Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To gain insight into the cellular and molecular mechanisms underlying cell interactions in the early postnatal mouse cerebellum, Ca2+-dependent and -independent aggregation mechanisms were characterized using single cell suspensions under conditions that allow discrimination between the two mechanisms. When cerebellar cells were derived from newborn to 10-day-old mouse cerebellum, both mechanisms were active and showed no major change in activity during this time period. Mg2+ could not replace Ca2+ in the Ca2+-dependent mechanism. In contrast to the Ca2+-independent mechanisms, the Ca2+-dependent mechanism was inactive at low temperatures, suggesting a necessity for molecular rearrangement within the surface membrane during aggregation. Neuraminidase, chondroitinase, heparinase or hyaluronidase treatment of cells did not influence the aggregation of cells under Ca2+-dependent and -independent conditions. Chondroitin sulfate inhibited and hyaluronic acid stimulated the Ca2+-dependent mechanism, whereas chondroitin sulfate only slightly and hyaluronic acid strongly inhibited the Ca2+-independent one. Dextran sulfate slightly inhibited both mechanisms, whereas heparin and fucoidan, a complex sulfated carbohydrate, did not influence cell aggregation, while they strongly inhibited attachment of cells to laminin. The polycation poly-L-lysine slightly stimulated the Ca2+-independent mechanism, but inhibited the Ca2+-dependent one. Interestingly, chondroitin sulfate and hyaluronic acid strongly stimulated cell aggregation under conditions where both mechanisms were almost destroyed or inactive. Dextran sulfate showed only a small effect under these conditions. These observations indicate that different molecular mechanisms are active in cell-cell versus cell-extracellular matrix interactions and suggest a hitherto unknown complexity in molecular mechanisms during early postnatal cerebellar development.
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PMID:Characterization of Ca2+-dependent and -independent aggregation mechanisms among mouse cerebellar cells. 246 13

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.
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PMID:Structural properties of the heparan sulfate proteoglycans of brain. 252 92

Neurons from embryonic (E18) rat hippocampus were chosen to identify and characterize neurite growth-stimulating proteins accumulating in serum-free conditioned media (CM) obtained from primary or secondary cultures of cerebral astrocytes (less than 5% nonglial cells) using a quantitative cell culture bioassay. CM were fractionated by FPLC on an anion exchange column (Mono Q) and by gel filtration (Superose 6). Column fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antibodies to laminin (LN) and fibronectin (FN). The neurite-promoting activity (NPA) was tested by incubating aliquots of the eluted fractions with poly-L-lysine precoated glass coverslips prior to addition of neurons suspended in chemically defined medium. We provide evidence that the NPA in astroglial CM could be assigned mainly to a negatively charged, highly sulfated LN complex consisting predominantly of the B-chains of LN and presumably a sulfated proteoglycan that was sensitive for chondroitinase and to a lower degree to heparinase degradation. In addition, a smaller proportion of the NPA was associated with uncomplexed LN and free FN. FN reached approximately 10 times the concentration of LN in astroglial CM. As revealed by immunofluorescence microscopy, both LN and FN are simultaneously expressed by cultured astrocytes; however, only the production of FN, measured by ELISA, increased during the time astrocytes were in culture, whereas the release of LN remained unchanged. We conclude that, besides the most active LN complex, FN bound to a polycationic matrix is able to induce neurite growth in hippocampal neurons in vitro.
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PMID:Astroglia-released neurite growth-inducing activity for embryonic hippocampal neurons is associated with laminin bound in a sulfated complex and free fibronectin. 252 80

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.
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PMID:Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor. 252 52

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.
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PMID:An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates. 262 58

Pro-inflammatory effects of cationic proteins secreted by human granulocytes include induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. We have shown previously that a series of synthetic polycationic amino acids produce a dose-, time- and Mr-dependent inhibition of [3H]leucine or [3H]thymidine incorporation into macromolecules by human umbilical vein endothelial cells, and that the extent of inhibition was correlated with changes in cell morphology, with release of cytoplasmic constituents and was irreversible. The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations. We have found that the extent of inhibition is related to both the identity of the monomer, for polymers of Mr 40,000 the order is ornithine greater than lysine greater than arginine, and to its configuration; poly-D-lysines are more potent inhibitors than poly-L-lysines of similar Mr. Only brief exposure to the agonist is required, 90% inhibition occurred after 10 min of exposure to poly-L-lysine (Mr 90,000). Treatment of endothelial cells with neuraminidase, heparinase, hyaluronidase, chondroitinase or trypsin did not reduce their susceptibility to polylysine. Inhibition of microtubule or microfilament formation also had no effect on polylysine cytotoxicity, indicating that internalisation of the polymer was not a prerequisite for the effect. Inhibition of protein synthesis or pretreatment with simple sugars likewise failed to block the effects of polylysine treatment. Natural cationic proteins exerted similar effects on endothelial cells, the extent of the effect apparently being related to the pI of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical characterisation of polycation-induced cytotoxicity to human vascular endothelial cells. 263 82

Three types (T1, T2, T3) of proteoglycan (PG) filaments, as demonstrated by cuprolinic blue (CB) under critical electrolyte concentration method in the epithelial-stromal interface of the guinea pig lateral prostate, were characterized cytochemically by using a number of glycosaminoglycan(GAG)-degrading enzymes and nitrous acid. The results showed that T1 filaments located in basement membranes of the epithelium, endothelium, and smooth muscle cells, were removed by nitrous acid, heparitinase, and pronase but resistant to chondroitinase (Ch)-ABC and Ch-AC, heparinase, neuraminidase, and Streptomyces (S) hyaluronidase. The T1 filaments, therefore, contain heparan sulfate. The T2 filaments closely linked to collagen fibrils were removed by Ch-ABC, Ch-ABC plus S-hyaluronidase, and pronase but were resistant to nitrous acid, heparitinase, heparinase, neuraminidase, and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulfate. The T3 filaments in the interstitial spaces and on the surface of fibroblasts were removed by Ch-ABC, Ch-AC, and pronase but were resistant to heparitinase, heparinase, hyaluronidase, neuraminidase, and nitrous acid. They are, therefore, rich in chondroitin sulfate.
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PMID:Cytochemical characterization of cuprolinic blue-stained proteoglycans in the epithelial-stromal interface of the guinea pig lateral prostate. 271 Jun 91

The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H. The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans. The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked to N-acetylglucosamine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC. These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.
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PMID:Ultracytochemical characterization of anionic sites in the wall of brain capillaries. 274 7

We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.
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PMID:A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences. 282 Sep 64

To clarify the relationship of the 290 and 145 kDa chains of the epidermolysis bullosa acquisita (EBA) antigen, we subjected urea extracts of skin basement membrane zone (BMZ) proteins and isolated 290 and 145 kDa chains of the EBA antigen cut out of sodium dodecyl sulfate polyacrylamide gels to treatment with clostridial collagenase. When the reaction products were electrophoresed, transblotted, and reacted with EBA patient sera or two monoclonal antibodies to the EBA antigen, the 290 kDa chain was degraded into the 145 kDa band that was resistant to cleavage with collagenase. The 145 kDa domain, isolated after collagenase treatment of the whole BMZ extract, was resistant to degradation by hyaluronidase, chondroitinase ABC, heparinase, and heparitinase but was readily degraded by V-8 protease. These data suggest that the EBA antigen consists of collagen and noncollagen domains of identical size (Mr 145,000), and that the 145 kDa noncollagen domain is generated via degradation of the native 290 kDa species by collagenase.
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PMID:Epidermolysis bullosa acquisita antigen: relationship between the collagenase-sensitive and -insensitive domains. 282 79


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