Biomedical subjects
Publications and source records attributed to T Stokke.
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The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).
This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.
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We studied the chromatin structure of rat thymocytes fixed in 70% ethanol at 0-44 degrees C by flow cytometry and gel electrophoresis. The fluorescence of the DNA-specific dye mithramycin increased by 93% when thymocytes were exposed at 44 degrees C in the fixative compared to cells kept at 0 degrees C. Antibody labeling (X-ANA) of the core histones was 65% lower for the 44 degrees C-treated cells compared to the control cells (0 degree C). The emission anisotropies of the DNA-specific dye Hoechst 33258 bound to chromatin were 0.341 and 0.318 for thymocytes fixed at 0 degree C and 44 degrees C, respectively. Increased mobility of DNA in chromatin of 44 degrees C-treated cells, as revealed by the emission anisotropy of Hoechst 33258, was not due to denaturation of DNA but was probably caused by removal of constraints situated at short intervals (less than or equal to 50 BP) along the DNA helix. The short intervals between these constraints in chromatin fixed at 0 degree C suggests that they were histones. PAGE of 0.5 N H2SO4-extracted histones showed that the 44 degrees C treatment reduced total core histone content by 65% and that the different histones were lost in unequal amounts. The loss was about 75% and 54% for the histone pairs H3/H4 and H2A/H2B, respectively. The amount of H1 was reduced by about 25% on temperature treatment. The temperature-induced change in the chromatin structure of the cells in 70% ethanol was biphasic. A change in the three-dimensional structure of chromatin occurred for temperatures up to 20 degrees C (no histones were released but binding of mithramycin increased by approximately 15%, whereas the binding of X-ANA decreased by the same amount). Sixty-five percent of core histones were released in the second phase (20-44 degrees C), which may explain the further increase and decrease in the binding of mithramycin and X-ANA, respectively.
Binding of the DNA-specific dye 7-aminoactinomycin D (7-AMD) in chromatin of human leucocytes was studied by flow cytometry. After formaldehyde fixation and permeabilization, monocytes bound 30-130% more 7-AMD than lymphocytes, while binding in granulocytes was 20-60% higher than in lymphocytes. Monocytes and lymphocytes bound similar amounts of 7-AMD when cells were permeabilized by detergent prior to fixation. Digestion of DNA in formaldehyde-fixed chromatin by DNase 1 was quantitated by measuring Hoechst 33258 (H33258) fluorescence of mononuclear cells. The monocyte/lymphocyte H33258 fluorescence ratio decreased with DNase 1 digestion to an asymptotic value of 0.74, showing that DNA in chromatin of monocytes was more susceptible to DNase 1 digestion. 7-AMD binding increased, reached a maximum and then decreased with extent of DNase 1 digestion in both mononuclear cell types. The monocyte/lymphocyte 7-AMD fluorescence ratio also decreased after DNase 1 digestion. RNA content and RNA synthesis were higher in monocytes than in lymphocytes. The results show that 7-AMD binding in chromatin of mononuclear leucocytes correlates with transcriptional activity as measured by DNase1 susceptibility and RNA synthesis. The staining procedure may be used for differential counting of mature myeloid cells in peripheral blood and bone marrow.
Complement activation was evaluated by assay of plasma C3dg and the terminal complement complex (TCC) in 19 patients with multiple injuries. In the nine patients with thoracic involvement, statistically significant increase of plasma TCC was found at first sampling (average 90 min post-trauma), and of C3dg after 24 hours. Such increase was not found in the ten patients without thoracic involvement. Heightened granulocyte elastase activity was found in bronchial lavage fluid 90 min after the trauma in three patients with thoracic injury. Pulmonary insufficiency (pO2/FiO2 less than 16 kPa on intermittent positive-pressure ventilation) arose in four patients. All four had raised plasma levels of TCC or C3dg on arrival at the hospital. Six patients with complement activation did not show pulmonary insufficiency. Although the series was relatively small, the results indicate that thoracic injury is particularly associated with complement activation, and that complement activation alone does not suffice to produce post-traumatic pulmonary insufficiency.
Fluorescence spectra of Hoechst 33258 bound to rat thymocytes were measured by flow cytometry. At low dye concentrations (less than or equal to 2 micrograms/ml) the fluorescence maximum was situated at 460 nm irrespective of solvent composition. With higher dye concentrations the fluorescence maximum was shifted upwards, the intensity decreased and the width of the fluorescence peak increased. Linear combinations of a spectrum obtained at a low dye concentration (0.5 microgram/ml, type 1 binding) and one obtained at a high dye concentration (42.4 micrograms/ml, type 2 binding) failed to reproduce spectra measured at intermediate dye concentrations (0.15 M NaCl). Hence, Hoechst 33258 forms at least three different fluorescing complexes with DNA in chromatin. The shift in the fluorescence maximum of the Hoechst 33258/chromatin complex towards higher wavelengths decreased with ionic strength. 25% ethanol in the 0.15 M NaCl staining buffer reduced the wavelength shift at high dye concentrations, indicating that the strength of type 2 binding depends on DNA conformation in addition to ionic strength. The fluorescence spectrum was independent of whether DNA in chromatin was complexed with histones or not. However, histone-depleted thymocytes fluoresced more intensely than cells in which DNA was complexed with histones, the difference being greater at low concentrations of Hoechst 33258. Hence, type 2 binding to DNA in chromatin appears to be less restricted by histones than type 1 binding.
Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.
The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 10(7) M-1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 less than or equal to bound dye/phosphate less than or equal to 0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (greater than or equal to 15 degrees) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (greater than or equal to 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient approximately 2) and the apparent binding constant was 2-3 X 10(5) M-1 as determined from quenching and precipitation data.(ABSTRACT TRUNCATED AT 250 WORDS)
In order to investigate the acute effects on lung morphology, lung function, hemodynamic and blood coagulation system, elastase (330 units (U) kg-1 h-1) was continuously infused into 16 anesthetized and mechanically ventilated minipigs. Elastase infusion induced a disturbance of blood coagulation leading to hypocoagulability, a pulmonary leukostasis, interstitial edema, a progressive respiratory failure with prompt increase in pulmonary vascular resistance, decrease in systemic vascular resistance, increased venous admixture, and increased dead space ventilation. Agranulocytosis prevented interstitial edema but not disturbances in pulmonary or hemodynamic function or hypocoagulability. The results clearly indicate that elastase may be involved in the pathophysiology of acute lung failure and defects in the blood coagulation system.
Measurements of pulmonary capillary blood flow by a nitrous oxide rebreathing technique (QN2O) were performed in 21 anesthetized and artificially ventilated minipigs with normal lungs and in nine minipigs with thrombin-induced (75-150 U kg-1 h-1) lung pathology. QN2O was calculated with the Hook-Meyer-formula and compared to cardiac output measurements (thermodilution, QT, or direct Fick's principle, QFick). The coefficient of variation in double QN2O measurements was 0.05. If the tidal volume to dead space ration (VD/VT) is normal, the nitrous oxide method works well, but when the efficacy of ventilation worsens, this gas uptake method fails to detect the circulation of the poorly ventilated parts of the lung. The mean ratio QN2O/QFick in pigs with normal lungs (58 measurements) was 1.00 +/- 0.10 (mean +/- s.d.). During thrombin infusion, the mean ratio QN2O/QT was 0.84 +/- 0.17 (n = 49). After corrections for shunt perfusion (Qs), the mean ration QN2O/(QT-Qs) was 0.89 +/- 0.17 (n = 49). QN2O/QT-Qs) decreased with increasing VD/VT. In measurements during thrombin infusion with VD/VT less than 0.33, the mean ratio QN2O/(QT-Qs) was 0.97 +/- 0.11 (n = 21), with a VD/VT between 0.33 and 0.44, the mean ratio QN2O (QT-QS) was 0.90 +/- 0.08 (n = 20), and with a VD/VT greater than or equal to 0.45, this ratio was 0.62 +/- 0.18 (n = 8). In the presence of only moderate functional inhomogeneities, this noninvasive rebreathing method will offer reliable data on pulmonary perfusion.
The decay of fluorescence of Tb3+ bound to DNA was measured in the absence and presence of adriamycin and actinomycin D. The decay for Tb3+ bound to DNA was mainly exponential (lifetime: tau = 0.96 ms). In the presence of adriamycin or actinomycin D, the Tb3+ fluorescence decayed much faster, indicating that excitation energy was transferred from Tb3+ to the drugs. Extrapolation of the decay curves to zero time showed that the number of strongly emitting, DNA-bound terbium ions was not reduced by the presence of adriamycin or actinomycin D. Hence, these drugs do not seem to displace Tb3+ bound to DNA.
The potential role of granulocyte proteinases on experimentally induced ARDS was evaluated. In order to investigate the acute effects on lung function, elastase (330 U kg-1h-1) or thrombin (75-150 U kg-1 h-1) was continuously infused into anaesthetized and mechanically ventilated mini pigs. Both elastase as well as thrombin induced a progressive respiratory failure with prompt increase in pulmonary vascular resistance, and decrease of cardiac output, further a pulmonary leukostasis, and a disturbance of blood coagulation leading to hypocoagulability. High proteolytic activity selectively in the lung indicates a possible role of proteinases released from sequestered polymorphonuclear neutrophils. Similar results following elastase infusion were however obtained in leukopenic animals pretreated with a single dose of dimethylmyleran (5 mg kg-1) which depleted the granulocytes totally. These results offer the possibility that elastase itself may cause respiratory failure and lung tissue damage. On the other hand the digestion pattern of phosphorylase kinase by lung tissue homogenates of thrombin- or elastase-infused mini pigs clearly indicates that elastase is only one of several mediators which may cause experimentally induced ARDS even in the absence of granulocytes.
Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.
Among annually 10 treatments with continuous arteriovenous haemofiltration (CAVH) during the last years there were some individual instances of severe complications: occluding thrombus in the femoral artery, infection at the site where the arterial catheter was inserted and disconnection in the extracorporeal shunt. When choosing the method for compensating renal failure, the possibility of these complications must be considered. Patients who will tolerate intermittent hyperhydration between two dialysis sessions should be treated discontinuously with a technique not-employing arterial cannulation rather than with CAVH.
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We have reinvestigated the problem of blood-gas equilibration of CO2 in lungs during hypercapnia. Six dogs with chronic tracheostomy and exteriorized carotid artery were subjected in acute experiments to hypercapnic inspired mixtures [CO2 fraction of expired gas (FICO2) = 0.06; 0.08; 0.10]. Expired CO2 partial pressure (PCO2) was continuously measured with a respiratory mass spectrometer and compared with arterial PCO2 determined in blood samples that were collected during apparent steady-state conditions. Particular care was taken in using continuously recorded temperature in the right heart for correction of blood PCO2 measured by CO2 electrodes. In no animal was there a significant difference between arterial and alveolar PCO2. On the average, this difference was -0.1 Torr at FICO2 = 0.06; 0.0 Torr at FICO2 = 0.08, and -0.2 Torr at FICO2 = 0.10. The results are in agreement with the conventional view that PCO2 in pulmonary capillary blood approaches PCO2 in alveolar gas.