Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin. 相似文献
Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780). 相似文献
Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved
in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies
do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized
of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron
microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored
to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses
ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions.
TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa. 相似文献
Fumonisin B1 (FB1) is a harmful mycotoxin produced by Fusarium species, which results in oxidative stress leading to cell death in plants. FB1 perturbs the metabolism of sphingolipids and causes growth and yield reduction. This study was conducted to assess the role of ethylene in the production and metabolism of reactive oxygen species in the leaves of wild type (WT) and ethylene receptor mutant Never ripe (Nr) tomato and to elucidate the FB1-induced phytotoxic effects on the photosynthetic activity and antioxidant mechanisms triggered by FB1 stress. FB1 exposure resulted in significant ethylene emission in a concentration-dependent manner in both genotypes. Moreover, FB1 significantly affected the photosynthetic parameters of PSII and PSI and activated photoprotective mechanisms, such as non-photochemical quenching in both genotypes, especially under 10 µM FB1 concentration. Further, the net photosynthetic rate and stomatal conductance were significantly reduced in both genotypes in a FB1 dose-dependent manner. Interestingly, lipid peroxidation and loss of cell viability were also more pronounced in WT as compared to Nr leaves indicating the role of ethylene in cell death induction in the leaves. Thus, FB1-induced oxidative stress affected the working efficiency of PSI and PSII in both tomato genotypes. However, ethylene-dependent antioxidant enzymatic defense mechanisms were activated by FB1 and showed significantly elevated levels of superoxide dismutase (18.6%), ascorbate peroxidase (129.1%), and glutathione S-transferase activities (66.62%) in Nr mutants as compared to WT tomato plants confirming the role of ethylene in the regulation of cell death and defense mechanisms under the mycotoxin exposure.
Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices. 相似文献
Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application. 相似文献
Intermolecular Cu(I)-catalyzed azide-alkyne cycloadditions of 15β-azido-17β-hydroxy-5α-androstan-3β-yl acetate with different terminal alkynes under optimized reaction conditions were carried out to furnish 15β-triazolyl derivatives in good yields. Subsequent oxidation of the 'click' products with the Jones reagent afforded the corresponding 17-ketones. All the synthetized compounds were tested on three malignant human cell lines (HeLa, MCF7 and A431) in order to investigate their antiproliferative activities in vitro. Evidence of cell cycle blockade and apoptosis induction was obtained for the most effective five selected compounds by means of flow cytometry and microscopic techniques. The 15β-triazolyl-5α-androstane framework may be considered an appropriate base for the design of steroidal antiproliferative agents. 相似文献
Siboglinids are symbiotic polychete annelids having hemoglobins as essential oxygen- and sulfide-carriers for their endosymbiotic bacteria. We analyzed the structure of the hemoglobins from two species of siboglinids: the monilifera Sclerolinum contortum and the frenulata Oligobrachia webbi (i.e. haakonmosbiensis) from Norwegian cold seeps. Measured by Multi-Angle Laser Light Scattering (MALLS), Sclerolinum shows a 3190 ± 50 kDa hexagonal bilayer hemoglobin (HBL-Hb) and a 461 ± 46 kDa ring-Hb, just as vestimentifera, whereas Oligobrachia has a 409 ± 3.7 kDa ring-Hb only. Electrospray Ionization-Mass Spectrometry (ESI-MS) showed Sclerolinum HBL-Hb composed of seven monomeric globins (15–16 kDa), three disulfide-bonded globin heterodimers and three linkers. The heterodimers always contain globin-b (15814.4 ± 1.5 Da). Sclerolinum ring-Hb is composed of globins and dimers with identical masses as its HBL-Hb, but lacks linkers. Oligobrachia ring-Hb has three globin monomers (14–15 kDa) only, with no disulfide-bonded dimers. Comparison of Sclerolinum hemoglobins between Storegga and Haakon Mosby Mud Volcano, using the normalized height of deconvoluted ESI-MS peaks, shows differences in globin monomers abundances that could reflect genetic differences or differential gene expression between distinct seep populations. The discovery of HBL-Hb in Sclerolinum is a new element supporting the hypothesis of monilifera being phylogenetically more closely related to vestimentifera, than to frenulata. 相似文献