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141.
Coyne CB Gambling TM Boucher RC Carson JL Johnson LG 《American journal of physiology. Lung cellular and molecular physiology》2003,285(5):L1166-L1178
Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction. 相似文献
142.
An atomic model for actin binding by the CH domains and spectrin-repeat modules of utrophin and dystrophin 总被引:1,自引:0,他引:1
Sutherland-Smith AJ Moores CA Norwood FL Hatch V Craig R Kendrick-Jones J Lehman W 《Journal of molecular biology》2003,329(1):15-33
Utrophin and dystrophin link cytoskeletal F-actin filaments to the plasmalemma. Genetic strategies to replace defective dystrophin with utrophin in individuals with muscular dystrophy requires full characterization of these proteins. Both contain homologous N-terminal actin-binding motifs composed of a pair of calponin-homology (CH) domains (CH1 and CH2) that are connected by spectrin-repeat modules to C-terminal membrane-binding sequences. Here, electron microscopy and 3D reconstruction of F-actin decorated with utrophin and dystrophin actin-binding constructs were performed using Utr261 (utrophin's CH domain pair), Utr416 (utrophin's CH domains and first spectrin-repeat) and Dys246 (dystrophin's CH domain pair). The lozenge-like utrophin CH domain densities localized to the upper surface of actin subdomain 1 and extended azimuthally over subdomain 2 toward subdomains 3 and 4. The cylinder-shaped spectrin-repeat was located at the end of the CH domain pair and was aligned longitudinally along the cleft between inner and outer actin domains, where tropomyosin is present when on thin filaments. The connection between the spectrin-repeat module and the CH domains defined the orientation of CH1 and CH2 on actin. Resolution of utrophin's CH domains and spectrin-repeats permitted docking of crystal structures into respective EM densities, leading to an atomic model where both CH and spectrin-domains bind actin. The CH domain-actin interaction for dystrophin was found to be more complex than for utrophin. Binding assays showed that Utr261 and Utr416 interacted with F-actin as monomers, whereas Dys246 appeared to associate as a dimer, consistent with a bilobed Dys246 structure observed on F-actin in electron microscope reconstructions. One of the lobes was similar in shape, position and orientation to the monomeric CH domains of Utr261, while the other lobe apparently represented a second set of CH domains in the dimeric Dys246. The extensive contact made by dystrophin on actin may be used in vivo to help muscles dissipate mechanical stress from the contractile apparatus to the extracellular matrix. 相似文献
143.
Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL 总被引:3,自引:0,他引:3
Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate. 相似文献
144.
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146.
Chakrabarti S Kobayashi KS Flavell RA Marks CB Miyake K Liston DR Fowler KT Gorelick FS Andrews NW 《The Journal of cell biology》2003,162(4):543-549
Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease. 相似文献
147.
Resources in the Great Basin of western North America often occur in pulses, and plant species must rapidly respond to temporary increases in water and nutrients during the growing season. A field study was conducted to evaluate below ground responses of Artemisia tridentata and Agropyron desertorum, common Great Basin shrub and grass species, respectively, to simulated 5-mm (typical summer rain) and 15-mm (large summer rain) summer rainfall events. The simulated rainfall was labeled with K(15)NO(3) so that timing of plant nitrogen uptake could be monitored. In addition, soil NH(4)(+) and NO(3)(-) concentrations and physiological uptake capacities for NO(3)(-) and NH(4)(+) were determined before and after the rainfall events. Root growth in the top 15 cm of soil was monitored using a minirhizotron system. Surprisingly, there was no difference in the amount of labeled N acquired in response to the two rainfall amounts by either species during the 7-day sample period. However, there were differences between species in the timing of labeled N uptake. The N label was detected in above ground tissue of Agropyron within 1 h of the simulated rainfall events, but not until 24 h after the rainfall in Artemisia. For both Agropyron and Artemisia, root uptake capacity was similarly affected by the 5-mm and 15-mm rainfall. There was, however, a greater increase in uptake capacity for NH(4)(+) than for NO(3)(-), and the 15-mm event resulted in a longer response. No root growth occurred in either species in response to either rainfall event during this 8-day period. The results of this study indicate that these species are capable of utilizing nitrogen pulses following even small summer rainfall events during the most stressful period of the summer and further emphasize the importance of small precipitation events in arid systems. 相似文献
148.
The serine protease factor Xa (FXa) is inhibited by ecotin with picomolar affinity. The structure of the tetrameric complex of ecotin variant M84R (M84R) with FXa has been determined to 2.8 A. Substrate directed induced fit of the binding interactions at the S2 and S4 pockets modulates the discrimination of the protease. Specifically, the Tyr at position 99 of FXa changes its conformation with respect to incoming ligand, changing the size of the S2 and S4 pockets. The role of residue 192 in substrate and inhibitor recognition is also examined. Gln 192 from FXa forms a hydrogen bond with the P2 carbonyl group of ecotin. This confirms previous biochemical and structural analyses on thrombin and activated protein C, which suggested that residue 192 may play a more general role in mediating the interactions between coagulation proteases and their inhibitors. The structure of ecotin M84R-FXa (M84R-FXa) also reveals the structure of the Gla domain in the presence of Mg(2+). The first 11 residues of the domain assume a novel conformation and likely represent an intermediate folding state of the domain. 相似文献
149.
We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos. 相似文献
150.
Zhou J Wang J Penny D Monget P Arraztoa JA Fogelson LJ Bondy CA 《Biology of reproduction》2003,69(1):22-29
It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth. 相似文献