Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.36 (hyaluronidase)
 4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue culture conditions can modulate apparent levels of incorporation of the radiolabeled precursor [3H]glucosamine into hyaluronic acid in cells. A careful study was made on the effects of culture conditions on human skin fibroblasts. A newly described technique to measure hyaluronic acid was utilized based on incorporation of [3H]glucosamine into cetylpyridinium chloride-precipitable hyaluronidase-digestible material. The precipitate was collected on glass fiber filters using a manifold suction apparatus. A six-fold greater level of incorporation occurred in rapidly growing preconfluent than in confluent fibroblasts. Ascorbic acid stimulated incorporation with a maximum at 25 micrograms/ml. The same ascorbic acid optimum was observed for collagen prolylhydroxylation. When beta-hydroxybutyrate was used as an energy source instead of D-glucose, a 3.5-fold increase in levels was observed. All tissue-culture media examined supported comparable levels of incorporation, except for Roswell Park Memorial Institute Media-1640, in which cells had only half the level. Fetal calf serum supported high levels of incorporation in a dose-dependent manner, while newborn calf and calf sera supported much lower levels of incorporation. Under serum-free conditions, lactalbumin hydrolysate was best able to support incorporation of hyaluronic acid. In the search for mechanisms that modulate hyaluronic acid, it is critical to consider the tissue culture conditions under which incorporation of radiolabeled precursors are being examined.
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PMID:Levels of [3H]glucosamine incorporation into hyaluronic acid by fibroblasts is modulated by culture conditions. 237 20

We have isolated a syngeneic monoclonal antibody (HepSS-1) reactive to a murine methylcholanthrene-induced fibrosarcoma, Meth-A. HepSS-1 also bound to a wide variety of established and fresh normal cells derived from not only mice but also other species such as human, monkey, rat, hamster, and chicken. Immunoprecipitation of surface iodinated Meth-A cell extract with HepSS-1, as well as Sepharose 4B gel chromatography of Meth-A cell extract and detection of antigens recognized by HepSS-1 by a sandwich-type radioimmunoassay revealed that the HepSS-1 antigens were composed of several molecular species, with one as large as approximately 10(6) daltons. The following evidence indicates that HepSS-1 specifically recognizes an epitope present in heparan sulfate glycosaminoglycan (HS-GAG). First, treatment of Meth-A cells with heparitinase or heparinase, but not with chondroitinase ABC or hyaluronidase, resulted in the loss of HepSS-1 binding. Second, HS-GAG but not seven other types of GAG (hyaluronic acid, heparin, chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and keratan sulfate) inhibited HepSS-1 binding to Meth-A cells. Third, HepSS-1 bound with HS-GAG but not with the seven other types of GAG. From the binding analysis of HepSS-1 to various modified HS-GAG and whale omega-heparin, it is additionally suggested that HepSS-1 recognizes an epitope closely related to O-sulfated and N-acetylated glucosamine. We found that NIH 3T3 cells expressed more HepSS-1 epitopes at a low cell density than at confluency and in G2 + M than in G1, whereas NIH 3T3 cells transformed with Kirsten-ras oncogene or SV-40 expressed high levels of HepSS-1 epitopes and ceased to show the density-dependent change in the amount of HepSS-1 epitopes. These observations were also reproduced by using NIH 3T3 cells transformed with a temperature sensitive Kirsten murine sarcoma virus maintained at permissive and non-permissive temperatures. Thus HepSS-1 is a first monoclonal antibody to HS-GAG and seems to be useful to elucidate changes in cell surface HS-GAG in normal cell growth and cell transformation.
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PMID:A syngeneic monoclonal antibody to murine Meth-A sarcoma (HepSS-1) recognizes heparan sulfate glycosaminoglycan (HS-GAG): cell density and transformation dependent alteration in cell surface HS-GAG defined by HepSS-1. 243 Oct 47

The effect of hyaluronidase treatment on the incorporation of [3H]glucosamine into hyaluronate in human skin fibroblast cultures was investigated. Fourth passage cells in confluent cultures were treated with hyaluronidase from bovine tests, Streptomyces and leech in Dulbecco's minimum essential medium in the presence of 3% fetal calf serum. The medium was removed from the control (non-treated) and the treated cultures and the washed cell layers were incubated with [3H]glucosamine and [35S]sulfate. [3H]Hyaluronate was separated by DEAE Trisacyl chromatography and identified by specific enzymic assays. Hyaluronidase treatment induced an increase in the amount of labelled hyaluronate secreted into the medium and into the pericellular compartment. This amount reached a plateau with increasing enzyme concentration and with the time of treatment. Oligosaccharides derived from hyaluronate did not produce this effect. The maximal increase was about 3-fold, and was not inhibited by exogenous hyaluronate (25-100 micrograms/ml) or by oligosaccharides from hyaluronate. Cycloheximide (0.03 mM) inhibited hyaluronate synthesis by 18% or less in the control cells and by 50% in the hyaluronidase-pretreated fibroblasts. No significant difference was found in the hyaluronate synthase activity between control and treated cells, at 60 min following treatment, indicating the reversibility of the effect. The persistence of the stimulation required the presence of hyaluronidase. The treatment of cells with specific hyaluronidases (from Streptomyces and leech) or with testicular hyaluronidase did not modify the labelling of the sulfated glycosaminoglycans. The incorporation kinetics of the [3H]glucosamine into labeled hyaluronate and the increased amount of non-labelled hyaluronate determined by radiometric assay indicated a specific stimulation of hyaluronate synthesis in the hyaluronidase-pretreated fibroblast cultures.
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PMID:Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture. 251 Aug 27

Colon cancer cells in culture synthesize and secrete mucin glycoproteins, which carry a number of cancer-associated antigens. However, the structures and mechanisms of biosynthetic processing are not well understood. Mucins synthesized and secreted by LS174T human colon cancer cells were compared to those in LS174T xenografts in athymic mice. Mucins radiolabeled with glucosamine or sulfate were purified by gel filtration and cesium chloride density gradient centrifugation. The mucins were of high molecular weight and were resistant to chondroitinase ABC, hyaluronidase and HNO2 treatment. They were, however, susceptible to pronase digestion and mild alkaline treatment. Using radiochemical precursors, the cellular mucin was shown to contain fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, and sulfate. Oligosaccharides released by beta-elimination had N-acetylgalactosaminitol as the reduced amino sugar and also unreduced galactosamine, indicating that there is N-acetyl-galactosamine O-glycosidically attached to protein core and also peripheral N-acetyl-galactosamine not directly linked to protein. DEAE-cellulose chromatography of mucins showed two major peaks with both intracellular and secreted mucins, but xenograft mucins also had more acidic components. Sulfate-labeled mucins were shifted to less acidic peaks by neuraminidase digestion, which indicates that the same mucin molecules are both sialylated and sulfated. We conclude that the intracellular mucins of cultured colon cancer cells, those secreted into the medium, and those in nude mouse xenografts are chemically similar, but differ in sialic acid and sulfate content. This experimental model system, LS174T cells maintained in culture and as nude mouse xenografts, may be useful for further biosynthetic and structural studies of colon cancer mucin.
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PMID:Comparison of metabolically labeled mucins of LS174T human colon cancer cells in tissue culture and xenograft. 273 49

We have determined whether changes in lung hyaluronan content affect extravascular water in lungs of unanesthetized rabbits. Three groups of experiments were performed. In group 1 (n = 12), no infusions were given; in group 2, nine pairs of rabbits received either intravenous hyaluronidase (750 U.kg-1.min-1) or an equivalent volume of saline; in group 3, nine pairs of rabbits received either hyaluronidase or saline, followed by intravenous saline infusion amounting to 24% of body weight. At the end of each experiment, one lung was analyzed for extravascular lung water by the wet-dry method. Except for group 3, in all animals the other lung was analyzed for hyaluronan content by a method that involved hydrolyzing lung hyaluronan with fungal hyaluronidase to release reducing N-acetyl glucosamine groups, which were quantified. In group 1, lung hyaluronan, which varied from 50 to 159 micrograms/g dry wt (mean 106 +/- 35 micrograms/g dry wt), significantly correlated with variation in extravascular lung water (mean 4.2 +/- 0.3 g/g dry wt). In group 2 rabbits given hyaluronidase, lung hyaluronan was 40% lower and extravascular lung water was 14.6% lower than in paired controls (P less than 0.01). In group 3, volume expansion did not affect lung water, except after hyaluronidase when lung water was 47% higher than paired controls. We conclude that in the lung the content of hyaluronan is one of the determinants of extravascular water content.
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PMID:Hyaluronan affects extravascular water in lungs of unanesthetized rabbits. 274 21

Rabbit corneal proteoglycans were labeled by intrastromal injection of 3H-glucosamine and 35S-sulfate 1 and 2 weeks after partial-thickness radial scalpel incisions. Proteoglycans were extracted with guanidine-HCl and purified by ion exchange chromatography. Wounding caused a marked decrease in the total incorporation of labeled precursors into proteoglycans. The labeled proteoglycans were more readily extracted with guanidine-HCl after wounding. Labeled proteoglycans from wounded corneas had a larger molecular size on gel filtration chromatography than did proteoglycans from control corneas, a result of an increased amount of keratan sulfate in the large molecular size fractions. Analysis of labeled glycosaminoglycan (GAG) from guanidine-extracted proteoglycans and from the corneal tissue after guanidine-HCl extraction showed an increase in the relative amount of heparan sulfate and keratan sulfate after wounding, and a decrease in relative amount of dermatan sulfate. The 35S:3H ratio of heparan and dermatan sulfates increased after wounding, and that of keratan sulfate decreased, suggesting changes in sulfation. Degradation of labeled dermatan sulfate with hyaluronidase and with periodate revealed a 2-fold increase in iduronic acid content and 2-4-fold increase in hyaluronidase-resistant dermatan sulfate in the wounded corneas. Reduction in proteoglycan content, reduced sulfation of keratan sulfate, and accumulation of a high-sulfate, high-iduronic acid dermatan sulfate are previously reported properties of proteoglycan in scar tissue from perforating corneal wounds. Demonstration of these properties in proteoglycan after wounds similar to radial keratotomy incisions suggests that deposition of scar tissue can result from wounds which do not damage Descemet's membrane.
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PMID:Proteoglycans of rabbit corneas with nonperforating wounds. 292 15

Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of erythrocytes. The fibrotic vitreous removed from experimental animals were then incubated with [3H]glucosamine at 37 degrees C for 24 h. The newly synthesized 3H-labeled glycosaminoglycans were isolated by 4 M guanidium hydrochloride extraction followed by pronase digestion. The 3H-labeled glycosaminoglycans were then characterized by gel filtration column chromatography and by specific enzymatic degradation, i.e., hyaluronidase, chondroitinase AC, and/or chondroitinase ABC. The disaccharides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. We previously demonstrated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. Our present results indicate that in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 26%, whereas the synthesis of chondroitin sulfate increased to 59% of the total newly synthesized glycosaminoglycans. These results suggest that cells present in fibrotic vitreous resemble fibroblasts with respect to their activities in glycosaminoglycans synthesis.
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PMID:Change in the synthesis of glycosaminoglycans by fibrotic vitreous induced by erythrocytes. 308 30

It appears that hyaluronate is associated with cell migration and the chondroitin sulphates with differentiation during morphogenesis of the chick embryo. The aim of this study was to see if such a correlation could be made for chondrocranium morphogenesis. Specifically, the purpose of this study was to determine the proportion of extracellular matrix (ECM) to cell area and total head mesenchymal area during chondrocranium morphogenesis; and to identify the location, types, and relative amounts of glycosaminoglycans (GAG) being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time. Morphometric analyses were made on median and parasagittal sections of heads of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. Sections of whole heads were stained with alcian blue at low and high pH as well as digested prior to staining with hyaluronidase (Streptomyces and testicular). Identification of GAGs was done by pulse labelling embryos with [3H]glucosamine, digesting homogenates with hyaluronidase (Streptomyces or testicular), precipitating the undigested GAGs with cetylpyridinium chloride and counting the dissolved precipitates using scintillation spectrophotometry. The types and relative amounts of GAGs present in the presumptive chondrocranium were determined by comparing the amount of radioactivity in the precipitates of the non-digested GAG with the counts in the precipitates of the predigested GAGs. This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Little change in cell density and individual cell area as well as in the relative proportion of total area allocated to cells and ECM occurs. GAGs are localized exclusively in the presumptive chondrocranium. These GAGs are restricted to the ventral half of the presumptive chondrocranium. Within this region, the GAGs are further localized to the presumptive facial area, perichordal region, ethmoid, sphenoid and periotic regions. The types of GAG being synthesized in the head mesenchyme of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAGs (dermatan, keratin, heparin and heparan sulphate).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of glycosaminoglycans in the chondrocranium of the chick embryo before and at the onset of chondrogenesis. 309 Jan 90

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.
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PMID:Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units. 311 58

Fibroblast cultures established from explants of mature scar and skin tissue were analyzed with regard to extracellular glycosaminoglycan (GAG) composition and response to interleukin-1 (IL-1). Following a serum-free 48 hour label with [3H]glucosamine, pericellular and medium GAGs were isolated by precipitation with cetylpyridinium chloride (CPC) and analyzed by cellulose acetate electrophoresis. In addition, susceptibility of the precipitates to Streptomyces hyaluronidase, chondroitinase ABC and heparitinase was determined. Labeled conditioned medium from the scar-derived cells contained both dermatan sulfate (DS) and hyaluronate (HA), as compared to medium from the control (skin-derived) cells which contained predominantly DS. IL-1 induced the appearance of chondroitin 4-sulfate (C4-S) in the medium of the scar cells with no concurrent effect on either DS or HA, and increased the amount of HA in the medium fraction of normal skin cells. The pericellular fraction of the scar-derived cells contained chondroitin 6-sulfate (C6-S) and DS; addition of IL-1 resulted in a shift from DS to heparan sulfate (HS), and the emergence of a pericellular GAG profile similar to that of normal dermal fibroblasts.
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PMID:Interleukin-1-induced changes in extracellular glycosaminoglycan composition of cutaneous scar-derived fibroblasts in culture. 313 46


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