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1.
Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations. 总被引:11,自引:10,他引:1
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K E Finberg T R Muth S P Young J B Maken S M Heitritter A N Binns L M Banta 《Journal of bacteriology》1995,177(17):4881-4889
The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell. Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein. Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction. Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation. Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction. The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4. The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells. 相似文献
2.
Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa 总被引:15,自引:11,他引:4
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To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion. 相似文献
3.
Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm 总被引:19,自引:15,他引:4
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The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function. 相似文献
4.
Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red
and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium. Journal of Industrial Microbiology & Biotechnology (2002) 28, 201–203 DOI: 10.1038/sj/jim/7000222
Received 12 July 2001/ Accepted in revised form 22 October 2001 相似文献
5.
Nanocrystalline SrS phosphors doped with Ce3+ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce3+ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce3+ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
6.
S Patyar R Joshi DS Prasad Byrav A Prakash B Medhi BK Das 《Journal of biomedical science》2010,17(1):21
Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative
cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited
toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic
bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver
tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying
selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores
also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising
cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy.
Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach
in cancer treatment. 相似文献
7.
Jim M Dunwell Mike J Wilkinson Stephen Nelson Sri Wening Andrew C Sitorus Devi Mienanti Yuzer Alfiko Adam E Croxford Caroline S Ford Brian P Forster Peter DS Caligari 《BMC plant biology》2010,10(1):1-25
Background
Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.Results
Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC4F2 segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.Conclusions
Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection. 相似文献8.
The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels
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Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures. 相似文献
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10.
Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine
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A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism. 相似文献