The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Radiation-induced red cell damage: role of reactive oxygen species

BACKGROUND: Cellular blood components are irradiated to prevent graft- versus-host disease in transfusion recipients at risk for this syndrome. Because gamma radiation can result in the production of reactive oxygen species, the role of reactive oxygen species was investigated in radiation-induced red cell damage. STUDY DESIGN AND METHODS: Whole blood from normal donors was exposed to various doses of t-butyl hydroperoxide (0-1 mM) and/or to gamma-radiation (0-50 Gy). Oxidative damage was assessed by the extent of lipid peroxidation (measured by thiobarbituric acid-reactive substances [TBARS]) and hemoglobin oxidation. Fresh blood was divided into three parts-one initially irradiated and stored, another stored with portions irradiated weekly, and a third stored without irradiation. TBARS and hemoglobin oxidation were measured weekly. RESULTS: As expected, t- butyl hydroperoxide induced TBARS formation and hemoglobin oxidation in a dose-dependent fashion. The gamma-radiation not only increased hemoglobin oxidation and TBARS formation, but also enhanced the t-butyl hydroperoxide effect on red cells. Red cell storage increased TBARS generation and hemoglobin oxidation in a time-dependent fashion. When radiation was administered either initially or after weekly storage, TBARS production and hemoglobin oxidation were increased over that measured in unirradiated paired controls. CONCLUSION: Gamma radiation at clinically used doses increases lipid peroxidation and hemoglobin oxidation in human red cells. The effect of gamma-radiation is accentuated by blood storage and induces damage independent of time of storage.     

Somatic mutation processes at a human minisatellite

Germline instability at human minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at minisatellites. An electrophoretic size-enrichment strategy was therefore developed at minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quantitated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over.      

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.