| 1. |
- Brynhildsen, Jan, 1962-, et al.
(författare)
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Attitudes among students and teachers on vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum
- 2002
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Ingår i: Medical teacher. - : Informa UK Limited. - 0142-159X .- 1466-187X. ; 24:3, s. 286-288
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Tidskriftsartikel (refereegranskat)abstract
- Important elements in the curriculum at the Faculty of Health Sciences in Link÷ping are vertical integration, i.e. integration between the clinical and basic science sections of the curriculum, and horizontal integration between different subject areas. Integration throughout the whole curriculum is time-consuming for both teachers and students and hard work is required for planning, organization and execution. The aim was to assess the importance of vertical and horizontal integration in an undergraduate medical curriculum, according to opinions among students and teachers. In a questionnaire 102 faculty teachers and 106 students were asked about the importance of 14 different components of the undergraduate medical curriculum including vertical and horizontal integration. They were asked to assign between one and six points to each component (6 points = extremely important for the quality of the curriculum, 1 point = unimportant). Students as well as teachers appreciated highly both forms of integration. Students scored horizontal integration slightly but significantly higher than the teachers (median 6 vs 5 points, p=0.009, Mann-Whitney U-test), whereas teachers scored vertical integration higher than students (6 vs 5, p=0.019, Mann-Whitney U-test). Both students and teachers considered horizontal and vertical integration to be highly important components of the undergraduate medical programme. We believe both kinds of integration support problem-based learning and stimulate deep and lifelong learning and suggest that integration should always be considered deeply when a new curriculum is planned for undergraduate medical education.
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| 2. |
- Dahle, L. O., et al.
(författare)
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Pros and cons of vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum : examples and experiences from Linköping, Sweden
- 2002
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Ingår i: Medical teacher. - : Taylor & Francis. - 0142-159X .- 1466-187X. ; 24:3, s. 280-285
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Tidskriftsartikel (refereegranskat)abstract
- Problem-based learning (PBL), combined with early patient contact, multiprofessional education and emphasis on development of communications skills, has become the basis for the medical curriculum at the Faculty of Health Sciences in Link ping (FHS), Sweden, which was started in 1986. Important elements in the curriculum are vertical integration, i.e. integration between the clinical and basic science parts of the curriculum and horizontal integration between different subject areas. This article discusses the importance of vertical integration in an undergraduate medical curriculum, according to experiences from the Faculty of Health Sciences in Link ping, and also give examples on how it has been implemented during the latest 15 years. Results and views put forward in published articles concerning vertical integration within undergraduate medical education are discussed in relation to the experiences in Link ping. Vertical integration between basic sciences and clinical medicine in a PBL setting has been found to stimulate profound rather than superficial learning, and thereby stimulates better understanding of important biomedical principles. Integration probably leads to better retention of knowledge and the ability to apply basic science principles in the appropriate clinical context. Integration throughout the whole curriculum entails a lot of time and work in respect of planning, organization and execution. The teachers have to be deeply involved and enthusiastic and have to cooperate over departmental borders, which may produce positive spin-off effects in teaching and research but also conflicts that have to be resolved. The authors believe vertical integration supports PBL and stimulates deep and lifelong learning.
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| 3. |
- Ekman, Anna-Karin, et al.
(författare)
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IL-17 and IL-22 Promote Keratinocyte Stemness in the Germinative Compartment in Psoriasis
- 2019
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Ingår i: Journal of Investigative Dermatology. - : ELSEVIER SCIENCE INC. - 0022-202X .- 1523-1747. ; 139:7, s. 1564-
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Tidskriftsartikel (refereegranskat)abstract
- Psoriasis is an inflammatory skin disorder characterized by the hyperproliferation of basal epidermal cells. It is regarded as T-cell mediated, but the role of keratinocytes (KCs) in the disease pathogenesis has reemerged, with genetic studies identifying KC-associated genes. We applied flow cytometry on KCs from lesional and nonlesional epidermis to characterize the phenotype in the germinative compartment in psoriasis, and we observed an overall increase in the stemness markers CD29 (2.4-fold), CD44 (2.9-fold), CD49f (2.8-fold), and p63 (1.4-fold). We found a reduced percentage of cells positive for the early differentiation marker cytokeratin 10 and a greater fraction of CD29(+) and involucrin thorn cells in the psoriasis KCs than in nonlesional KCs. The up-regulation of stemness markers was more pronounced in the K10(+) cells. Furthermore, the psoriasis cells were smaller, indicating increased proliferation. Treatment with IL-17 and IL-22 induced a similar expression pattern of an up-regulation of p63, CD44, and CD29 in normal KCs and increased the colony-forming efficiency and long-term proliferative capacity, reflecting increased stem cell-like characteristics in the KC population. These data suggest that IL-17 and IL-22 link the inflammatory response to the immature differentiation and epithelial regeneration by acting directly on KCs to promote cell stemness.
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| 4. |
- Gréen, Anna, 1973-
(författare)
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Histone H1 : Subtypes and phosphorylation in cell life and death
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genetic information of a human diploid cell is contained within approximately 2 metres of linear DNA. The DNA molecules are compacted and organized in various ways to fit inside the cell nucleus. Various kinds of histones are involved in this compaction. One of these histones, histone H1 is the topic of the present thesis. In addition to its structural role, H1 histones have been implicated in various processes, for example gene regulation and inhibition of chromatin replication.H1 histones, also termed linker histones, are relatively conserved proteins, and the various subtypes seem to have different and important functions even though redundancy between the subtypes has been demonstrated. Despite the sequence conservation of H1 subtypes, two sequence variations were detected within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid chromatographic separation of H1 proteins from K562 and Raji cell lines in Paper I in the present thesis. The variations were confirmed by genetic analysis, and the H1.2 sequence variation was also found in genomic DNA of normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence variation was concluded to be Raji specific. The significance of H1 microsequence variants is unclear, since the physiological function of H1 histones remains to be established.H1 histones can be phosphorylated at multiple sites. Changes in H1 phosphorylation has been detected in apoptosis, the cell cycle, gene regulation, mitotic chromatin condensation and malignant transformation. Contradictory data have been obtained on H1 phosphorylation in apoptosis, and many results indicate that H1 dephosphorylation occurs during apoptosis. We and others hypothesized that cell cycle effects by the apoptosis inducers may have affected previous studies. In Paper II, the H1 phosphorylation pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle effects into account. In receptor-mediated apoptosis, apoptosis occurs with a mainly preserved phosphorylation pattern, while Camptothecin induced apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating that H1 dephosphorylation is not a general event in apoptosis, but may occur upon apoptosis induction via the mitochondrial pathway. The dephosphorylation may also be a result of early cell cycle effects or signalling.Therefore, the H1 phosphorylation pattern in the cell cycle of normal activated T cells was investigated in Paper IV in this thesis. Some studies, which have been made using cancer cell lines from various species and cell synchronization, have indicated a sequential addition of phosphate groupsacross the cell cycle. Normal T cells and cell sorting by flow cytometry were used to circumvent side-effects from cell synchronization. The data demonstrate that a pattern with phosphorylated serines is established in late G1/early S phase, with some additional phosphorylation occurring during S, and further up-phosphorylation seems to occur during mitosis. Malignant transformation may lead to an altered G1 H1 phosphorylation pattern, as was demonstrated using sorted Jurkat T lymphoblastoid cells.During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages, while H1.2 was detected in the nucleus during prophase and telophase, and primarily in the cytoplasm during metaphase and early anaphase. H1.5 was located mostly to chromatin during prophase and telophase, and to both chromatin and cytoplasm during metaphase and anaphase. Phosphorylated H1 was located in chromatin in prophase, and in both chromatin and cytoplasm during metaphase, anaphase and telophase, indicating that the mechanism for a possible H1 subtype relocation to the cytoplasm is phosphorylation.In conclusion, data obtained during this thesis work suggest that H1 histones and their phosphorylation may participate in the regulation of events in the cell cycle, such as S-phase progression and mitosis, possibly through altered interactions with chromatin, and/or by partial or complete removal of subtypes or phosphorylated variants from chromatin.
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| 5. |
- Gréen, Anna, 1943-, et al.
(författare)
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Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis
- 2008
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Ingår i: Biochemistry. - : ACS Publications. - 0006-2960 .- 1520-4995. ; 47, s. 7539-7547
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Tidskriftsartikel (refereegranskat)abstract
- Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have been linked to the apoptotic process. Such conflicting results may depend on which various apoptosis-inducing agents cause apoptosis via different apoptotic pathways and often interfere with cell proliferation. Therefore, we investigated the relation between apoptosis and H1 phosphorylation in Jurkat cells after apoptosis induction via both the extrinsic and intrinsic pathways and by taking cell cycle effects into account. After apoptosis induction by anti-Fas, no significant dephosphorylation, as measured by capillary electrophoresis, or cell cycle-specific effects were detected. In contrast, H1 subtypes were rapidly dephosphorylated when apoptosis was induced by camptothecin. We conclude that histone H1 dephosphorylation is not connected to apoptosis in general but may be coupled to apoptosis by the intrinsic pathway or to concomitant growth inhibitory signaling.
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| 6. |
- Gréen, Anna, et al.
(författare)
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Histone H1 interphase phosphorylation becomes largely established in G(1) or early S phase and differs in G(1) between T-lymphoblastoid cells and normal T cells
- 2011
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Ingår i: Epigenetics & Chromatin. - : BioMed Central. - 1756-8935. ; 4:15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G(1). This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G(1), S and G(2)/M populations. less thanbrgreater than less thanbrgreater thanResults: We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G(1) or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G(1) compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G(1) in Jurkat cells. less thanbrgreater than less thanbrgreater thanConclusion: Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G(1) or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G(1) may be affecting the G(1)/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.
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| 7. |
- Gréen, Anna, 1973-, et al.
(författare)
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Histone H1 interphase phosphorylation pattern becomes largely established during G1/S transition in proliferating cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Histone H1 is an important constituent of chromatin, and is believed to be involved in regulation of chromatin structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones during the cell cycle. However, many of these experiments have been performed in non-human and human cancer cell lines, and by the use of cell synchronization techniques and cell cycle-arresting drugs. In this study, we have investigated the H1 subtype composition and phosphorylation pattern in the cell cycle. Exponentially growing normal human activated T cells and Jurkat lymphoblastoid cells were sorted by fluorescence activated cell sorting into G1, S and G2/M populations, without the use of cell cycle arresting drugs. We found that the H1.5 protein level increased after T-cell activation. Our data indicate that serine phosphorylation of H1 subtypes occurred to a large extent in late G1 phase or early S, while some additional serine phosphorylation took place during S, G2 and M phases. Furthermore, our data suggest that the newly synthesized H1 molecules during S phase also achieve a similar phosphorylation pattern as the previous ones. Jurkat cells showed more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. In conclusion, our data is consistent with a model where a major part of interphase H1 serine phosphorylation takes place within a narrow time window during the G1/Stransition. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication.
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| 8. |
- Gréen,, Anna, 1973-, et al.
(författare)
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Translocation of Histone H1 Subtypes Between Chromatin and Cytoplasm During Mitosis in Normal Human Fibroblasts
- 2010
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 77A:5, s. 478-484
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Tidskriftsartikel (refereegranskat)abstract
- Histone H1 is an important constituent of chromatin which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3 and H1.5 during mitosis. H1.2 was found in chromatin during prophase, and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase it appeared in both chromatin and cytoplasm, and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but some H1.5 remained situated in chromatin during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or their phosphorylated variants, may be signalling molecules in mitosis or that they leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.
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| 9. |
- Klang Årstrand, Hanna
(författare)
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Phosphoproteomic analysis of Arabidopsis thaliana ribosomes
- 2012
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribosomes serve as the site of protein synthesis in all living cells. Ribosomes were discovered in 1955 by George E. Palade when he was studying the endoplasmic reticulum which is covered by ribosomes. He received the Nobel Prize in Physiology or Medicine in 1974 for this discovery. Ribosomes are large protein and rRNA complexes which are made up from one small and one large subunit that work together to translate mRNA into a protein chain. Eukaryotic translation is mainly controlled during the initiation, which involves protein phosphorylation. In plants there is a general increase of protein synthesis during the day in order to synthesize proteins needed for photosynthesis. Phosphorylation can alter protein function and localization and is reversibly added and removed by kinases and phosphatases, respectively.The aim of the studies in this thesis was to elucidate the phosphorylation status of ribosomal proteins in the Arabidopsis thaliana 80S ribosome. I have focused on comparing ribosomal protein phosphorylation between different conditions and sub cellular locations, namely day/night conditions and cytosol/nucleus location.By using Fe3+IMAC to enrich phosphorylated peptides from cytosolic ribosomes followed by mass spectrometric analysis eight serine residues in six ribosomal proteins were found to be phosphorylated. Among these was a novel phosphorylation site in 40S ribosomal protein S6 at Serine 231. By using quantification with stable isotope labeling and mass spectrometry this phosphorylated residue and three other ribosomal phosphopeptides were found to have increased phosphorylation levels during day as compared to night ranging from 2 to 4 times. This phosphorylation increase can in turn effect the modulation of the diurnal protein synthesis in Arabidopis thaliana.Ribosome biogenesis involves shuttling of proteins and ribosomal subunits between the cell nucleus and cytoplasm. By purifying ribosomal proteins from these two cellular compartments and enriching for phosphopeptides using TiO2 affinity chromatography combined with mass spectrometry I was able to analyze their phosphorylation status. This method identified 13 phosphopeptides derived from 11 ribosomal proteins as well as phosphopeptides from two ribosomal associated proteins. 40S ribosomal protein S2-3 was found phosphorylated only in the cytoplasmic samples while 60S ribosomal protein L13-1 and the two ribosomal associated proteins were found only in the nuclear enriched samples.
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| 10. |
- Kostova-Koleva, Nora N., et al.
(författare)
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Histone H5chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation
- 2013
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Ingår i: Cytometry Part A. - : Wiley-Blackwell. - 1552-4922 .- 1552-4930. ; 83A:3, s. 273-279
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Tidskriftsartikel (refereegranskat)abstract
- We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
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| 11. |
- Kostova, NN, et al.
(författare)
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Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions
- 2004
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 58A:2, s. 132-139
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Tidskriftsartikel (refereegranskat)abstract
- Background: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. Methods: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. Results: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. Conclusions: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition. (C) 2004 Wiley-Liss, Inc.
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| 12. |
- Kostova, Nora, et al.
(författare)
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Immunohistochemical demonstration of histone H10 in human breast carcinoma
- 2005
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Ingår i: Histochemistry and Cell Biology. - : Springer Science and Business Media LLC. - 0948-6143 .- 1432-119X. ; 124:5, s. 435-443
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Tidskriftsartikel (refereegranskat)abstract
- Histone H10 is a linker histone subvariant present in tissues of low proliferation rate. It is supposed to participate in the expression and maintenance of the terminal differentiation phenotype. The aim of this work was to study histone H10 distribution in human breast carcinoma and its relationship with the processes of proliferation and differentiation. Most of the cells in carcinomas of moderate and high level of differentiation expressed histone H10 including cells invading connective and adipose tissues. In low differentiated tumours, the number of H10 expressing cells was considerably lower. Staining of myoepithelial cells, when seen, and of stromal fibroblasts was variable. The metastatic malignant cells in the lymph nodes also accumulated H10 but lymphocytes were always negative. All immunopositive malignant cells exhibited signs of polymorphism. Double H1 0/Ki-67 staining showed that the growth fraction in more differentiated tumours belonged to the H10-positive cells, while in poorly differentiated carcinomas it also included a cell subpopulation not expressing H10. If expressed, p27Kip1 was always found in H10-positive cells. These findings are inconsistent with the widespread view that histone H10 is expressed only in terminally differentiated cells. Rather, they suggest that the protein is expressed in cells in a prolonged intermitotic period irrespective of their level of differentiation. Double H10/Ki-67 immunostaining could be a useful tool in studying the growth fraction in tumours. © Springer-Verlag 2005.
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| 13. |
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| 14. |
- Koutzamani, Elisavet, 1973-
(författare)
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Chromatin, histones, and epigenetic tags
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues.The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin.The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.
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| 15. |
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| 16. |
- Koutzamani, Elisavet, et al.
(författare)
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Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 277:47, s. 44688-44694
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Tidskriftsartikel (refereegranskat)abstract
- The replacement linker histones H10 and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H10 dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H10 variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H10-2 is the only H10 subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.
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| 17. |
- Loborg, Helena, 1963-, et al.
(författare)
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Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe
- 2000
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Ingår i: Cytometry. - 0196-4763 .- 1097-0320. ; 40:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Background: It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure.Methods: Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4',6-diamidino-2- phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry.Results: The association between linker histones and chromatin was stronger in cultured human fibroblasts and rat smooth muscle cells than in both human T lymphocytes and granulocytes, and in particular, frog erythrocytes, which exhibited highly condensed chromatin. Our data indicate a lower affinity of linker histones for metaphase chromosomes than for interphase chromatin, and we observed a reduction in linker histone affinity during terminal differentiation of frog erythrocytes in vivo.Conclusions: Taken together, our findings imply that the affinity of linker histones for chromatin in situ is unrelated or inversely related to chromatin condensation.
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| 18. |
- Loborg, Helena, et al.
(författare)
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DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ
- 1997
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Ingår i: Cytometry. - 0196-4763 .- 1097-0320. ; 28:3, s. 212-219
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)–chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 μM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HCl treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53–68%. The 7-AAMD obviously showed higher sensitivity for H1–chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1–chromatin interactions in situ and that our method has a potential to provide new information on such interactions.
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| 19. |
- Loborg, Helena, et al.
(författare)
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High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes
- 1995
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 20:4, s. 296-306
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Tidskriftsartikel (refereegranskat)abstract
- The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.
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| 20. |
- Rakar, Jonathan
(författare)
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Fibroblast Differentiation and Models of Human Skin
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis combines three publications and one manuscript, covering two principal topics: functional differentiation of human fibroblasts and laboratory models of human skin. The two topics favourably unite in the realm of tissue engineering. This thesis is therefore split into three main parts: 1. a discussion of phenotypic plasticity as it pertains to fibroblasts and the stem cell continuum; 2. a short review of engineered tissue, with particular focus on soluble factors and materials; and, 3. a motivated review of the biology, diversity and culture of skin, including skin construction.The intended goal of our research endeavor was to achieve the formulation of a bioactive therapy for skin regeneration. The main hypothesis was that fibroblast-to-keratinocyte differentiation would facilitate wound healing, and that the protocol for such a method could be adapted to clinical translation. The foundation for the hypothesis lay in the differentiation capabilities of primary dermal fibroblasts (Paper I). However, the goal has not yet been achieved. Instead, intermediate work on the construction of skin for the purpose of creating a model test-bed has resulted in two other publications. The use of excised human skin, a formidable reference sample for tissue engineered skin, has been used to investigate a gelatinbased material in re-epithelialization (Paper II). A first attempt at standardizing a constructed skin model also resulted in a publication: an evaluation of melanocyte influences on keratinocyte-mediated contraction (Paper III).The introduction of melanocytes into a skin model raised questions about other appendages of the integumentary system. Our previous experience with preadipocyte isolation and identification, and our attempts at constructing three-dimensional adipose tissue, motivated further investigations into fibroblast-to-adipocyte differentiation. We investigated the possibility of activating thermogenesis in fibroblasts, a property otherwise reserved for cells of the adipogenic and myogenic lineages. Our attempts were successful, and are presently in manuscript form (Paper IV). Some further experiments and optimizations are necessary before establishing a reproducible protocol for thermogenic induction.The knowledge obtained through these scientific inquiries have moved us closer to achieving our goals, but methodological advances are still necessary. In the meantime, we have new test-beds for investigating different interactions in skin, and that enables many new questions to be asked and answered.
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| 21. |
- Rundquist, Ingemar, et al.
(författare)
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Analyses of linker histone - chromatin interactions in situ
- 2006
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Ingår i: Biochemistry and Cell Biology. - 0829-8211 .- 1208-6002. ; 84:4, s. 427-436
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.
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| 22. |
- Rundquist, Ingemar, 1947-
(författare)
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Cytofluorometry : technique and applications
- 1981
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aims of this investigation were to examine the conditions for rapid fluorescence measurements on cellular material, to improve the performance of measurement systems for microscope fluorometry, and to apply this technique in studies on mast cell biology. Fluorescence fading, a general complication of cytofluorometry, was studied during illumination times in the millisecond range, and a new fading phenomenon characterized by short duration and rapid recovery was described. The findings of the study formed the basis for the construction of two instfurnent systems for microscope fluorometry based on Leitz MPV I and MPV II microscope photometers. Rapid fluorescence measurements were performed by a completely automatic measuring sequence, except forselectionandfocusing of the objects to be measured. Automation was mainly achieved by the integration of computers in the measurement systems, which also resulted in easily interchangeable programdetermined measuring routines and proper data processing and presentation of results. The systems were mainly used for rapid analysis of cell populations. The precision of the measurements was improved by different standardization techniques, and the measuring speed, about 500 cells per h on well prepared specimens, was high enough to permit analysis of relatively large cell populations within a reasonable time.The cytofluorometric technique was applied to studies on the biology of the connective tissue mast cell. Rat peritoneal mast cells were used for this purpose. The proliferation of mast cells was estimated by cytofluorometric measurements of DNA in mast cell populations after staining with the fluorescent dye Hoechst 33258. Formaldehyde-induced fluorescence was used to study the uptake and turnover of dopamine in mast cells in vivo. Measurements of the mast cell content of heparin, a constituent of the mast cell granule matrix, were performed by a combination of microscope fluorometry, which permits visual identification of the cells, and flow cytofluorometry, by which rapid measurements of large populations can be made.
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| 23. |
- Sarg, Bettina, et al.
(författare)
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Characterization of sequence variations in human histone H1.2 and H1.4 subtypes
- 2005
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Ingår i: The FEBS Journal. - : Wiley InterScience. - 1742-464X .- 1742-4658. ; 272:14, s. 3673 -3683
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Tidskriftsartikel (refereegranskat)abstract
- In humans, eight types of histone H1 exist (H1.1–H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC→GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA→AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC→GTC shift, indicating that this is a relatively frequent polymorphism. The AAA→AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions.
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| 24. |
- Sarg, Bettina, et al.
(författare)
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Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 277:42, s. 39195-39201
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Tidskriftsartikel (refereegranskat)abstract
- Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono- and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.
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| 25. |
- Vegfors, Jenny, 1984-
(författare)
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Psoriasin For Better or for Worse in Sickness and in Health : The Role of Psoriasin in Angiogenesis and Differentation of Epithelial Cells
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is highly expressed in high-grade ductal carcinoma in situ (DCIS) and in the benign hyper-proliferative skin disorder psoriasis. Both breast cancer and psoriasis are diseases which are characterized by hyperproliferation and a disturbed differentiation of the epithelial cells as well as a pronounced angiogenesis. The potential role of psoriasin in angiogenesis and the epithelial differentiation remain unclear.The aim of this thesis was to investigate the cellular effects of psoriasin in angiogenesis and the differentiation processes, with special emphasis on breast cancer and psoriasis.We found that psoriasin expression was induced in mammary epithelial cells and keratinocytes by oxidative stress. Psoriasin expression was shown to induce vascular endothelial growth factor (VEGF) expression and several other pro-angiogenic factors in epithelial cells. Upon down-regulation of psoriasin, H2O2-induced expression of VEGF was decreased as well as the pro-angiogenic factors heparin-binding EGF-like growth factor (HBEGF) and matrix metalloproteinase (MMP)-1. Extracellular psoriasin contributed to the subsequent induction of proliferation, migration and tube formation of endothelial cells. The proliferative effect of psoriasin was shown to be mediated by the receptor for advanced glycation end products (RAGE). Furthermore, psoriasin induced reactive oxygen species (ROS) in both endothelial and epithelial cells through the action of RAGE, and contributed to the expression of the pro-angiogenic factors in endothelial cells.The expression of psoriasin was up-regulated in mammary epithelial cells and keratinocytes in response to differentiation-inducing stimuli and was shown to be regulated by pathways involved in epithelial cell differentiation. Upon psoriasin down-regulation the shift towards a more differentiated CD24+-phenotype of mammary epithelial cells was abolished. Furthermore, the expression of the differentiation markers involucrin, desmoglein 1, transglutaminase 1 and CD24 was decreased in keratinocytes upon down-regulation of psoriasin expression. In vivo we demonstrated a gradient of psoriasin expression in the psoriatic epidermis, with intense expression in the suprabasal differentiated layers, and a similar staining pattern between psoriasin and the differentiation marker CD24 in DCIS tumors.In conclusion, our findings describe psoriasin as a mediator in the angiogenic process and a contributor of epithelial cell differentiation. Consequently, psoriasin is possibly a contributor to the development and progression of breast cancer and psoriasis and a potential target in the treatment of these diseases.
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