Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry

Summary Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method).An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescence in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value.The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.Partly supported by Alexander von Humboldt-Stiftung     

Plasma endothelin levels during myocardial ischemia and reperfusion

Endothelin, an endothelium-derived vasoconstrictive peptide, has a strong potency of coronary artery constriction. However, the role of endogeneous endothelin under pathophysiological conditions has not yet been known. In this study, we examined plasma endothelin concentration in dogs with myocardial ischemia and reperfusion. Anesthetized open-chest dogs underwent either 45 minutes occlusion of the left anterior descending coronary artery followed by 3 hours reperfusion, or 4-10 hours of continuous occlusion. Plasma concentration of endothelin from the central vein was measured by the highly sensitive enzyme-immunoassay. Plasma endothelin concentration increased 2.2-fold with the peak level at 60 minutes after release of the ligated artery, but occlusion per se caused no remarkable change. These data suggest that reperfusion of the occluded artery might be needed to increase the plasma concentration of endothelin in case of myocardial infarction.     

Molecular cloning, genetic mapping, and expression of the mouse Sf3b1 (SAP155) gene for the U2 snRNP component of spliceosome

SAP155, a subunit of the U2 snRNP, is essential for prespliceosome assembly and splicing catalysis of the major spliceosome. Moreover, the protein has been identified in the minor (U12-dependent) spliceosome. These facts strongly suggest that SAP155 is shared by two distinct complexes owing to its importance in the removal of any type of intron. Here we have isolated a cDNA encoding the 146-kDa mouse homolog, designated Sf3b1. The amino acid sequence of Sf3b1 is very highly conserved among homologs from Schizosaccharomyces pombe (52.4% identity) to human (99.6%), and the C-terminal 825 residues of these Sf3b1 homologs show even higher identities. This C-terminal region shows significant similarity to the PR65 subunit of protein phosphatase 2A, which is composed of 15 tandem repeats of a 39 amino acid sequence. Mouse genome analyses showed Sf3b1 to be a single-copy gene mapping to the central part of Chromosome (Chr) 1. Northern blot analysis and whole mount in situ hybridization revealed Sf3b1 to be ubiquitously expressed in a variety of adult tissues and mid-gestation embryos. Received: 14 June 2000 / Accepted: 19 October 2000