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81.
BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles. METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy. RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts. Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans. Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell. CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors. 相似文献
82.
Absence of accessory subunit in the DNA polymerase gamma purified from yeast mitochondria 总被引:1,自引:0,他引:1
In yeast and animals, replication of the mitochondrial genome is carried out by the DNA polymerase gamma. In mammals this polymerase is composed of a catalytic and an accessory subunit. Yeast DNA polymerase gamma was purified over 6600-fold from mitochondria. The catalytic polypeptide of this enzyme was identified as a 135-kDa protein by a photochemical crosslinking procedure and its native molecular weight was estimated between 120 and 140 kDa by gel filtration and glycerol gradient sedimentation. These results indicate that yeast DNA polymerase gamma contains only one subunit and thus has a different quaternary structure from its counterpart in animals. 相似文献
83.
With a view to visualizing the ultrastructural distribution of syringyl lignins in secondary plant cell walls, a polyclonal antibody raised from a synthetic DHP polymer consisting only of syringyl propane units was prepared. To test the reactivity of the antiserum, a mini-dot-blot immunoassay reducing the amounts of substrates and antiserum was developed. A characteristic attribute of the S-antiserum appears to be its specific recognition of sequences of three or more consecutive syringyl units. On ultra-thin sections of model plants of Arabidopsis thaliana, Populus and tobacco, the antiserum allowed us to demonstrate a higher concentration of syringyl epitopes in fibres than in vessels. Variations in the distribution pattern of these epitopes between the three plants examined suggest that the synthesis of syringyl lignins in angiosperms depends on the species. 相似文献
84.
Hobbs JP Munday PL Jones GP 《Proceedings. Biological sciences / The Royal Society》2004,271(1553):2109-2114
Labile maturation and sex determination should be advantageous where the probability of finding a mating partner is unpredictable. Here we tested the hypothesis that the presence of a potential mating partner induces maturation and sex determination in a coral-dwelling fish, Gobiodon erythrospilus. In natural populations at Lizard Island (Great Barrier Reef), single individuals were less likely to be mature than paired individuals and they matured at a larger size, indicating plasticity in the timing of maturation. By manipulating group structure we demonstrated that both the timing of maturation and the sex of maturing individuals are socially controlled. Single juveniles did not mature, but maturation was rapidly induced by the presence of an adult partner. In addition, sex determination was found to be labile, with juveniles maturing into the opposite sex of the partner encountered. To our knowledge, this is the first experimental demonstration of social induction of maturation in conjunction with labile sex determination at maturation in vertebrates. This flexibility enables individuals to maximize their reproductive success in an environment where the timing of mate acquisition and the sex of their future partner are unpredictable. 相似文献
85.
EFA6, exchange factor for ARF6, regulates the actin cytoskeleton and associated tight junction in response to E-cadherin engagement
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Luton F Klein S Chauvin JP Le Bivic A Bourgoin S Franco M Chardin P 《Molecular biology of the cell》2004,15(3):1134-1145
We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin-mediated cell adhesion, EFA6 is recruited to a Triton X-100-insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability. 相似文献
86.
Targets of fibroblast growth factor 1 (FGF-1) and FGF-2 signaling involved in the invasive and tumorigenic behavior of carcinoma cells 总被引:3,自引:0,他引:3
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Fibroblast growth factor (FGF)-1 and -2 have potent biological activities implicated in malignant tumor development. Their autocrine and nonautocrine activity in tumor progression of carcinoma was investigated in the NBT-II cell system. Cells were manipulated to either produce and be autocrine for FGF-1 or -2 or to only produce but not respond to these factors. The autocrine cells are highly invasive and tumorigenic and the determination of specific targets of FGF/fibroblast growth factor receptor (FGFR) signaling was assessed. In vitro studies showed that nonautocrine cells behave like epithelial parental cells, whereas autocrine cells have a mesenchymal phenotype correlated with the overexpression of urokinase plasminogen activator receptor (uPAR), the internalization of E-cadherin, and the redistribution of beta-catenin from the cell surface to the cytoplasm and nucleus. uPAR was defined as an early target, whereas E-cadherin and the leukocyte common antigen-related protein-tyrosine phosphatase (LAR-PTP) were later targets of FGF signaling, with FGFR1 activation more efficient than FGFR2 at modulating these targets. Behavior of autocrine cells was consistent with a decrease of tumor-suppressive activities of both E-cadherin and LAR-PTP. These molecular analyses show that the potential of these two growth factors in tumor progression is highly dependent on specific FGFR signaling and highlights its importance as a target for antitumor therapy. 相似文献
87.
Regulation of Cre recombinase by ligand-induced complementation of inactive fragments 总被引:5,自引:1,他引:4
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Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals. 相似文献
88.
VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration 总被引:5,自引:0,他引:5
van Wetering S van den Berk N van Buul JD Mul FP Lommerse I Mous R ten Klooster JP Zwaginga JJ Hordijk PL 《American journal of physiology. Cell physiology》2003,285(2):C343-C352
Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the 1- and 2-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation. human umbilical vein endothelial cells; vascular-endothelial cadherin; F-actin; reactive oxygen species; p38 mitogen-activated protein kinase; vascular cell adhesion molecule 相似文献
89.
Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis 总被引:4,自引:0,他引:4
BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain. 相似文献
90.
Gobin S Thuillier L Jogl G Faye A Tong L Chi M Bonnefont JP Girard J Prip-Buus C 《The Journal of biological chemistry》2003,278(50):50428-50434
Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders. 相似文献