共查询到20条相似文献,搜索用时 15 毫秒
1.
Mohammed Akaaboune Martine Verdire-Sahuqu Sylvie Lachkar Barry W. Festoff Daniel Hantaï 《Journal of cellular physiology》1995,165(3):503-511
The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, α1-antichymotrypsin (α1-ACT), and Kunitz-inhibitor containing forms of the β-amyloid precursor protein (βAPP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-α1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-βAPP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both α1-ACT and βAPP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, α1-ACT and βAPP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both α1-ACT and βAPP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for α1-ACT and βAPP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation. © 1995 Wiley-Liss Inc. 相似文献
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Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β1 integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α5β1) or collagen (a ligand for α2β1) coated plates after 4 h exposure to cyclic strain. β3 integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α5 and α2 integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α5, α2, and β1 did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125FAK occurred concomitant with the reorganization of β1 integrin. We concluded that α5β1 and α2β1 integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc. 相似文献
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The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 310-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-Cα-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc. 相似文献
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The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β1 family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α2β1 integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α2β1 integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α2β1 integrin on their membranes. Using [35S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α2β1 integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α2β1 integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd. 相似文献
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HEIZABURO KATAYAMA FUMIHIRO MORISHITA OSAMU MATUSHIMA KOJI YAMADA 《Pigment cell & melanoma research》1990,3(4):192-199
The effects of β-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these β-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α-and β-adrenoceptors, and that the activation of the β-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two β1-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β1-receptors. By contrast, the pigment-dispersing effects of two β2-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β1- and β2-adrenoceptors in the melanophores. The relative numbers of β1- and β2-adrenoreceptors as a percentage of the total population of β-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β-agonists on the melanophores in the presence of butoxamine or metoprolol. 相似文献
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Central (hypothalamic) control of bone mass is proposed to be mediated through β2‐adrenergic receptors (β2‐ARs). While investigations in mouse bone cells suggest that epinephrine enhances both RANKL and OPG mRNA via both β‐ARs and α‐ARs, whether α‐ARs are expressed in human bone cells is controversial. The current study investigated the expression of α1‐AR and β2‐AR mRNA and protein and the functional role of adrenergic stimulation in human osteoblasts (HOBs). Expression of α1B‐ and β2‐ARs was examined by RT‐PCR, immunofluorescence microscopy and Western blot (for α1B‐ARs). Proliferation in HOBs was assessed by 3H‐thymidine incorporation and expression of RANKL and OPG was determined by quantitative RT‐PCR. RNA message for α1B‐ and β2‐ARs was expressed in HOBs and MG63 human osteosarcoma cells. α1B‐ and β2‐AR immunofluorescent localization in HOBs was shown for the first time by deconvolution microscopy. α1B‐AR protein was identified in HOBs by Western blot. Both α1‐agonists and propranolol (β‐blocker) increased HOB replication but fenoterol, a β2‐agonist, inhibited it. Fenoterol nearly doubled RANKL mRNA and this was inhibited by propranolol. The α1‐agonist cirazoline increased OPG mRNA and this increase was abolished by siRNA knockdown of α1B‐ARs in HOBs. These data indicate that both α1‐ARs and β2‐ARs are present and functional in HOBs. In addition to β2‐ARs, α1‐ARs in human bone cells may play a role in modulation of bone turnover by the sympathetic nervous system. J. Cell. Physiol. 220: 267–275, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
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Silvana Molossi Mariano Elices Thomas Arrhenius Marlene Rabinovitch 《Journal of cellular physiology》1995,164(3):620-633
We previously reported infiltration of immune-inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)-1b?. We now investigate, using a porcine endothelial-smooth muscle cell co-culture system, whether IL-1b?-stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL-2, were characterized by fluorescence-activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL-1b?. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking α4b?1 and α5b?1 integrin receptors on lymphocyte surfaces, respectively. A 55-70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium; we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine-mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface α4b?1 α5b?1 integrins. © 1995 Wiley-Liss, Inc. 相似文献
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Fahmy A. Mamuya Yan Wang Victoria H. Roop David A. Scheiblin Jocelyn C. Zajac Melinda K. Duncan 《Journal of cellular and molecular medicine》2014,18(4):656-670
Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α‐smooth muscle actin (α‐SMA)‐expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of αV integrin and its interacting β‐subunits β1, β5, β6, β8 are up‐regulated concomitant with α‐SMA in LCs following surgery. To test the hypothesis that αV integrins are functionally important in PCO pathogenesis, we created mice lacking the αV integrin subunit in all lens cells. Adult lenses lacking αV integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up‐regulated the expression of α‐SMA, β1‐integrin, fibronectin, tenascin‐C and transforming growth factor beta (TGF‐β)–induced protein within 48 hrs, while LCs lacking αV integrins exhibited much less cell proliferation and little to no up‐regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of αV integrins in latent TGF‐β activation as αV integrin null lenses do not exhibit detectable SMAD‐3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF‐β in fibrotic PCO. These data suggest that therapeutics antagonizing αV integrin function could be used to prevent fibrotic PCO following cataract surgery. 相似文献
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In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were deteced by Western ligand blot (WLB) with125I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse.Special issue dedicated to Dr. Sidney Ochs. 相似文献
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Jenson Lim Aurélien G. Dupuy David R. Critchley Emmanuelle Caron 《Journal of cellular biochemistry》2010,111(4):999-1009
The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin αMβ2 in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with αMβ2, we show that Rap1 acts on the β2 and not the αM chain, and that residues 732–761 of the β2 subunit are essential for Rap1‐induced RBC binding. Activation of αMβ2 by Rap1 was dependent on W747 and F754 in the β2 tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of αMβ2 in macrophages shares both common and distinct features from Rap1 activation of αIIbβ3 expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
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Hui-Jun Zhu F. Patrick Ross Xu Cao Steven L. Teitelbaum 《Journal of cellular biochemistry》1996,61(3):420-429
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) transactivates the avian β3 integrin gene whose promoter contains at least two vitamin D response elements, one of which is in close proximity to a candidate AP1 site (TGACTCA). Since fos/jun and steroid hormones interact to regulate gene expression, we asked whether phorbol-12-myristate-13-acetate (PMA), which stimulates binding of fos/jun to AP1 sites, transactivates the avian β3 integrin gene and, if so, does the phorbol ester modulate 1,25(OH)2D3 induction of the gene. We find the candidate AP1 sequence comigrates with the consensus AP1 sequence on electromobility shift assay when incubated with recombinant c-jun protein. Furthermore, PMA prompts expression of β3 integrin mRNA in the avian monocytic line, HD11. The increase in message reflects transactivation of the β3 gene and is mirrored by plasma membrane appearance of the integrin heterodimer αvβ3. Moreover, attesting to the functional significance of PMA-enhanced αvβ3 expression, cells treated with concentrations of the phorbol ester that induce the β3 gene, spread extensively on plastic, an event blocked by an anti-αv antibody and a peptide mimetic known to inhibit αvβ3-mediated cell attachment. Interestingly, co-addition of 1.25(OH)2D3 and PMA prompts greater expression of αvβ3 than when the cells are exposed to either agent alone and PMA enhances 1,25(OH)2D3-induced β3 integrin mRNA expression. Thus, PMA and 1,25(OH)2D3 impact on the avian β3 integrin gene independently and in combination. © 1996 Wiley-Liss, Inc. 相似文献
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Friederike Rohn Claus Kordes Tobias Buschmann Doreen Reichert Marianne Wammers Gereon Poschmann Kai Stühler Amelie S. Benk Fania Geiger Joachim P. Spatz Dieter Hussinger 《Aging cell》2020,19(4)
Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5/β1 is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5/β1 in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration. 相似文献
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Sophie Ringeard Jean Harb Fabien Gautier Jean Menanteau Khaled Meflah 《Journal of cellular biochemistry》1996,62(1):40-49
Malignant cell transformation is generally accompanied by changes in their interactions with environing matrix proteins in a way to facilitate their migration and generate invasion. Our results show the binding of rat colon adenocarcinoma PROb cells to fibronectin strongly reduced when compared to normal rat intestine epithelial cells. This decrease was not due to the level of α(s)β1 integrins expressed at the surface of the cell line. However, β1- and α(s)-associated subunits appeared to be structurally altered as shown by immunoprecipitation followed by electrophoresis. Pulse chase experiments using 35S methionine evidenced differences in the biosynthesis of β1- and α (s) associated integrins: normal epithelial IEC18 cells required 16 h for maximal biosynthesis of the completely mature β1 subunit, while PROb cells did it within 4-6 h. Studies using endoglycosidases O, H, D, and N glycanase confirmed that the molecular weight alterations were due to abnormal glycosylation and suggested that α(s)β1 integrins of PROb cells could bear both mature complex and immature high mannose types while IEC18 cells borne only mature complex type oligosaccharidic chains. Treatment of both cell types with castanospermine, an inhibitor of N-glycosylation, reduced the differences observed in their adhesion to the fibronectin without significantly affecting β1 receptors expression at the cell surface. These results strongly suggest a role of the glycosylation of β1 receptors in the adhesion of rat colon adenocarcinoma PROb cells to fibronectin substrata. © 1996 Wiley-Liss, Inc. 相似文献
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Deborah L. Webb Patricia J. Conrad Lan Ma Marie-Luise Blue 《Journal of cellular biochemistry》1996,61(1):127-138
We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc. 相似文献
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13C-, 1H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 310-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures. 相似文献
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Marion Flechtner‐Mors Christopher P. Jenkinson Andreas Alt Hans K. Biesalski Guido Adler Herwig H. Ditschuneit 《Obesity (Silver Spring, Md.)》2004,12(4):612-620
Objective: To investigate the involvement of α1‐adrenoceptors in the sympathetic regulation of glucose uptake in human adipocytes. Research Methods and Procedures: Twenty‐four severely obese subjects participated in this study. The microdialysis technique was used to determine interstitial glucose concentration after stimulation of abdominal subcutaneous adipose tissue with the α1‐agonist norfenefrine, the α1, 2β‐agonist norepinephrine, and both agents in combination with the α1‐antagonist urapidil. The effect of β‐adrenoceptor stimulation was assessed by orciprenaline. Changes in local blood flow were determined using the ethanol escape technique. Results: Both norfenefrine and norepinephrine induced a concentration‐dependent decrease of interstitial glucose concentration, with a greater decrease observed with norepinephrine. Preperfusion of adipose tissue with urapidil inhibited glucose decrease. The inhibition was overcome with high concentrations of norfenefrine and norepinephrine, respectively. Both adrenergic agents induced tachyphylaxia. Urapidil enhanced extracellular glucose level at high concentration. Blood flow decreased in the presence of norfenefrine and norepinephrine but increased in response to urapidil. The accelerated blood flow due to urapidil was counteracted by norepinephrine and norfenefrine. Orciprenaline decreased interstitial glucose concentration and increased nutritive blood flow. The observed changes in blood flow induced by adrenergic agents were not related to glucose uptake. Discussion: The stimulatory effect of the sympathetic nerves on glucose uptake in subcutaneous adipose tissue appears to be mediated by the α1‐adrenoceptor. Norepinephrine enhances glucose entry into adipocytes independently of insulin action. In obese subjects with insulin resistance, the α1‐adrenergic receptor may provide an important alternative pathway for glucose uptake. 相似文献