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1.
Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells 总被引:2,自引:0,他引:2
Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region. 相似文献
2.
Production of IGF-binding proteins by vascular endothelial cells 总被引:2,自引:0,他引:2
R S Bar L C Harrison R C Baxter M Boes B L Dake B Booth A Cox 《Biochemical and biophysical research communications》1987,148(2):734-739
Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host. 相似文献
3.
Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture. 总被引:1,自引:1,他引:0
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Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells. 相似文献
4.
J N Beresford N S Fedarko L W Fisher R J Midura M Yanagishita J D Termine P G Robey 《The Journal of biological chemistry》1987,262(35):17164-17172
Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent Mr = 600,000, 400,000, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2% Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein).(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
5.
6.
B Tyree E A Horigan D L Klippenstein J R Hassell 《Archives of biochemistry and biophysics》1984,231(2):328-335
Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain. 相似文献
7.
Isolation and characterization of proteoglycans synthesized by cultured mesangial cells 总被引:3,自引:0,他引:3
E Yaoita K Oguri E Okayama K Kawasaki S Kobayashi I Kihara M Okayama 《The Journal of biological chemistry》1990,265(1):522-531
Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells. 相似文献
8.
N Ghinea A Fixman D Alexandru D Popov M Hasu L Ghitescu M Eskenasy M Simionescu N Simionescu 《The Journal of cell biology》1988,107(1):231-239
Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine. 相似文献
9.
10.
Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture 总被引:1,自引:0,他引:1
D J Klein T R Oegema T S Fredeen F van der Woude Y Kim D M Brown 《Archives of biochemistry and biophysics》1990,277(2):389-401
Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans. 相似文献
11.
Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process.
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Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity. 相似文献
12.
Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture 总被引:15,自引:6,他引:9
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Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans. 相似文献
13.
Ariane I. de Agostini Marie-Andre Ramus Robert D. Rosenberg 《Journal of cellular biochemistry》1994,54(2):174-185
The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface–associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface–associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface–associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface–associated and soluble aHSPGs. These observations indicate that different cells types can preferentially accumulate aHSPGs as cell surface–associated or soluble forms which could reflect alternate biological functions. 相似文献
14.
Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. 总被引:48,自引:0,他引:48
J Tran J Rak C Sheehan S D Saibil E LaCasse R G Korneluk R S Kerbel 《Biochemical and biophysical research communications》1999,264(3):781-788
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has been shown to act as an endothelial cell mitogen as well as a vascular permeability factor. Several recent reports have also implicated VEGF as a major survival factor for endothelial cells during angiogenesis and vasculogenesis along with other growth factors such as bFGF and angiopoietin-1. VEGF has been shown to mediate this additional function, at least in part through the induction of bcl-2 and the activation of the PI3 kinase-Akt/PKB signaling pathway. We report here that VEGF can also mediate the induction/upregulation of members of a newly discovered family of antiapoptotic proteins, namely the Inhibitors of Apoptosis (IAP), in vascular endothelial cells. We show that VEGF(165) leads to the induction of XIAP (2.9-fold) and survivin (19.1-fold) protein in human umbilical vein endothelial cells (HUVECs). In contrast, bFGF had little effect on XIAP expression, but produced approximately a 10-fold induction on survivin. VEGF-dependent upregulation of survivin could be prevented by cell cycle arrest in the G1 and S phases. These findings implicate that the survival and mitotic functions of VEGF in an angiogenic context may be more intrinsically related than previously anticipated. Moreover, they also raise the possibility of therapeutically targeting XIAP or survivin in antiangiogenic therapy as a means of suppressing tumor growth, in addition to directly targeting tumor cells which express these survival proteins. 相似文献
15.
Characterization of heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells in culture 总被引:4,自引:0,他引:4
Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled using [35S]sulfate, [3H]serine, [3H]glucosamine, or [3H]mannose as precursors. A species of heparan sulfate proteoglycan was purified using DEAE-Sephacel chromatography under dissociative conditions in the presence of detergent. The heparan sulfate proteoglycan, which constituted approximately 15% of the 35S-labeled proteoglycans in the culture medium has a similar hydrodynamic size (Kd = 0.62 on Sepharose CL-2B) and buoyant density distribution in CsCl density gradients as the low buoyant density dermatan sulfate proteoglycan synthesized by the same granulosa cells and described in the accompanying report (Yanagishita, M., and Hascall, V. C. (1983) J. Biol. Chem. 258, 12847-12856). The heparan sulfate chains (average Mr = 28,000) have an average of 0.8-0.9 sulfate groups/repeating disaccharide, of which 50% are N-sulfate, 30% are alkaline-labile O-sulfate (presumably on the 6-position of glucosamine residues), and 20% are alkaline-resistant O-sulfate groups. Alkaline borohydride treatment released both N-linked oligosaccharide-peptides containing mannose, glucosamine, and sialic acid, and O-linked oligosaccharides. Trypsin digestion of the proteoglycan generated fragments which contain (a) glycosaminoglycan-peptides with an average of 2 heparan sulfate chains/peptide; (b) clusters of O-linked oligosaccharides on peptides; and (c) N-linked oligosaccharide-peptides, which are as small as single N-linked oligosaccharides. The compositions of the O-linked and N-linked oligosaccharides and the trypsin fragments of this heparan sulfate proteoglycan were very similar to those of the low buoyant density dermatan sulfate proteoglycan synthesized by the same cells. 相似文献
16.
Analysis of the collagen types synthesized by bovine corneal endothelial cells in culture. 总被引:3,自引:2,他引:1
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Bovine corneal endothelial cells synthesize in culture predominantly type III collagen, with lesser amounts of types I and V and apparently little if any type IV. This pattern of synthesis is observed in both dividing and post-confluent cultures and irrespective of whether cells are attached to plastic or collagen-coated surface. 相似文献
17.
W W Wright M Parvinen N A Musto G L Gunsalus D M Phillips J P Mather C W Bardin 《Biology of reproduction》1983,29(1):257-270
Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time. 相似文献
18.
The fibroblast growth factor (FGF) receptor of human umbilical vein-derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin-A does not inhibit the binding of 125l-bFGF to HUE cell-surface receptors, whereas it inhibits bFGF binding to BHK-21 cell-surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti-angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor. 相似文献
19.
Acquisition of hyaluronate-binding affinity in vivo by newly synthesized cartilage proteoglycans. 总被引:3,自引:1,他引:2
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We have studied the hyaluronate-binding properties of aggregating cartilage proteoglycans synthesized in vivo by immature (6-week), mature (25-week) and aged (75-week) rabbits. Precursor isotope (35SO4) was given by intra-articular injection and articular cartilage was removed from rabbits after periods ranging from 1.5 h to 168 h. Proteoglycans were extracted with 4 M-guanidinium/HCl and monomers were isolated by CsCl gradient centrifugation under dissociative conditions. The percentages of both radiolabelled and total tissue monomers with a high affinity for hyaluronate [that is, capable of forming aggregates on Sepharose CL-2B in the presence of 0.8% (w/w) hyaluronate] were then determined. For all samples about 30% of the tissue monomers were high-affinity; however, less than 5% of the radiolabelled monomers were high-affinity at 1.5 h after injection, and this figure increased gradually with time in vivo. The increase was rapid in immature rabbits, such that after 24 h, about 30% of the radiolabelled monomers were high-affinity; on the other hand for mature and aged rabbits the increase was markedly slower such that 30% high-affinity was attained only after about 72 h. The results show that aggregating cartilage proteoglycans are secreted in vivo in a 'precursor' form with a low affinity for hyaluronate, and suggest that conversion of these monomers to a form with a higher binding affinity occurs with a half-time of about 12 h in immature cartilages but greater than 24 h in mature cartilages. The possible relationship of these findings to the process of proteoglycan aggregation in vivo is discussed. 相似文献
20.
Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures. 相似文献