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181.
We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly. 相似文献
182.
183.
Kitamura K Takeda K Koya T Miyahara N Kodama T Dakhama A Takai T Hirano A Tanimoto M Harada M Gelfand EW 《Journal of immunology (Baltimore, Md. : 1950)》2007,178(1):480-488
The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR. 相似文献
184.
Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway 总被引:1,自引:0,他引:1
Minami K Tambe Y Watanabe R Isono T Haneda M Isobe K Kobayashi T Hino O Okabe H Chano T Inoue H 《Journal of virology》2007,81(20):11106-11115
GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense. 相似文献
185.
Huang M Ida H Arima K Nakamura H Aramaki T Fujikawa K Tamai M Kamachi M Kawakami A Yamasaki H Origuchi T Eguchi K 《Life sciences》2007,81(19-20):1461-1466
Our recent report demonstrated that apoptosis-specific autoantibodies against granzyme B-induced cleavage fragments of SS-B (La) were found in the sera from patients with primary Sj?gren's syndrome. The objective of this study was identified by the intracellular redistribution of La autoantigen during granzyme B-induced apoptosis. We developed green fluorescence protein (GFP)-La and GFP-LaDelta220 (generation of granzyme B-specific cleavage of La protein) fusion proteins. GFP-La protein was localized in the nucleus, whereas the GFP-LaDelta220 protein predominantly existed in the cytoplasm in transformed A293T cells. Nuclear GFP-La protein was translocated to cytoplasm after granzyme B enriched YT cells incubation. La protein in human salivary grand HSG cells is cleaved and translocated from the nucleus to the cytoplasm after YT cell co-cultivation. These results suggest that La protein is cleaved by granzyme B and N-terminal La fragment (27 kD) translocated to the cytoplasm, thus leading to a novel autoantibody production during granzyme B-mediated cytotoxicity. 相似文献
186.
Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts. 相似文献
187.
Iino N Matsunaga T Harada T Igarashi S Koyama I Komoda T 《Cell and tissue research》2007,328(2):355-363
Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been
detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant
aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues
of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were
detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated
protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant
aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates
from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected
in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant
aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions
from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type
of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and
adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has
thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous
cell carcinoma tissue. 相似文献
188.
189.
Yoshida T Murai M Abe M Ichimaru N Harada T Nishioka T Miyoshi H 《Biochemistry》2007,46(36):10365-10372
Natural antibiotic polyene amides such as myxalamides are potent inhibitors of mitochondrial complex I. Because of the significant instability of this series of compounds due to an extended pi-conjugation skeleton, a detailed characterization of their inhibitory action has not been performed. To elucidate the action mechanism as well as binding manner of polyene amides with complex I, identification of the roles of each functional group in the inhibitory action is needed. We here synthesized a series of amide analogues and carried out structure-activity studies with bovine heart mitochondrial complex I. With respect to the left-hand portion, the natural pi-conjugation skeleton common to many natural products is not required for the inhibition and can be substituted with a simpler substructure such as a conjugated diene. The geometry and shape of the left-hand portion were shown to be important for the inhibition, suggesting that this portion may bind to a narrow hydrophobic pocket in the enzyme rather than merely partitioning into the lipid membrane phase. Concerning the right-hand portion of the inhibitor, the presence of the 2-methyl, amide NH, and (S)-1'-methyl groups was crucial for the activity, suggesting that both methyl groups neighboring the amide group finely adjust the hydrogen-bonding ability of the amide group. In contrast, modifications of the 2'-OH group did not significantly influence the activity, suggesting that the role of this functional group is not to serve as a hydrogen bond donor to the enzyme but to act as a hydrophilic anchor directing the right-hand portion at or near the membrane surface. Detailed characterization of the action mechanism indicated that the polyene amides share a common binding domain with other complex I inhibitors, though their binding position (or manner) within the domain may differ considerably from that of other inhibitors. 相似文献
190.
Hayashi A Chiba T Hayashi H Sasayama S Ishiguro T Onozaki K 《Cancer immunology, immunotherapy : CII》2007,56(4):545-553
In order to study the effect of glycosylation on its biological activities, and to develop TNFα with less deleterious effects,
recombinant human TNFα was chemically coupled with N-acetylneuraminic acid (NeuAc). NeuAc with C9 spacer was coupled to TNFα by acyl azide method. Two glycosylated TNFαs, designated
L NeuAc-TNFα and H NeuAc-TNFα, were purified by anion-exchange chromatography. NeuAc coupling to TNFα was confirmed by lectin
blotting. Average number of carbohydrate molecules introduced per molecule of L NeuAc-TNFα and H NeuAc-TNFα were estimated
to be 1.0 and 1.5, respectively. We examined a variety of TNFα activities in vitro, including antiproliferative or cytotoxic
activities to tumor cells, proliferative effect on fibroblast cells, stimulatory effects on IL-6 production by melanoma cells
and NF-κB activation in hepatoma cells. L NeuAc-TNFα and H NeuAc-TNFα exhibited reduced activities about 1/3 and 1/10 as compared
to native TNFα in all the activities performed in vitro. 相似文献