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991.
992.
Kockx M Rye KA Gaus K Quinn CM Wright J Sloane T Sviridov D Fu Y Sullivan D Burnett JR Rust S Assmann G Anantharamaiah GM Palgunachari MN Katz SL Phillips MC Dean RT Jessup W Kritharides L 《The Journal of biological chemistry》2004,279(25):25966-25977
Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE. 相似文献
993.
The alveolar epithelium is lined by surfactant, a lipoprotein complex that both reduces surface tension and mediates several innate immune functions including bacterial aggregation, alteration of alveolar macrophage function, and regulation of bacterial clearance. Surfactant protein-D (SP-D) participates in several of these immune functions, and specifically it enhances the clearance of the pulmonary pathogen Pseudomonas aeruginosa, a common cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa secretes a variety of virulence factors including elastase, a zinc-metalloprotease, which degrades both SP-A and SP-D. Here we show that SP-D is cleaved by elastase to produce a stable 35-kDa fragment in a time-, temperature-, and dose-dependent manner. Degradation is inhibited by divalent metal cations, a metal chelator, and the elastase inhibitor, phosphoramidon. Sequencing the SP-D degradation products localized the major cleavage sites to the C-terminal lectin domain. The SP-D fragment fails to bind or aggregate bacteria that are aggregated by intact SP-D. SP-D fragment is observed when normal rat bronchoalveolar lavage (BAL) is treated with Pseudomonas aeruginosa elastase, and SP-D fragments are present in the BAL of CF lung allograft patients. These data show that degradation of SP-D occurs in the BAL environment and that degradation eliminates many normal immune functions of SP-D. 相似文献
994.
Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite different, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850cm(-1) and the attenuation of a peak around 1205cm(-1). Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote beta sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction. 相似文献
995.
Adipocyte differentiation of multipotent cells established from human adipose tissue 总被引:31,自引:0,他引:31
Rodriguez AM Elabd C Delteil F Astier J Vernochet C Saint-Marc P Guesnet J Guezennec A Amri EZ Dani C Ailhaud G 《Biochemical and biophysical research communications》2004,315(2):255-263
In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist > beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism. 相似文献
996.
Expert-Bezançon A Sureau A Durosay P Salesse R Groeneveld H Lecaer JP Marie J 《The Journal of biological chemistry》2004,279(37):38249-38259
Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them. 相似文献
997.
998.
Marques JP Schattat MH Hause G Dudeck I Klösgen RB 《Journal of experimental botany》2004,55(403):1697-1706
Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway. 相似文献
999.
Cheron A Peltier J Perez J Bellocq A Fouqueray B Baud L 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(12):7677-7683
1000.
CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi
Martins GA Tadokoro CE Silva RB Silva JS Rizzo LV 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(8):4893-4901
Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection. 相似文献