ABSTRACT
Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.
Subject(s)
Apoptosis , Chromosome Breakage , Molecular Biology/methods , Nucleic Acid Conformation , Oligonucleotide Probes , Animals , Biotin/analysis , Child, Preschool , Humans , Kidney/chemistry , Male , Microscopy, Confocal , Rats , Rats, Sprague-Dawley , Thymus Gland/chemistry , Wilms Tumor/geneticsABSTRACT
In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.
Subject(s)
Adamantane/analogs & derivatives , DNA Probes/analysis , Digoxigenin/analysis , Adamantane/metabolism , Alkaline Phosphatase/metabolism , Animals , Cattle , Luminescent Measurements , RNA, Complementary/analysis , Steroid 17-alpha-Hydroxylase/genetics , Time FactorsABSTRACT
Changes in the content of lipid peroxidation (LP) products and activities of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in myocardium of rats after experimental infarction as well as after pretreatment with antioxidant ionol, beta-adrenoblocker inderal and verapamil, an inhibitor of slow Ca2+-channels have been studied. In the left ventricles of the control animals decreased levels of LP-products (Schiff bases and lipid hydroperoxides) have been registered as compared with right ventricles, accompanied by increased activity of antioxidant enzymes in the left ventricles. In experimental infarction the level of LP products increases and activity of antioxidant enzymes decreases both in ischemic and nonischemic regions of the heart. In nonischemic zone these changes can be prevented by pretreatment with inderal and ionol but not with verapamil.
Subject(s)
Lipid Peroxides/metabolism , Myocardial Infarction/metabolism , Myocardium/metabolism , Animals , Butylated Hydroxytoluene/therapeutic use , Catalase/metabolism , Drug Evaluation, Preclinical , Glutathione Peroxidase/metabolism , Male , Myocardial Infarction/drug therapy , Oxidation-Reduction/drug effects , Propranolol/therapeutic use , Rats , Superoxide Dismutase/metabolism , Verapamil/therapeutic useABSTRACT
Rats with experimental myocardial infarction demonstrated decrease in the activity of superoxide dismutase and catalase and increase in the content of lipid peroxidation (LPO) products and Schiff bases both in and outside the area of necrosis. The combined ischemic damage and hyperbaric oxygenation resulted in the over additive effect of accumulation of LPO products in and outside the area of infarction. The data suggest that it is desirable to use antioxidants during hyperbaric oxygenation.