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991.
992.
Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. 总被引:18,自引:28,他引:18 下载免费PDF全文
We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding. 相似文献
993.
Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit 总被引:15,自引:0,他引:15
We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein. 相似文献
994.
Jeonghan Lee Michael T. Nelson Kirstin E. Rose Slobodan M. Todorovic 《Molecular neurobiology》2013,48(2):274-280
T-type calcium channels in the dorsal root ganglia (DRG) have a central function in tuning neuronal excitability and are implicated in sensory processing including pain. Previous studies have implicated redox agents in control of T-channel activity; however, the mechanisms involved are not completely understood. Here, we recorded T-type calcium currents from acutely dissociated DRG neurons from young rats and investigated the mechanisms of CaV3.2 T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-penicillamine rapidly reduced T-type current amplitudes. GSNO did not affect voltage dependence of steady-state inactivation and macroscopic current kinetics of T-type channels. The effects of GSNO were abolished by pretreatment of the cells with N-ethylmaleimide, an irreversible alkylating agent, but not by pretreatment with 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor, suggesting a potential effect of GSNO on putative extracellular thiol residues on T-type channels. Expression of wild-type CaV3.2 channels or a quadruple Cys-Ala mutant in human embryonic kidney cells revealed that Cys residues in repeats I and II on the extracellular face of the channel were required for channel inhibition by GSNO. We propose that SNO-related molecules in vivo may lead to alterations of T-type channel-dependent neuronal excitability in sensory neurons and in the central nervous system in both physiological and pathological conditions such as neuronal ischemia/hypoxia. 相似文献
995.
自噬(autophagy)是一种溶酶体依赖性的细胞内降解途径,其主要功能是将生物大分子(蛋白质、多糖等)或细胞器(线粒体等)回收至溶酶体中并将其降解为单糖、氨基酸等小分子以重复利用。发现HOPS复合体中的两个基因vps39和vps41的缺失会导致酵母内GFP-ATG8大量积累。进一步研究表明,积累的原因是GFP-ATG8与液泡不能发生融合。而在HOPS复合体中的另外两个基因vps16和vps18缺失的情况下,自噬融合没有受到影响;在vps16和vps18双敲除的菌株中,自噬融合同样没有受到影响。该实验结果为理解HOPS复合体的功能和自噬体与液泡融合的过程提供了新的线索。 相似文献
996.
The immune response of male DSN hamsters to a primary infection with Ancylostoma ceylanicum 总被引:3,自引:0,他引:3
The immune response of hamsters to a chronic hookworm infection has been investigated. Ancylostoma ceylanicum caused long term infections in hamsters which were associated with prominent changes in secondary lymphoid organs. The mesenteric lymph nodes and spleens increased rapidly in size stabilizing at approximately 3-4 times the weight in control animals by weeks 3-7. Cells from both the mesenteric lymph node and spleen, after an initial period of increased blast cell activity, became less reactive in the latter stages of infection. Serum antibody responses were marked, commencing in weeks 3-4 and increasing in intensity throughout the 10 week period of measurement. The results are discussed in relation to their contribution to the understanding of human hookworm infection. 相似文献
997.
Rose M. Jones Sabrina Hedrich D. Barrie Johnson 《Extremophiles : life under extreme conditions》2013,17(5):841-850
Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed. 相似文献
998.
999.
Role of caveolin-1 and cytoskeletal proteins, actin and vimentin, in adipogenesis of bovine intramuscular preadipocyte cells 总被引:6,自引:0,他引:6
We investigated the involvement of caveolin-1 and the cytoskeletal proteins, actin and vimentin, in the adipogenesis of bovine intramuscular preadipocyte (BIP) cells. Immunoblot analysis demonstrated that levels of caveolin-1 and actin gradually increased during adipose conversion in BIP cells, whereas a slight decrease was observed for vimentin. We found that part of the vimentin was clearly distributed to caveolin-1-enriched membrane fractions in BIP cells, but actin was not. During adipogenesis of BIP cells, treatment with the tubulin depolymerizer, nocodazole, significantly increased intracellular triglyceride accumulation compared to non-treated cells. Immunocytochemical analysis showed that actin microfilaments were significantly disrupted in nocodazole-treated cells. Also, a decrease in the localization of vimentin in caveolin-1-enriched fractions and a failure of vimentin to co-immunoisolate with caveolin-1 were observed in nocodazole-treated cells. These results suggest that a rearrangement of cytoskeletal proteins has a role in the intracellular accumulation of lipid droplets during adipogenesis of BIP cells. 相似文献
1000.
Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates 总被引:3,自引:0,他引:3
Tyurin VA Tyurina YY Borisenko GG Sokolova TV Ritov VB Quinn PJ Rose M Kochanek P Graham SH Kagan VE 《Journal of neurochemistry》2000,75(5):2178-2189
Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury. 相似文献