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991.
Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization 下载免费PDF全文
The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented. 相似文献
992.
993.
Plant regeneration from thin cell layers in Spinacia oleracea 总被引:1,自引:1,他引:0
Leguillon Stéphanie Charles Gilbert Branchard Michel 《Plant Cell, Tissue and Organ Culture》2003,74(3):257-265
Caulogenesis and somatic embryogenesis were induced from transverse thin cell layers (tTCLs) of two European (Spinacia oleracea L.) spinach genotypes. Regeneration occurred mostly when tTCLs had been excised from seedlings grown on a preconditioning medium consisting of White's macroelements, Nitsch's microelements, Murashige and Skoog's (MS) vitamins, 6 g l–1 agar and 20 g l–1 glucose. The explants were cultured on MS medium supplemented with sucrose (10, 30, 50 or 80 g l–1) or fructose (5, 10 or 30 g l–1) and several combinations of indole-3-acetic acid (IAA), -naphtalene acetic acid (NAA), 6-benzylaminopurine (BAP) and gibberellic acid (GA3). Most of the regeneration events were obtained from root explants of the cultivar Carpo. The best result was observed on MS medium supplemented with 50 g l–1 sucrose, 100 M NAA, 1 M BAP and 10 M GA3. After an 8-week culture, the calluses were transferred onto MS medium where shoots and somatic embryos appeared 1 week later. The best root development was obtained on MS medium supplemented with 4.9 M indole-3-butyric acid (IBA) and 8 g l–1 Phytagel. The plantlets were, then, transferred to soil and developed into well-conformed, fertile plants. 相似文献
994.
Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation. 相似文献
995.
Mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17). Functionally, about half of the known mutations increase the alternative mRNA splicing of exon 10 of the tau gene, resulting in the overproduction of tau isoforms with four microtubule-binding repeats. The other mutations reduce the ability of tau to interact with microtubules, with some mutations also increasing the propensity of tau to assemble into filaments. Here we have examined the functional effects of the recently described tau gene mutations deltaN296 and N296H. Both mutations reduced the ability of tau to promote microtubule assembly, without having a significant effect on tau filament formation. By exon trapping, they increased the splicing of exon 10. DeltaN296 and N296H thus define a class of tau mutations with effects at both the RNA and the protein level. 相似文献
996.
Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells 总被引:3,自引:0,他引:3
Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells,
since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect
of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect
of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate
concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated
at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in
the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration.
Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min,
suggesting that actin filaments might be organized at this region.
Received July 31, 2001 Accepted December 14, 2001 相似文献
997.
998.
Shikama Y Shen L Yonetani M Miyauchi J Miyashita T Yamada M 《Biochemical and biophysical research communications》2002,291(3):484-493
Caspase-8 and -10 are thought to be involved in a signaling pathway leading to death receptor-mediated apoptosis. The prodomains of these caspases are known to form fibrous structures in the perinuclear region when overexpressed, though the meaning of the structures remains unclear. In a previous study we showed that the overexpressed caspase-8 or -10 prodomain (PDCasp8 or PDCasp10) did not induce cell death, and we hypothesized that these prodomains interfere with the receptor-mediated cell death signaling pathway. Indeed, in 293, HeLa and Jurkat cells, cell death mediated by agonistic anti-Fas antibody, TRAIL or overexpression of full-length caspase-8 was significantly inhibited by overexpression of PDCasp8 or PDCasp10 which colocalized with the Golgi complex and with overexpressed FADD. However, when about 20 amino acid residues were deleted from either terminus of the caspase-10 prodomain (amino acid residue 1 to 219), the ability to inhibit Fas-mediated cell death was lost. Interestingly, these deletion mutants also lost the ability to make fibrous structures and to bind FADD, suggesting that FADD binding is important for their function, and that PDCasp8 and PDCasp10 act as dominant-negative inhibitors. 相似文献
999.
1000.
Ma Z Thomas KS Webb DJ Moravec R Salicioni AM Mars WM Gonias SL 《The Journal of cell biology》2002,159(6):1061-1070
The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked. 相似文献