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91.
Malisauskas M Darinskas A Zamotin VV Gharibyan A Kostanyan IA Morozova-Roche LA 《Biochemistry. Biokhimii?a》2006,71(5):505-512
In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses. 相似文献
92.
Shahdat Hossain† Michio Hashimoto Masanori Katakura Koji Miwa Toshio Shimada‡ Osamu Shido 《Journal of neurochemistry》2009,111(2):568-579
The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ1–42 ), Aβ1–42 -oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ1–42 -fibrill formation with a concomitant reduction in the levels of soluble Aβ1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ1–42 -induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease. 相似文献
93.
Chalermchai Mitrpant Sue Fletcher Patrick L. Iversen Steve D. Wilton 《The journal of gene medicine》2009,11(1):46-56
Background
Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein‐truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg‐Dmdmdx–4Cv/J (4CV) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.Methods
A series of 2′‐O‐methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.Results
Compared to the original mdx mouse studies, induction of exon skipping from the 4CV dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near‐full length dystrophin protein in cultured 4CV myogenic cells and in vivo, after a single intramuscular injection.Conclusions
By‐passing the protein‐truncating mutation in the 4CV mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo. Copyright © 2008 John Wiley & Sons, Ltd. 相似文献94.
利用超临界CO2萃取微孔草籽油,并对籽油进行了HPLC/MS分析。实验确定的最佳超临界CO2流体萃取条件是:萃取温度45℃,萃取压力20MPa,CO2流量为35-40kg/h,萃取时间120min,在此条件下白刺籽油的萃取率为16.12%。利用HPLC/MS对微孔草籽油分析,发现其不饱和脂肪酸的相对含量高达73.19%。比较了超临界CO2萃取微孔草籽油油样和石油醚萃取微孔草籽油油样的理化性质,发现超临界CO2流体萃取的籽油质量优于传统溶剂萃取的籽油。 相似文献
95.
96.
Inna Kuperstein Kerensa Broersen Iryna Benilova Jef Rozenski Wim Jonckheere Maja Debulpaep Annelies Vandersteen Ine Segers-Nolten Kees Van Der Werf Vinod Subramaniam Dries Braeken Geert Callewaert Carmen Bartic Rudi D'Hooge Ivo Cristiano Martins Frederic Rousseau Joost Schymkowitz Bart De Strooper 《The EMBO journal》2010,29(19):3408-3420
The amyloid peptides Aβ40 and Aβ42 of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates. 相似文献
97.
Expression,solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4
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Mitic LL Unger VM Anderson JM 《Protein science : a publication of the Protein Society》2003,12(2):218-227
The tight junction tetraspan protein claudin-4 creates a charge-selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin-4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro-octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin-4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique. 相似文献
98.
Van Montfort RL Bateman OA Lubsen NH Slingsby C 《Protein science : a publication of the Protein Society》2003,12(11):2606-2612
Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions. 相似文献
99.
100.
The pungent principle of the aerial parts and roots of Matricaria pubescens has shown to be the known thienyl-hexadien-isobutylamide. The structure was confirmed by 13C NMR analysis. In addition, the roots afforded small amounts of decadien-isobutylamide, and the aerial parts afforded large amounts of herniarin. The chemosystematic significance of amide accumulation within the tribe Anthemideae is briefly discussed. 相似文献