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- Buch, Charlotta
(författare)
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Dynamic protein trafficking of the nuclear membrane and in peroxisomes
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell nucleus is enclosed by the nuclear envelope (NE), a double lipid membrane separating the nucleoplasm from the cytoplasm. Transport of macromolecules between the nucleus and the cytoplasm takes places through nuclear pore complexes (NPCs) in a selective and energy dependent manner. The inner nuclear membrane (INM) contains transmembrane proteins that interact with the nuclear lamina and chromatin. In addition to being a barrier between the nucleoplasm and cytoplasm, an emerging view is that the NE has an active role in chromatin organization and gene regulation.In order to study structural and functional organization of the NE in live cells, we have used green fluorescent protein (GFP)-labeled proteins and laser scanning confocal microscopy (LSCM). In order to investigate dynamic properties of specific proteins or protein complexes we have used photobleaching techniques. In order to understand the organization of the NPC it is essential to study components necessary for NPC biogenesis and maintenance. We have investigated the possible alterations in the NPC in cells naturally lacking one of the integral membrane proteins of the NPC, gp210. Despite the lack of gp210, we observed no difference in distribution or density of pores. Neither did cell cycle progression nor generation time differ between cells having or lacking gp210. In addition, targeting or dynamic properties of the NPC proteins POM121, Nup107 or Nup153 were unaltered in the absence of gp210. We conclude that gp210 can not be essential for NPC biogenesis or maintaining stability of the NPC.The steps involved in onset of nuclear apoptosis are unclear. We studied nuclear alterations during apoptosis. We show that the nucleocytoplasmic barrier is disrupted early in apoptosis at the same time as chromatin collapses against the nuclear periphery but prior to nucleosomal DNA fragmentation. In addition, the disruption of nucleocytoplasmic transport correlates with caspase-3 dependent cleavage of POM121 at aspartate-531.The INM is estimated to contain ~70 uncharacterized transmembrane proteins. We characterized a novel putative mammalian NE protein that we termed Samp1. We show that Samp1 is an integral membrane protein specifically localized to the inner nuclear membrane during interphase. Interestingly, during mitosis a sub fraction of Samp1 distributed in the polar region of the mitotic spindle, colocalizing with tubulin and a lipid marker. However, another inner nuclear membrane protein, emerin, was excluded from this area. Thus Samp1 appears to define a specific membrane domain associated with the mitotic machinery.The distribution of peroxisomal fatty acid metabolizing enzymes have been reported to vary in different tissues. We investigated whether photobleaching techniques could be used to study the distribution of peroxisomal matrix proteins. We used GFP-labeled peroxisomal proteins and fluorescence recovery after photobleaching to show that peroxisomal matrix proteins become “trapped” inside peroxisomes after import. Thus we conclude that fluorescence loss in photobleaching can be used to distinguish between a strictly cytoplasmic localization and a dual localization when a protein is present both in the cytoplasm and in peroxisomes. Using this technique we determined that GFP-BAAT (bile acid-CoA:amino acid N-acyltransferase) is exclusively localized to the cytoplasm in HeLa cells.
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| 2. |
- Nishio, Akiyosho, et al.
(författare)
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Comparative studies of mitochondrial autoantibodies in sera and bile in primary biliary cirrhosis
- 1997
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Ingår i: Hepatology. - : Ovid Technologies (Wolters Kluwer Health). - 0270-9139 .- 1527-3350. ; 25:5, s. 1085-1089
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Tidskriftsartikel (refereegranskat)abstract
- Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of intrahepatic bile ducts. Although the pathogenesis of this disease is still unknown, high titers of antimitochondrial autoantibodies (AMA) have long been recognized in patient sera. However, little is known about the presence of AMA in bile. In this study, we investigated bile and sera from patients with PBC and healthy controls for the presence of AMA and mitochondrial autoantigens. AMA were detected in the bile of 17 of 19 patients (89.4%) with PBC; they were specifically directed against the pyruvate dehydrogenase complex (PDC-E2) in 15 of 19 patients (78.9%), to the branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) in 6 of 19 patients (31.6%), and to the 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) in 1 of 19 patients (5.3%). In a comparative study of sera from the same patients, anti-PDC-E2 antibodies were found in 19 of 19 patients (100%), anti-BCOADC in 9 of 19 patients (47.3%), and anti-OGDC-E2 in 4 of 19 patients (21.1%) patients. AMA in bile were always found together with antibodies of corresponding specificities in the serum from the same patient. Immunoglobulin (Ig)A AMA were found in the bile of 9 of 19 patients (47.7%) with PBC; they were specifically directed against PDC-E2 in 8 of 19 patients (42.1%) and to BCOADC in 2 of 19 patients (10.5%). Epitope mapping of IgA anti-PDC-E2 antibodies indicated that, like serum autoantibodies, the immunodominant epitope is directed against the inner lipoyl domain of PDC-E2. The prevalence and antigen reactivity of IgA AMA in sera correlated completely with IgA AMA in bile. Autoantibodies against nuclear envelope pore proteins (gp210) were found in 1 of 8 (12.5%) sera of patients with PBC, but not in bile. Furthermore, and of particular interest, we detected the autoantigens, PDC-E2, OGDC-E2, and BCOADC-E2, in the bile of 12 of 19 patients (63.2%), 9 of 19 patients (47.4%), and 9 of 19 patients (47.4%), respectively; PDC-E2 was found in only 1 of 17 (5.9%) disease controls. Although the presence of AMA in bile may merely reflect the presence of these antibodies in sera, the simultaneous detection of mitochondrial autoantigens in bile suggests an increase of mitochondrial autoantigens at inflammatory sites. Such autoantigens, coupled with AMA, may augment the local immune response and disease progression.
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