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.

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.

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.

Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe.

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.

DNA binding fluorochromes as probes for histone H1-chromatin interactions in situ.

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 microM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HC1 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.

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.

High affinity binding of 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole to human neutrophilic granulocytes and lymphocytes.

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 method 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.

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.

Equilibrium binding of DAPI and 7-aminoactinomycin D to chromatin of cultured cells.

The binding of 4',6-diamidino-2-phenylindole (DAPI) and 7-aminoactinomycin D (7-AAMD) to chromatin of intact cultured fibroblast nuclei was studied by cytofluorometry. Staining was performed at equilibrium at varying dye concentrations. By using large volumes of dye solutions, the free dye concentration became approximately equal to total dye concentration, and the estimation of affinity and number of binding sites was performed by Scatchard analysis. Since all Scatchard plots showed a definite curvature, indicating more than one class of binding sites, a two component regression was performed, although only the highest affinity binding sites were determined. The observed affinity constants did not change substantially between different preparational procedures for any of the dyes. The number of DAPI binding sites increased about 30% after detergent extraction. No such increase was observed for 7-AAMD. Fixation in formaldehyde, instead of ethanol, after detergent extraction had no effect on DAPI binding, but decreased the number of 7-AAMD binding sites with about 50%. Extraction of basic proteins with HCl resulted in about 100% increase of the number of binding sites for both dyes. It should be feasible to use this technique as an assay for the identification of proteins or other nuclear components important for the maintenance of chromatin structure.

The use of acridine orange cytofluorometry in the study of macrophage lysosomal exocytosis.

We present a rather simple cytofluorometric technique for the study of exocytosis of lysosomal contents from individual cultured cells. It is based on the use of the lysosomotropic weak base acridine orange (AO) which, in its stacked form, as it occurs within lysosomes, emits red fluorescence when excited by blue light. Mouse peritoneal macrophages were cultured for 48 h and, after 2 h in serum-free medium, stained with AO. The cells were then exposed to F10-medium with or without newborn calf serum (NCS), zymosan A (Z) or cytochalasin B (CB) for different times at 20 or 37 degrees C. After staining, the macrophages showed no change in red fluorescence intensity, if stored at room temperature in the dark. If, however, the cells were kept in the incubator at 37 degrees C, the cells showed slightly decreasing red fluorescence intensity with time. This decrease was markedly potentiated by the presence of NCS, Z or CB, which are known to induce secretion of lysosomal enzymes from macrophages in vitro. Selective lysosomal enzyme release was confirmed biochemically during treatment with zymosan A. The technique presented here may be of value in further studies on the stimulation of, and the mechanisms behind, lysosomal exocytosis in cultured cells.

Estimation of pH in individual alveolar macrophage phagolysosomes.

A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions.

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.

Increased breakdown of phosphatidylinositol 4,5-bisphosphate is not an initiating factor for actin assembly in human neutrophils.

The present study examined the possible role of increased phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) breakdown in the regulation of actin assembly in human neutrophils. Tetracaine, a local anesthetic, was used since it has recently been proposed to inhibit the phosphorylation of phosphatidylinositol 4-phosphate to form PtdIns(4,5)P2. Surprisingly, it was found that incubation with tetracaine alone increased the breakdown of PtdIns(4,5)P2, measured as total inositol trisphosphate formation. This occurred without any rise above basal in the cellular content of filamentous actin. However, in the presence of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), tetracaine potentiated the chemotactic-induced increase of both inositol trisphosphate formation and actin polymerization. To further explore the relationship between increased PtdIns(4,5)P2 breakdown and actin polymerization, the activity of phospholipase C was depressed by lowering the cytosolic free calcium ion level or by incubating the cells with ionomycin. In these cells, fMet-Leu-Phe stimulation still raised the cellular content of filamentous actin to a level similar to levels in nontreated cells, despite the absence of PtdIns(4,5)P2 hydrolysis. Consequently, increased breakdown of PtdIns(4,5)P2 alone is not enough to initiate actin polymerization, nor is the polymerization of actin dependent on an increased PtdIns(4,5)P2 breakdown. However, we cannot exclude the possibility that increased turnover of phosphoinositides might act as a modulator of actin assembly.

Use of cytofluorometry to evaluate binding of antibodies to the cytoskeleton of cultured cells.

Immunocytochemistry is routinely used to examine the occurrence and distribution of cytoskeletal proteins in cells, but the results are usually evaluated visually and subjectively. Little use has been made of the potential the method offers for quantitative work. Here we report on application of cytofluorometry to quantify binding of antibodies to the cytoskeleton of U-251 MG human malignant glioma cells in culture. The results show that cytofluorometry is a simple and reliable procedure for: (a) determining the optimal concentrations of primary and secondary antibodies and other labeling reagents; (b) evaluating the binding specificity of commercial secondary antisera; and (c) evaluating the effect of different preparatory procedures on preservation of and binding of antibodies to cytoskeletal structures. Experiments with a monoclonal antibody to tubulin show that preservation of tubulin is very sensitive to the preparatory procedures used. Maximum labeling of tubulin in intact cells was obtained when the cells were pre-fixed with formaldehyde before permeabilization with solvent. Maximum labeling of tubulin in Triton-extracted cytoskeletons was achieved by pre-fixing the cells with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate), extracting with Triton in a microtubule-stabilizing buffer, and post-fixing with formaldehyde. GTP was not required to preserve tubulin in cytoskeletons.

Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt.

We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.

Flow cytofluorometry of lysosomal acridine orange uptake by living cultured cells. Effect of trypsinization and starvation.

The uptake of the fluorescent, lysosomotropic weak base acridine orange (AO) by living cells in culture was studied by flow cytofluorometry. A mouse myeloma cell line (SP 2/0), growing in suspension, and an anchorage-dependent human malignant glioma cell line (U-251 MG), brought into suspension by trypsinization, were used. The consequences of trypsinization were also studied using static cytofluorometry. The lysosomal accumulation of AO by myeloma cells growing in suspension was found to be only moderately affected by starvation (i.e. incubation without medium change) for a period of up to five days. Trypsinization of the glioma cells after staining with AO caused pronounced release of the fluorescent dye while trypsinization before staining with AO did not significantly change the average lysosomal concentration of AO. We did, however, notice certain side effects of trypsinization in the form of both increased cellular green fluorescence and greater intercellular variability that reduce the validity of data obtained from cells detached by routine trypsinization. In conclusion, the condition of the lysosomal vacuome of living cultured cells growing in suspension may be studied by flow cytofluorometry after vital staining with the lysosomotropic weak base AO. Anchorage-dependent trypsinized cells, however, yield unsatisfactory results when examined in a flow cytofluorometer system and are better studied while still attached to their substratum, using static cytofluorometry.

DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study.

Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease. In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974. Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch. Thirty-micron sections were cut from each plug for dewaxing and disintegration. The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry. In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern. No apparent correlation was found between the histopathological grade and the DNA ploidy. Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy. The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.

Sciatic nerve blood flow measured by laser Doppler flowmetry and [14C]iodoantipyrine.

Blood flow was examined in sciatic nerves of pentobarbital-anesthetized rats by means of laser Doppler flowmetry (LDF) and intravenous [14C]iodoantipyrine infusion. Continuous LDF signals demonstrated slow oscillations and acute, pressure-related changes in flow. The steady-state LDF signal was related linearly to nerve blood flow, as measured with [14C]iodoantipyrine, in intact nerves and nerves stripped of the epineurium. In 14 intact nerves, nerve blood flow averaged 0.27 +/- 0.03 (SE) ml X min-1 X g-1, whereas it averaged 0.13 +/- 0.01 in 5 stripped nerves. Autoradiographs of [3H]-nicotine-infused nerves and intra-arterial injection of 57Co-labeled microspheres demonstrated that flow was not uniform throughout the nerve cross section. The results indicate that LDF can be used to examine nerve blood flow in vivo, demonstrate a linear relation between the LDF signal and flow, and establish absolute values for blood flow in intact and stripped nerves of the anesthetized rat.

Cytofluorometric quantitation of acridine orange uptake by cultured cells.

The vacuolar accumulation of the lysosomotropic weak base acridine orange (AO) within living cells in culture was studied by cytofluorometry. Mouse peritoneal macrophages, malignant human glioma cells, and normal human glial cells were utilized. Exposure to AO resulted in granular bright red fluorescence, as well as a diffuse weak green background fluorescence. To obtain reproducible "staining" conditions, the red granular fluorescence was measured as a function of dye concentration and staining time. Exposure to high concentrations of AO (greater than 10 micrograms/ml) was found to cause cell damage in combination with markedly changed fluorescence distribution for the cell population with reduced mean fluorescence and increased variability. Granular uptake of AO was pH-dependent and almost zero at pH 5.5. AO fluorescence, as measured by cytofluorometry, was found to be roughly linear to the amount of AO present in the cells, as measured by spectrofluorometry after cell solubilization, indicating negligible fluorescence quenching. AO labelling of living cells might serve as a useful indicator of the condition of the cellular vacuolar (lysosomal) apparatus.