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1.
Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor. 相似文献
2.
Sclerostin (SOST) is a glycoprotein having many important functions in the regulation of bone formation as a key negative regulator of Wnt signaling in bone. Surface plasmon resonance (SPR), which allows for a direct quantitative analysis of the label-free molecular interactions in real-time, has been widely used for the biophysical characterization of glycosaminoglycan (GAG)-protein interactions. In the present study, we report kinetics, structural analysis and the effects of physiological conditions (e.g., salt concentrations, Ca2+ and Zn2+concentrations) on the interactions between GAGs and recombinant human (rh) and recombinant mouse (rm) SOST using SPR. SPR results revealed that both SOSTs bind heparin with high affinity (rhSOST-heparin, KD~36 nM and rmSOST-heparin, KD~77 nM) and the shortest oligosaccharide of heparin that effectively competes with full size heparin for SOST binding is octadecasaccharide (18mer). This heparin binding protein also interacts with other highly sulfated GAGs including, disulfated-dermatan sulfate and chondroitin sulfate E. In addition, liquid chromatography-mass spectrometry was used to characterize the structure of sulfated GAGs that bound to SOST. 相似文献
3.
The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways. 相似文献
4.
Davide Vigetti Manuela Rizzi Paola Moretto Sara Deleonibus Jonathan M. Dreyfuss Evgenia Karousou Manuela Viola Moira Clerici Vincent C. Hascall Marco F. Ramoni Giancarlo De Luca Alberto Passi 《The Journal of biological chemistry》2011,286(40):34497-34503
Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2. 相似文献
5.
The bacterial surfaces of enterococci are not uniform. This fact is confirmed by several studies and by our results when great
differences between individual strains with regard to their cell surface hydrophobicity, binding of eight ECM (extracellular
matrix) molecules immobilized on latex beads and four selected ECM molecules in microtiter plates were observed. The strains
expressing high binding of ECM molecules (e.g., HJ 18, HJ 23, HJ 24, HJ 26, HJ 28, HJ 36, etc.) were found among Enterococcus faecalis and E. faecium by PAA (particle agglutination assay). On the other hand, weak ECM binders (e.g., HJ 21, HJ 32, HJ 34, HJ 38, HJ 39, HJ 42,
HJ 43) were also found. A direct correlation was found between porcine mucin and fetuin binding ability of eight selected
strains tested in microtiter plates and by PAA. Moreover, the influence of tunicamycin treatment was different because significant
(P < 0.001) blocking effect of tunicamycin was observed with two selected strains (HJ 26 and HJ 36), whereas two strains (HJ 18 and HJ
22) were not significantly affected in their fetuin binding. The treatment of six enterococcal strains with proteolytic enzymes,
pronase P, and trypsin, and with sodium metaperiodate also significantly (P < 0.001) decreased their fetuin binding. This suggests that both protein and carbohydrate moieties are involved in the binding of
immobilized fetuin. However, the influence of these chemicals on the fetuin binding by individual strains was different.
Received: 24 May 2002 / Accepted: 2 August 2002 相似文献
6.
Tomasz W. Zare¸ba Corina Pascu Waleria Hryniewicz Torkel Wadström 《Current microbiology》1997,34(1):6-11
Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of 125I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity.
Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin,
human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin
and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic
digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding
of 125I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers
such as dextran sulfate (M
r
5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding
of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine,
but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface.
Received: 22 April 1996 / Accepted: 29 June 1996 相似文献
7.
Kurt R. Brunden Nancy J. Richter-Cook Nishith Chaturvedi Robert C. A. Frederickson 《Journal of neurochemistry》1993,61(6):2147-2154
The seinile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain β-amyloid peptide (Aβ) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A/β, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with Aβ and its precursor. Here we have examined the ability of synthetic Aβ s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. Aβ(1–28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both Aβ(1–28) and Aβ(1–40) to bind glycosaminoglycans is pH-dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin-induced aggregation of Aβ(1–28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the Aβ sequence reveals a consensus heparin-binding domain at residues 12–17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans. This hypothesis is supported by the following observations: (a) Aβ(13–17) binds tightly to a heparin affinity column at pH 4.0, but not at pH 8.0; and (b) an Aβ(13–17) in which histidine residues 13 and 14 have been replaced with serines does not bind to a heparin column at either pH 8.0 or 4.0. Together, the data indicate that Aβ is capable of binding to the glycosaminoglycan chains of proteoglycans, and such an interaction may be relevant to the etiology and pathology of Alzheimer's disease. 相似文献
8.
An in vitro system to measure the adhesion of bacteria to human, eukaryotic cells was devised. Adhesion indices for test strains of bacteria could be calculated. Significant differences were then observed between various strains of Escherichia coli from a variety of sources, in their ability to adhere. The possible applications of the test, especially for the routine screening of bacteria for adhesion and for inhibitors of attachment, were considered. 相似文献
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10.
Beatriz Salvador Nicole R. Sexton Ricardo Carrion Jr. Jerritt Nunneley Jean L. Patterson Imke Steffen Kai Lu Marcus O. Muench David Lembo Graham Simmons 《Journal of virology》2013,87(6):3295-3304
Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells. 相似文献
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12.
Igor Štyriak Andrea Lauková Corina Fallgren Torkel Wadström 《Current microbiology》1999,39(6):327-335
Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were
immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating
S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays.
According to the absorbance readings (A570nm), 11 strains were classified as nonadherent (A570nm < 0.1), 10 strains as weakly adherent (0.1 < A570nm > 0.3), and 2 strains as strongly adherent (A570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A570nm readings of microtiter plate assays. Binding of 125I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of 125I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of
125I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated
hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf
binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains
had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes.
Received: 28 April 1999 / Accepted: 26 July 1999 相似文献
13.
Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds. 相似文献
14.
Clifford Barnes Lucia Speroni Kyle P. Quinn Mael Montevil Kurt Saetzler Gbemisola Bode-Animashaun George McKerr Irene Georgakoudi C. Stephen Downes Carlos Sonnenschein C. Vyvyan Howard Ana M. Soto 《PloS one》2014,9(4)
Background
Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.Results
The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.Conclusions
Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs. 相似文献15.
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18.
Lukasz Wujak Miroslava Didiasova Dariusz Zakrzewicz Helena Frey Liliana Schaefer Malgorzata Wygrecka 《The Journal of biological chemistry》2015,290(11):7027-7039
Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis. 相似文献
19.
The outcome of microbial infections in mammals, including humans, is affected by the age, sex, and reproductive status of the host suggesting a role for sex steroid hormones. Testosterone, estradiol, and progesterone, signaling through their respective steroid receptors, affect the functioning of immune cells to cause differential susceptibility to parasitic, bacterial, and viral infections. Microbes, including fungi, bacteria, parasites, and viruses, can also use sex steroid hormones and manipulate sex steroid receptor signaling mechanisms to increase their own survival and replication rate. The multifaceted use of sex steroid hormones by both microbes and hosts during infection forms the basis of this review. In the arms race between microbes and hosts, both hosts and microbes have evolved to utilize sex steroid hormone signaling mechanisms for survival. 相似文献
20.
Formation and Identification of Interfacial-Active Glycolipids from Resting Microbial Cells 总被引:6,自引:1,他引:5
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Zu-Yi Li Siegmund Lang Fritz Wagner Ludger Witte Victor Wray 《Applied microbiology》1984,48(3):610-617
Resting cells of Arthrobacter sp. strain DSM2567 incubated in the presence of various mono-, di-, or trisaccharides biosynthesized different glycolipids. All eight glycolipids, containing the corresponding carbohydrate moiety and one, two, or three α-branched β-hydroxy fatty acids, were produced when mannose, glucose, cellobiose, maltose, and maltotriose were used as carbon sources in a simple phosphate buffer. The structures of the compounds were elucidated by means of 1H and 13C nuclear magnetic resonance spectroscopy and by chemical ionization mass spectroscopy. In high-salinity solution, the substances showed different surfactant properties. Cellobiose and maltose monocorynomycolates reduced the interfacial tension from 42 to 1 mN/m at critical micelle concentrations below 20 mg/liter. 相似文献