transferred sulfateto position 6 of galactose residue which was glycosidicallylinked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamineresidue. Northern blot analysis using poly (A)⁺ RNA from 12-d-oldchick embryos indicated that the message of C6ST was expressednot only in the cartilage but also in the cornea in which keratansulfate is actively synthesized. chondroitin sulfate keratan sulfate glycosaminoglycan sulfotransferase hydrazinolysis deaminative cleavage