| 1. |
- Khotin, M.G., et al.
(författare)
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Analysis of nuclear protein complexes comprising a-actinin-4 by 2D-electrophoresis and mass-spectrometry
- 2009
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Ingår i: Tsitologiya. - : SP MAIK Nauka/Interperiodica. - 0041-3771. ; 51:8, s. 684-690
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Tidskriftsartikel (refereegranskat)abstract
- Actin-binding protein a-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of a-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with a-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear a-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. ß-Actin, a- and ß-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with ß-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with a-actinin-4 may suggest that a-actinin-4 is involved in transcription and splicing. Presence of a-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDITOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against a-tubulin confirmed association of a-actinin-4 with a-tubulin in the protein complex. Nuclear a-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with a-tubulin. These data suggest that a-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.
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| 2. |
- Turoverova, L.V., et al.
(författare)
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Analysis of extracellular matrix proteins produced by cultured cells
- 2009
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Ingår i: Cell and Tissue Biology. - : SP MAIK Nauka/Interperiodica. - 1990-519X .- 1990-5203. ; 3:5, s. 497-502
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.
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| 3. |
- Turoverova, L.V., et al.
(författare)
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Analysis of extracellular matrix proteins produced by cultured cells
- 2009
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Ingår i: Tsitologiya. - St Petersburg, Russian Federation : Sankt-Peterburgskaya Izdatel'skaya Firma Nauka. - 0041-3771. ; 51:8, s. 691-696
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.
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| 4. |
- Ahlstrand, Elisabeth, 1947-, et al.
(författare)
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Teacher Knowledge Development in a Social and Individual Context
- 1996
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Ingår i: Changing Research and Practice. Teachers' Professionalism, Identities and Knowledge. - London, Washington : Falmer Press. - 075070585X - 0750705868
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 5. |
- Babakov, V.N., et al.
(författare)
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RelA/NF-?B transcription factor associates with a-actinin-4
- 2008
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 314:5, s. 1030-1038
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Tidskriftsartikel (refereegranskat)abstract
- The NF-?B/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-?B in the cytoplasm and the transport mechanism to the nucleus. We found that NF-?B is associated with the actin-binding protein a-actinin-4. NF-?B and a-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-a, a-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-a led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-?B co-immunoprecipitated a-actinin-4 from A431 cell lysates and nuclear extracts, but a-actinin-1 and ß-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-?B can bind to matrix-bound chicken gizzard a-actinin. We suggest that the a-actinin-4 is important for the NF-?B nuclear translocation and its functions inside the nucleus. © 2007 Elsevier Inc. All rights reserved.
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| 6. |
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| 7. |
- Magnusson, Mattias, et al.
(författare)
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Importance of CpG dinucleotides in activation of natural IFN-alpha-producing cells by a lupus-related oligodeoxynucleotide
- 2001
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Ingår i: Scandinavian Journal of Immunology. - : John Wiley & Sons. - 0300-9475 .- 1365-3083. ; 54:6, s. 543-550
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Tidskriftsartikel (refereegranskat)abstract
- The oligodeoxyribonucleotide (ODN) 5-TTTTCAATTCGAAGATGAAT-3 (ODN H), identified in systemic lupus erythematosus (SLE) serum, induced the production of interferon (IFN)-alpha in human peripheral blood mononuclear cells (PBMC) when combined with lipofectin. Flow cytometric analysis with staining for surface antigens and intracellular IFN-alpha, showed that the IFN-alpha -producing cells (IPC) were the natural IPC, also termed type 2 dendritic cell precursors (pDC2) or plasmacytoid monocytes. The importance of unmethylated CpG dinucleotides for the interferogenic activity of ODN was studied. Methylation of CpG impaired the activity of single-stranded (ss) ODN H, but increased that of the complementary ssODN I. Furthermore, CpG-methylated double-stranded (ds) ODN H-met-I-met lost, but hemimethylated dsODN H-I-met retained interferogenic activity. Inversion of the CpG to GpC had no effect on the interferogenic activity of ssODN H, increased that of ssODN I, however abolished the activity of dsODN H-I. Alteration of the CpG in ODN H to ApG and in the ODN I to CpT destroyed their activity. The induction of IFN-alpha is therefore sequence-specific, but unmethylated CpGs are not always required, especially not in ssODNs. Interferogenic DNA sequences could therefore be more frequent in eukaryotic genomes than previously thought and their capacity to activate natural IPC may have implications for immune responses to microbial antigens and nuclear autoantigens.
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| 8. |
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| 9. |
- Baniulis, Danas, et al.
(författare)
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Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox
- 2005
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Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 74:4, s. 337-347
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Tidskriftsartikel (refereegranskat)abstract
- Human phagocyte flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the β-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the characterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91phox, of the oxidase protein. This antibody recognizes gp91phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91phox/p22 phox heterodimer, prepared on anti-p22phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135-DPYSVALSELGDR of gp91phox. The antibody was used to probe for the presence of gp91phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic granulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91 phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91phox from membranes of Cytb-producing cells. © Blackwell Munksgaard 2005.
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| 10. |
- Baniulis, Danas, et al.
(författare)
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Unusual polyclonal anti-gp91phox peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4
- 2005
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Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 74:3, s. 241-249
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Tidskriftsartikel (refereegranskat)abstract
- To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91phox regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91phox in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91phox gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91phox homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.
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