DAPI

Abstract: Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.

Abstract: DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

Abstract: 4′-6-DIAMIDINO-2-PHENYLINDOLE (DAPI), which was first synthesised by Dann et al.1 as a trypanocide related to Berenil, has been shown to possess useful DNA binding properties2. Thus, DAPI will bind differentially to yeast mitochondrial and nuclear DNA forming highly fluorescent complexes and enhancing the separation of the two DNAs in caesium chloride gradients2. DAPI can also be used as a highly specific fluorescent stain for both nuclear and mitochondrial DNA in yeast. It seems to be highly sensitive and probably permits detection of a single yeast mitochondrial DNA molecule (D.H.W., and D. J. Fennell, unpublished).

Abstract: Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces apoptotic cell death in the myocardium in vivo. Hyperglycemia-induced myocardial apoptosis is mediated, at least in part, by activation of the cytochrome c-activated caspase-3 pathway, which may be triggered by ROS derived from high levels of glucose.