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1.
The islet amyloid polypeptide (IAPP) and insulin are coproduced by the β-cells of the pancreatic islets of Langerhans. Both peptides can interact with negatively charged lipid membranes. The positively charged islet amyloid polypeptide partially inserts into these membranes and subsequently forms amyloid fibrils. The amyloid fibril formation of insulin is also accelerated by the presence of negatively charged lipids, although insulin has a negative net charge at neutral pH-values. We used water-polymer model interfaces to differentiate between the hydrophobic and electrostatic interactions that can drive these peptides to adsorb at an interface. By applying neutron reflectometry, the scattering-length density profiles of IAPP and insulin, as adsorbed at three different water-polymer interfaces, were determined. The islet amyloid polypeptide most strongly adsorbed at a hydrophobic poly-(styrene) surface, whereas at a hydrophilic, negatively charged poly-(styrene sulfonate) interface, the degree of adsorption was reduced by 50%. Almost no IAPP adsorption was evident at this negatively charged interface when we added 100 mM NaCl. On the other hand, negatively charged insulin was most strongly attracted to a hydrophilic, negatively charged interface. Our results suggest that IAPP is strongly attracted to a hydrophobic surface, whereas the few positive charges of IAPP cannot warrant a permanent immobilization of IAPP at a hydrophilic, negatively charged surface at an ionic strength of 100 mM. Furthermore, the interfacial accumulation of insulin at a hydrophilic, negatively charged surface may represent a favorable precondition for nucleus formation and fibril formation. 相似文献
2.
本文观察了表皮生长因子(EGF)对小鼠胚胎成纤维细胞C_3H/10T_1/2CL_8(简称C_3H/10)细胞周期的影响。结果表明:EGF使S期提前,细胞周期缩短。进一步探讨了EGF对细胞周期影响的机制,发现EGF可活化在细胞周期调节中起重要作用的P34~(cdc2)激酶(简称CD2K),使CD2K在周期中活性高峰出现的时间提前,提示EGF对细胞周期的影响可能通过作用于CD2K实现的。 相似文献
3.
Eric Sterner Sayaka Masuko Guoyun Li Lingyun Li Dixy E. Green Nigel J. Otto Yongmei Xu Paul L. DeAngelis Jian Liu Jonathan S. Dordick Robert J. Linhardt 《The Journal of biological chemistry》2014,289(14):9754-9765
Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ∼40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model. 相似文献
4.
Karlo Komorowski Annalena Salditt Yihui Xu Halenur Yavuz Martha Brennich Reinhard Jahn Tim Salditt 《Biophysical journal》2018,114(8):1908-1920
We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca2+ or Mg2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ~1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering. 相似文献
5.
Anton?P. Le?Brun Cathryn?L. Haigh Simon?C. Drew Michael James Martin?P. Boland Steven?J. Collins 《Biophysical journal》2014,107(10):2313-2324
The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. 相似文献
6.
Christine Kuch Dirk Winnekendonk Stefan Butz Ursula Unvericht Rolf Kemler Anna Starzinski-Powitz 《Experimental cell research》1997,232(2):331
M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK−cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with α-catenin/β-catenin or with α-catenin/plakoglobin, as E- and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence. 相似文献
7.
Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete α-catenin from the developing mouse eye to show that it is required for the successful closing of the optic fissure. In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans. 相似文献
8.
Wen Zhao Wenwen Yu Jiawei Zheng Ying Wang Zhiyuan Zhang Dongsheng Zhang 《Cellular and molecular neurobiology》2014,34(1):43-50
This study investigated the in vitro cytocompatibility of carbon nanotubes (CNTs) in a chitosan/collagen-based composite. Mouse fibroblasts were cultured on the surface of a novel material consisting of CNTs in a chitosan/collagen-based composite (chitosan/collagen+CNTs group). Chitosan/collagen composites without CNTs served as the control material (chitosan/collagen group) and cells cultured normally in tissue culture plates served as blank controls (blank control group). Cell adhesion and proliferation were observed, and cell apoptosis was measured. The doubling time (DT1) of cells was significantly shorter in the chitosan/collagen+CNTs group than in the chitosan/collagen group, and that in the chitosan/collagen group was shorter than in the blank control group. The CNTs in the chitosan/collagen-based composites promoted mouse fibroblast adhesion, producing a distinct cytoskeletal structure. At 24 h after culture, the cytoskeleton of the cells in the chitosan/collagen+CNTs group displayed typical fibroblastic morphology, with clear microfilaments. Cells in the chitosan/collagen group were typically round, with an unclear cytoskeleton. The blank control group even had a few unattached cells. At 4 days after incubation, no early apoptosis of cells was detected in the blank control group, whereas early apoptosis of cells was observed in the chitosan/collagen+CNTs and chitosan/collagen groups. No significant difference in the proportion of living cells was detected among the three groups. After entering the plateau stage, the average cell number in the chitosan/collagen+CNTs group was similar to that in the chitosan/collagen group and significantly smaller than that in the blank control group. Early apoptosis of cells in the blank control group was not detectable. There were significant differences in early apoptosis among the three groups. These results suggest that CNTs in a chitosan/collagen-based composite did not cause significant cytotoxic effects on mouse fibroblasts. Compared with chitosan/collagen composites, early adhesion and proliferation of fibroblasts were increased on chitosan/collagen+CNTs. However, at relatively high cell densities, the CNTs in the chitosan/collagen-based composite might exert an inhibitory effect on mouse fibroblast proliferation by inducing apoptosis. 相似文献
9.
Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings ≥90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings ≤50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength. 相似文献
10.
Brenna Hogan Avin Babataheri Yongyun Hwang Abdul?I. Barakat Julien Husson 《Biophysical journal》2015,109(2):209-219
We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results. 相似文献
11.
白细胞与内皮细胞的粘附 总被引:1,自引:0,他引:1
白细胞与内皮细胞相互作用由粘附分子介导.整合素、免疫球蛋白及选择素家族的粘附分子在这两种细胞的粘附中起关键作用.粘附的起始阶段由选择素介导,随后由CD11/CD18复合物与ICAM-1形成更为紧密的结合.多种细胞因子及炎症反应可诱导粘附.抗粘附分子单抗、药物等可抑制粘附. 相似文献
12.
Alexandre Chenal Lydia Prongidi-Fix Aurélie Perier Grégory Vernier Giovanna Fragneto Daniel Gillet Michel Ferrand 《Journal of molecular biology》2009,391(5):872-346
Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. 相似文献
13.
基因重组人碱性成纤维细胞生长因子的遗传毒性研究 总被引:2,自引:0,他引:2
用Ames试验、CHL细胞染色体畸变试验、小鼠骨髓PCE微核试验和小鼠致畸试验对基因重组人碱性成纤维细胞生长因子(rh-bFGF)进行研究。结果显示,rh-bFGF(0.1~500μg/皿)对TA97、TA98、TA100和TA102菌株在±S9mix条件下无致突变作用。各剂量组的微核细胞率与溶剂组比较,P>0.05。4个剂量组CHL细胞染色体畸变率均<5%。在妊娠母鼠6~15d P0给药,各剂量组活胎率90.2%~95.9%,与溶剂组95%比较,P>0.05。对胎鼠外观、骨骼和内脏无致畸作用。但0.03264mg/kg b.w吸收胎与溶剂组比较,P<0.01,显示一定的胚胎毒性。
Abstract: The genotoxicity of recombinant human basic fibroblast growth factor(rh-bFGF)was studied by Ames test,Chromosome aberration assay of mammalian cell(CHL)in vitro,mouse marrow micronucleus assay and teratogenesis assay.The results showed that rh-bFGF at the dose level of(0.1~500μg/plate)did not induce positive mutations in TA97,TA98,TA100,TA102 with or without S9mix of Ames test.It was control within the range(<5%)of chromosome aberration rates from rh-bFGF with 4 doses groups with or without S9min.The frequency of mouse micronuleus rate had no increase.It was divided into each groups of rh?bFGF received(p.o)dosages the 6th to 15 day of gestation mouse respectively in the teratology test.The frequency of live fetuses of each dose was between 90.2~95.9%,there were no significant difference as compared with 95% of solution control group.It did not cause deformity of the fetus appearances,bone and internal organs.But there were significant differences from solution control in rf-bFGF group of absorfoetus at 0.03264mg/kg(P<0.01).The results showed certainly dembyotoxicity. 相似文献
14.
Physical Properties of Bacterial Outer Membrane Models: Neutron Reflectometry & Molecular Simulation
Arwel V. Hughes Dhilon S. Patel Göran Widmalm Jeffery B. Klauda Luke A. Clifton Wonpil Im 《Biophysical journal》2019,116(6):1095-1104
The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM. 相似文献
15.
Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 103 M−1 s−1 and 10−4 s−1, respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity. 相似文献
16.
Envelope of Mouse Mammary Tumor Virus Studied by Freeze-Etching and Freeze-Fracture Techniques 总被引:4,自引:3,他引:1
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Joel B. Sheffield 《Journal of virology》1973,12(3):616-624
As part of a study of the cell surface changes associated with the production of murine mammary tumor virus, the structure of the envelope of this virus has been examined by using freeze-fracture techniques. Both fracture and deep-etch surfaces were examined. The fracture faces contain 10-nm spheres comparable to those observed on fractured plasma membranes, although fewer in number. Surfaces exposed by etching possess a highly regular hexagonal array of pits 25 nm apart. By examining freeze-fracture and freeze-etch preparations of virus with ferritin covalently bound to its surface, it has been determined that the surface exposed by etching is the outer surface of the virus. The pitted exterior surface of the mammary tumor virus appears to be a unique surface structure. 相似文献
17.
目的:探讨三磷酸腺苷(ATP)对小鼠骨骼肌成纤维细胞的迁移作用及其可能机制。方法:细胞划痕实验检测1μM、10μM、100μMATP对NOR-10细胞愈合率的影响;细胞迁移小室实验检测空白对照组、100μM ATP组、30μM PPADS+100μM ATP组、100μM RB2+100μM ATP组细胞的迁移率。结果:细胞划痕实验及迁移实验表明高浓度ATP能够促进NOR-10细胞的迁移能力,100μM ATP促细胞迁移能力最强(P0.05),并且其促迁移作用能被30μM PPADS,100μM RB2所抑制(P0.05),但100μM RB2的抑制作用更强(P0.05)。结论:高浓度ATP(10μM)能够促进NOR-10细胞的迁移能力,并且其促迁移可能通过激活P2Y受体作用大于P2X受体。 相似文献
18.
Mihailescu M Soubias O Worcester D White SH Gawrisch K 《The Journal of membrane biology》2011,239(1-2):63-71
A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, (2)H-NMR and (13)C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 ? away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid-water interface. Addition of cholesterol partially redistributes DHA density from near the lipid-water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C(8)-C(18) is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin. 相似文献
19.
Abstract. In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (Do , 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (Do , 0.43 Gy) than the other phases of the cell cycle (Do , 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. the data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most. 相似文献
20.
Mapping the Mouse Cell Atlas by Microwell-Seq 总被引:3,自引:0,他引:3
Xiaoping Han Renying Wang Yincong Zhou Lijiang Fei Huiyu Sun Shujing Lai Assieh Saadatpour Ziming Zhou Haide Chen Fang Ye Daosheng Huang Yang Xu Wentao Huang Mengmeng Jiang Xinyi Jiang Jie Mao Yao Chen Chenyu Lu Guoji Guo 《Cell》2018,172(5):1091-1107.e17