Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites.

We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long-term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon-gamma and interleukin-5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release.     

The effects of exogenous histamine in isolated rat hearts

The role of histamine in cardiac physiology and pathophysiology is not clarified, but is dependent on species. The effects of exogenous histamine in Langendorff-perfused rat hearts were investigated. 1 mM, 100, 10, 1 and 0.1 M of histamine (n=7 each) as 15 min infusions were employed in a dose-response study, and compared to control perfused hearts (n=7). In another experimental series, 100 M histamine (n=15) was added during reperfusion after 25 min global ischemia, and compared to control ischemia-reperfusion (n=15). The maximal response to histamine in the dose-response study (100 M) was an increase of left ventricular developed pressure to 126±8% of initial value (mean±SEM, p<0.04), and increase of coronary flow to 152+6% (p<0.02) after 5 min infusion. 100 M histamine did not significantly influence heart rate or rhythm. The lowest concentration (0.1 M) did not have effects cardiac performance. Reperfusion with histamine for 2 min after ischemia reduced left ventricular developed pressure to 68±10% of initial value versus 116+17% in ischemic controls (p<0.05), and increased left ventricular end-diastolic pressure to 24±8 mmHg compared to 6±2 mmHg in controls (p<0.04). Left ventricular pressures were similar in hearts reperfused with histamine and in ischemic controls for the rest of the observation. Coronary flow increased during reperfusion in hearts given histamine. Histamine had a dose-dependent positive inotropic and vasodilatory effect in isolated rat hearts. Exogenous histamine had only minor effects on post-ischemic cardiac function.     

Effects of a Novel, Low-Molecular Weight Inhibitor of Lipid Peroxidation on Ischemia–Reperfusion Injury in Isolated Rat Hearts and in Cultured Cardiomyocytes

We investigated the effect of H290/51, a novel, low-molecular-weight inhibitor of lipid peroxidation, on cardiac ischemia–reperfusion injury. Lactate dehydrogenase (LD) release from cultured cardiomyocytes exposed to 1 h hypoxia and 4 h reoxygenation was measured after pretreatment with different concentrations of H290/51. In another series, Langendorff-perfused rat hearts were exposed to 30 min global ischemia and 60 min reperfusion (n = minimum 10 in each group): 1. Control ischemia–reperfusion. 2. Vehicle throughout the experiment. 3. Vehicle during stabilization, and H290/51 (10⁻⁶ mol/l) during reperfusion. 4. H290/51 throughout the experiments. During reoxygenation of isolated cardiomyocytes, H290/51 dose dependently inhibited LD release with an pIC₅₀ value of 7.2 ± 0.4 (mean ± SEM), with 10⁻⁶ mol/l as the lowest efficient concentration. In isolated hearts ischemia–reperfusion induced severe reperfusion arrhythmias, reduced left ventricular developed pressure (LVDP) and coronary flow (CF), and increased LV end-diastolic pressure (LVEDP). LD activity in the effluent increased. H290/51 throughout perfusion (group 4) reduced the occurrence of severe reperfusion arrhythmias (p < .0001), attenuated the decrease of LVDP (p < .008), and CF (p < .006), the increase of LVEDP (p < .008), and the release of LD (p < .002). Tissue contents of thiobarbituric acid-reactive substances did not increase during reperfusion in controls, but was reduced in group 4 (p < .004). H290/51 given only during reperfusion (group 3) tended to improve cardiac function, but significantly so only for increase of CF (p < .01). The lipid peroxidation inhibitor H290/51 attenuated cardiac injury induced by ischemia–reperfusion.     

Cell cycle-specific expression and nuclear binding of DNA polymerase alpha.

The expression and distribution of DNA polymerase alpha was measured by cytometry and confocal laser scanning microscopy. Expression was proportional to DNA content in proliferating cells, while only S-phase cells retained DNA polymerase alpha after detergent extraction. Nuclear DNA polymerase alpha binding may be one of the key events of S-phase entry.     

Establishment in monolayer culture and characterization of four human melanoma cell lines

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (T_c) during exponential growth ranged from 15 to 21 h. The differences among the lines in T_c were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90–100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

An electrophoretic device concentrating charged macromolecules to a predetermined final solution volume

An apparatus for electrophoretic concentration of charged macromolecules to a predetermined final solution volume has been developed. The concentration process has a yield of near 100%, which implies that it is possible to predetermine the final macromolecule concentration as well. Both the final macromolecule solution volume and concentration are nearly independent of the electrophoresis time when it exceeds a certain minimum value. The electric field strength across the boundary containing the concentrated macromolecule solution is very low. This considerably reduces macromolecule aggregation, adsorption, and denaturation at this boundary compared to conventional electrophoretic concentrator designs. Both one-stage and two-stage versions of the apparatus have been developed. The one-stage version easily yields a 10-fold and the two-stage version a 50-fold concentration of the macromolecules. Typical macromolecule solution start volumes are 20-50 ml.     

Neither adriamycin nor actinomycin D displaces Tb3+ from DNA

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.