Ingemar Rundquist

Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging.

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.

Pros and cons of vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum: examples and experiences from Linkoping, Sweden.

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 Linköping (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 Linköping, 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 Linköping. 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.

Attitudes among students and teachers on vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum

Important elements in the curriculum at the Faculty of Health Sciences in Linköping 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.

Characterization of sequence variations in human histone H1.2 and H1.4 subtypes

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 Burkitts 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.

Millisecond fading and recovery phenomena in fluorescent biological objects

SummaryCytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine organge the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.

Lipofuscin-induced autofluorescence of living neonatal rat cardiomyocytes in culture

Lipofuscin-specific autofluorescence of living, cultured rat neonatal myocardial myocytes was studied with respect to intensity and spectral characteristics. The autofluorescence emission spectrum changed over time indicating continuously ongoing intramolecular reorganisation of the pigment. Moreover, as compared with formaldehyde-fixed material native lipofuscin produced a considerable red-shifted autofluorescence, indicating formaldehyde-induced molecular modification or quenching of certain parts of the spectrum. The relationship between oxidative stress and lipofuscinogenesis was confirmed because the rate of lipofuscin accumulation was increased when cells were grown at increasing ambient oxygen concentrations.

Immunohistochemical demonstration of histone H10 in human breast carcinoma

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 H10/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.

Translocation of histone H1 subtypes between chromatin and cytoplasm during mitosis in normal human fibroblasts.

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 H1.5 was partitioned between chromatin and cytoplasm 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 theirphosphorylated forms, may leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation

DNA-protein interactions, mainly DNA-histone interactions, are thought to play an essential role in the packing mechanisms of chromatin as well as in transcriptional control. In this context it is important to develop new methods to study DNA-protein interactions and structural changes associated with them. Paraformaldehyde (PFA) has been shown to crosslink proteins, mainly histones, to DNA; and in this short study we have used a system with the DNA-binding dye 7-aminoactinomycin D (7-AAMD) as an indirect probe for PFA fixation. The aim was to investigate the dynamics of fixation on the binding of this dye to intact human lymphocytes. The results show a decrease in accessibility of 7-AAMD,initially affecting both the nonspecific binding of 7-AAMD and the high affinity binding sites, and thereafter mainly the high affinity binding sites. We conclude that fixation with PFA is a long-term reaction that requires a fixation time of several hours at a sufficient concentration to be completed. Our findings suggest that staining with a low concentration of 7-AAMD after extensive PFA fixation may be used to obtain information on DNA-protein interactions in intact cells.

Histone H1 interphase phosphorylation becomes largely established in G1 or early S phase and differs in G1 between T-lymphoblastoid cells and normal T cells

BackgroundHistone 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 G1. 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 G1, S and G2/M populations.ResultsWe 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 G1 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 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.ConclusionOur data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 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 G1 may be affecting the G1/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.