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1.
The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture 总被引:5,自引:0,他引:5
A Colombatti P Bonaldo D Volpin G M Bressan 《The Journal of biological chemistry》1988,263(33):17534-17540
Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents. 相似文献
2.
Distribution of urokinase-type plasminogen activator immunoreactivity in the mouse 总被引:25,自引:7,他引:18
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L I Larsson L Skriver L S Nielsen J Gr?ndahl-Hansen P Kristensen K Dan? 《The Journal of cell biology》1984,98(3):894-903
Immunocytochemistry, using rabbit antibodies to a urokinase-type 48- Kdalton Mr mouse plasminogen activator, showed that enzyme immunoreactivity is widely distributed in the normal mouse. Strong staining was obtained in widely disseminated connective tissue cells with a fibroblast-like morphology. Such cells occurred in high numbers in the lamina propria mucosae of the gastrointestinal tract, and in moderate numbers in the connective tissue septa of the pancreas. A few such cells were detected around the larynx, trachea, and bronchi. Immunoreactivity also occurred in epithelial cells of the proximal and distal kidney tubules, the ductus deferens, and in pulmonary pneumocytes. In addition, presumably extracellular staining was seen irregularly along the basement membrane and fibrillar structures in the lamina propria of the small and large intestines. Moreover, decidual cells of the mouse placenta stained strongly, and a moderate staining was observed in epithelial cells of involuting mammary glands, but not in those of noninvoluting glands. No immunoreactivity was observed in endothelial cells. Control experiments included absorption of the antibodies against highly-purified mouse plasminogen activator and the corresponding proenzyme, and the finding of a good correspondence between the number of immunoreactive cells and measurable enzymatic activity determined in adjacent tissue sections. Separation by SDS PAGE followed by immunoblotting revealed only one immunochemically stainable protein band with Mr approximately 48 Kdaltons in extracts from tissues showing immunoreactivity. 相似文献
3.
Phosphorylase phosphatase regulatory subunit. "Western" blotting with immunoglobulins against inhibitor-2 reveals a protein of Mr = 60,000 总被引:5,自引:0,他引:5
P A Gruppuso G L Johnson M Constantinides D L Brautigan 《The Journal of biological chemistry》1985,260(7):4288-4294
Rabbit muscle phosphorylase phosphatase has been isolated in different laboratories as an inactive complex of Mr = 70,000, composed of separate catalytic (Mr = 38,000) and regulatory (Mr = 31,000) proteins. The regulatory protein is identical to one of two heatstable inhibitors called inhibitor-2 (I2). Antiserum raised in sheep against I2 by repeated immunization potently blocked inhibitory activity, whereas preimmune serum did not. Immunoglobulins which blocked inhibitory activity were purified by affinity chromatography with I2 as the immobilized ligand. Using a "Western" immunoblotting procedure, as little as 1-5 ng of pure I2, obtained by electroelution of the Mr = 31,000 band of I2 from a polyacrylamide gel segment, were detected. Immunoblotting of the immunogen revealed only a band at Mr = 31,000, indicating the absence of contaminating antigenic proteins. When extracts of skeletal muscle and other rabbit tissues were denatured directly in dodecyl sulfate for immunoblotting the most intensely stained band was present at Mr = 60,000, rather than at Mr = 31,000 as expected. A small amount of I2 and other bands were detected, in particular at Mr = 36,000 and 25,000. Subsequent to heat treatment of the tissue extracts, there was an enrichment of I2 content relative to the Mr = 60,000 band. The results indicate the existence of a Mr = 60,000 protein related to I2. Activation of phosphorylase phosphatase in a muscle extract by treatment with Co2+ plus trypsin exactly coincided with digestion of the Mr = 60,000 immunoreactive protein. Available data indicate that this protein may function as a regulatory subunit of phosphorylase phosphatase. 相似文献
4.
Extracellular matrix of lymphoid tissues in the chick 总被引:2,自引:0,他引:2
A Colombatti A Poletti A Carbone D Volpin G M Bressan 《The journal of histochemistry and cytochemistry》1989,37(5):757-763
We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp. 相似文献
5.
L Heickendorff 《The International journal of biochemistry》1988,20(4):381-386
1. The intra- and extracellular distribution of fibronectin and laminin was studied by immunofluorescence in cultures of rabbit and human arterial smooth muscle cells. 2. Basement membrane (BM)-like material was isolated from the cell layer of arterial smooth muscle cells cultures and analysed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and immunoblotting. The major 220-240 kD component of arterial BM-like material was identified as fibronectin. Also a 200 kD fibronectin band was observed. 3. The 200 kD subunit of laminin was contained in isolated BM-like material, but no slower migrating laminin chains were detected. 4. Collagens were prepared from pepsinized BM-like material. The band pattern as resolved by SDS-PAGE and silver staining suggested that type IV collagen is the major collagen of arterial BM-like material. 相似文献
6.
NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin. 相似文献
7.
Identification of smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta with monoclonal antibody IIG10 总被引:2,自引:0,他引:2
M A Glukhova O I Ornatsky M G Frid A E Kabakov R R Adany L Muszbek V N Smirnov 《Tissue & cell》1987,19(5):657-663
We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta. 相似文献
8.
Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells. 相似文献
9.
M W Lark T K Yeo H Mar S Lara I Hellstr?m K E Hellstr?m T N Wight 《The journal of histochemistry and cytochemistry》1988,36(10):1211-1221
We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue. 相似文献
10.
A tetranectin-related protein is produced and deposited in extracellular matrix by human embryonal fibroblasts 总被引:5,自引:0,他引:5
I Clemmensen L R Lund L Christensen P A Andreasen 《European journal of biochemistry》1991,195(3):735-741
Tetranectin is a tetrameric human plasma protein that binds to plasminogen kringle 4. Its amino acid sequence is homologous with the C-terminal parts of asialoglycoprotein receptors and proteoglycan core proteins. In the present study, we have demonstrated that the human embryonal fibroblast cell line WI-38 produce a tetranectin-related molecule, which might, by several criteria, be similar to tetranectin from plasma. These criteria include immunoblotting analysis of conditioned cell medium revealing a protein band with Mr 17,000, indistinguishable from the Mr of plasma tetranectin. A preparation obtained by purification of conditioned medium by affinity chromatography on an anti-(plasma tetranectin) IgG column also contained the Mr 17,000 protein. This protein (partly purified from the conditioned medium) was shown by crossed immunoelectrophoresis to bind to heparin, CaCl2 and plasminogen kringle 4, as previously described for tetranectin in plasma. Importantly, this tetranectin-related protein is not only present in conditioned culture medium, but the Mr 17,000 protein reacting with anti-(plasma tetranectin) IgG was also present in the extracellular material, remaining after removal of WI-38 cells from the culture dishes, as demonstrated by immunoblotting analysis and immunocytochemical staining. We conclude that WI-38 cells produce a tetranectin-related protein and secrete it into the extracellular matrix. 相似文献
11.
大鼠降主动脉雌激素受体的免疫组织化学研究 总被引:1,自引:0,他引:1
冰冻切片微波修复后,用ER单克隆抗体免疫组织化学SP法及图像分析方法,观察雌激素受体(ER)在雌性与雄性大鼠降主动脉壁的分布,结果显示,ER阳性反应存在于血管壁的内皮细胞与平滑肌细胞中,阳性反应强度无性别差异。无段间差异(降主动脉胸段与腹段),血管周围的脂肪组织也可见大量呈阳性反应的细胞,结果表明,降主动脉壁的内皮细胞,血管平滑肌细胞中存在ER。降主动脉可能是雌激素的靶器官。 相似文献
12.
A fibroblast-associated antigen: characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells. 总被引:6,自引:0,他引:6
L R?nnov-Jessen J E Celis B Van Deurs O W Petersen 《The journal of histochemistry and cytochemistry》1992,40(4):475-486
Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10 immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells. 相似文献
13.
Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors. 相似文献
14.
Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000 总被引:6,自引:0,他引:6
An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000. 相似文献
15.
Identification of integrin-like matrix receptors with affinity for interstitial collagens 总被引:11,自引:0,他引:11
D Gullberg L Terracio T K Borg K Rubin 《The Journal of biological chemistry》1989,264(21):12686-12694
Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors. 相似文献
16.
Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis. 相似文献
17.
Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma 总被引:4,自引:0,他引:4
Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n = 11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in peripheral areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma. 相似文献
18.
Vascular smooth muscle caldesmon 总被引:10,自引:0,他引:10
T Clark P K Ngai C Sutherland U Gr?schel-Stewart M P Walsh 《The Journal of biological chemistry》1986,261(17):8028-8035
Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard. 相似文献
19.
Arlen R. Severson Ronald T. Ingram Lorraine A. Fitzpatrick 《In vitro cellular & developmental biology. Animal》1995,31(11):853-857
Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular
component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines,
chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque.
Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought
to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and
noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein,
osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I,
and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely
associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan.
The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth
muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix
involved in calcification of developing lesions. 相似文献
20.
Arteriosclerosis in rat aortic allografts: Dynamics of cell growth,apoptosis and expression of extracellular matrix proteins 总被引:4,自引:0,他引:4
Religa P Bojakowski K Gaciong Z Thyberg J Hedin U 《Molecular and cellular biochemistry》2003,249(1-2):75-83
Transplant vasculopathy is a key factor behind the late loss of transplanted organs. Since effective treatment is still lacking, a further understanding of the pathology of this process is important. Here, a rat model of aortic allografts was used and analyzed by immunohistochemistry and biochemical tests. Infrarenal aortic segments were transplanted from F344 to Lewis rats and analysed after 1–12 weeks using isografts as controls. After 1 week, endothelial cells gradually disappeared at the graft lumen as shown by von Willebrand factor staining and cellular activation was detected in the adventitia and intima using cellular retinol-binding protein-1 as a marker. Subsequently, proliferating smooth muscle cells, lymphocytes and macrophages accumulated in the intima as indicated by the appearance of staining for cell- and proliferation-specific antigens (smooth muscle -actin, CD45RC, ED1, cyclin D1 and proliferating cell nuclear antigen). After 4–8 weeks, TUNEL- and Fas-positive cells were observed in the media, denoting progressive apoptosis. In parallel, the developing neointima contained increased immunoreactivity for fibronectin and osteopontin. At the end of the observation period, an accumulation of macrophages and calcification was observed in the media and endothelial cells reappeared at the graft surface. The findings demonstrate major cellular and structural changes in the transplanted artery, including activation, proliferation and apoptosis of SMCs, and an altered composition of the extracellular matrix. Possibly, the observed changes in SMC phenotype, cell cycle and apoptosis during development of transplant arteriosclerosis are related to the expression of extracellular matrix proteins. 相似文献