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.     

Postanoxic Myoclonus: Two Case Presentations and Review of Medical Management

Postanoxic myoclonus is a rare manifestation after an anoxic event, with fewer than 150 cases reported in the literature. The condition is characterized by myoclonic jerks, which are worse on action than at rest, and postural lapses, ataxia, and dysarthria. The disability caused by postanoxic myoclonus can be profound, and treatment in the rehabilitation setting is exceptionally challenging. We present 2 patients who suffered from postanoxic myoclonus after an anoxic event, both of whom were successfully treated with a combination of levetiracetam, valproic acid, and clonazepam. These cases act as a framework for discussing the management of postanoxic myoclonus in the clinical setting.     

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%.      

Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.      

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media

Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF. Thus, effective supplementation of media with PAF requires inactivation of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera. When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10%. The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined. A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid- treatment protocol. The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.      

Hydrosalpinges adversely affect markers of endometrial receptivity

While in-vitro fertilization (IVF) was initially developed in women with tubal factor infertility, recent clinical studies have suggested that the presence of hydrosalpinges lowers implantation and pregnancy rates. We postulated that these hydrosalpinges cause impaired endometrial receptivity. A total of 103 women with hydrosalpinges were prospectively evaluated, and compared with 55 infertile and 44 fertile controls. All women had endometrial biopsies during the window of implantation, analysed by conventional histological criteria, and also stained for three integrin markers of endometrial receptivity (alpha1beta1, alpha4beta1 and alpha vbeta3). Women with hydrosalpinges (cases) expressed significantly less of the alpha vbeta3 integrin compared with controls. There was no difference in expression of alpha1beta1 or alpha4beta1 among groups. A significantly greater number of cases had out of phase histology and missing alpha vbeta3 (type I defects) and absent integrin expression despite normal histological maturation (type II) defects, compared with controls. Of 20 women with impaired endometrial receptivity who were also biopsied after hydrosalpinx surgery, 70% demonstrated increased alpha vbeta3 expression. Seventy-seven percent of type I and 57% of type II defects were corrected postoperatively. Using markers of endometrial receptivity, this study demonstrates that inflammatory hydrosalpinges have an adverse effect on endometrial receptivity, which in some cases may be overcome by surgical treatment of the hydrosalpinx.