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- Björkegren Sjögren, Camilla
(författare)
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Profilin in cell motility and signal transduction studies using site-specific mutagenesis
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When a cell is stimulated by signalling molecules such as growth factors, one immediate response is an increased motile activity of the cell. Membrane lamella and spikes appear at the cell surface and perform undulating movements. This is caused by the activation and reorganisation of the highly dynamic microfilament system, present beneath the cell membrane. The major component of the microfilament system is actin, a protein that can polymerise into filaments -microfilaments-, and several actin-binding proteins which act in concert to organise actin into different supramolecular arrangements. To understand how a signal is transmitted form the exterior of the cell to the microfilament system, it is important to reveal how the activities of different actin-binding proteins are regulated.This thesis focus on the actin-binding protein profilin, which binds actin monomers and regulates actin polymerisation. Profilin also interacts with polyphosphoinositides, which are important signalling molecules, and this association appears to regulate both the function of profilin and the turnover of polyphosphoinositides. Furthermore, profilin binds poly(L-proline) and proline-rich proteins. The latter interaction seems to be involved in the positioning of profilin within a cell, and may also be important for signal transduction-related processes. In the current investigation site-directed mutagenesis was combined with biochemical methods to further investigate the function of profilin.The replacement of two amino acid residues located in a hydrophobic patch on the surface of the profilin molecule was shown to abolish the interaction with poly(L-proline), and enabled the localisation of the poly(L-proline) binding-site to this part of profilin. A new purification method was developed for these profilin mutants and further biochemical characterisation showed that the two mutations also decreased the affinity of profilin for actin. As the poly(L-proline)-binding site is separated from the actin-binding surface of profilin this showed that these amino acid replacements in the poly(L-proline)-binding site also introduced long-range alterations in the profilin molecule.Profilin was shown to be phosphorylated on a serine and a tyrosine residue by an epidermal growth factor receptor complex isolated from stimulated cells. The phosphorylated residues were located to the poly(L-proline) binding site of profilin, and the phosphorylation interfered with the binding of profilin to poly(L-proline). This suggests that this phosphorylation regulate interactions between profilin and proline-rich proteins.The simultaneous deletion of two amino acid residues adjacent to the actin-binding site of profilin was shown to reduce the affinity of profilin for actin, without affecting the interaction of profilin with other ligands. The effect of this mutant profilin on the organisation of the microfilament system in living cells was compared to that of wild type profilin in microinjection experiments. While wild type profilin caused disruption of actin bundles within the cell, the profilin mutant had no apparent effect, showing that the derangement of the actin bundles by profilin is dependent on the stability of the profilin:actin complex.
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- Norell, Marie, 1955-
(författare)
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The influence of nutritional status and age on chromatin structure and transcriptional activity in liver and intestinal epithelial cells
- 1990
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All processes in mammals depend on regulated gene expression whichin turn depends on the amount and composition of the nutrient intake.Not only is the regulation of gene expression affected by nutrition butalso the structure of DNA and chromatin can be influenced. In thisthesis the effect of dietary methionine deficiency and methioninecysteinerestriction on chromatin structure was studied, as well asage-related modifications of the nuclear organization.Nuclei were isolated from rat liver and intestinal epithelial cells.After limited digestion with nucleases the chromatin was separated intofractions enriched in transcriptionally active and inactive structures.The deficient or restricted diets affected the chromatin byrendering it more condensed than the control chromatin. This wasmanifested as a diminished nuclease sensitivity, lowered DNA-dependentRNA polymerase activities and increased amounts of histone Hl in thenuclease resistant chromatin fraction. In intestinal epithelial cells,but not in liver, a more condensed chromatin structure appeared withage.In liver the amount of protein per DNA was the same in all dietaryand age groups; in the intestinal epithelial cells the ratioprotein/DNA increased after methionine-cysteine restriction and withage. The protein pattern obtained after gel electrophoresis was similarfor all the physiological conditions studied. Thus the amount ofproteins but not the composition was altered by nutritional regimen andage.The liver increased in size with age mainly by increasing the cellsize and not the cell number. The number of intestinal epithelial cellsincreased with age but cell size was unaffected.In conclusion this study demonstrates an alteration of the chromatinstructure after nutritional deprivation or as a consequence ofincreasing age. The differences in adaptation to nutritional conditionsand age were found to be tissue specific.
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- Beier, Frank, et al.
(författare)
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Localization of silencer and enhancer elements in the human type X collagen gene.
- 1997
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 66:2, s. 210-218
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Tidskriftsartikel (refereegranskat)abstract
- Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.
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- Frithz-Lindsten, Elisabet, et al.
(författare)
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Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.
- 1998
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 29:5, s. 1155-1165
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Tidskriftsartikel (refereegranskat)abstract
- Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.
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- Leary, Sophie E. C., et al.
(författare)
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Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague
- 1999
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Ingår i: Microbial Pathogenesis. - : Elsevier. - 0882-4010 .- 1096-1208. ; 26:3, s. 159-169
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Tidskriftsartikel (refereegranskat)abstract
- The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective.
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