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.     

A longitudinal study of otitis media with effusion among 2- to 5-year-old African-American children in child care

OBJECTIVE: To prospectively document the prevalence of otitis media with effusion (OME) in 86 African-American children between ages 2 and 5 years. STUDY DESIGN: Eighty-six children in center-based child care whose ear status had been followed from infancy continued to be observed. Middle ear status was assessed by pneumatic otoscopy and tympanometry biweekly. RESULTS: The prevalence of OME decreased as children became older. The mean proportion of examinations demonstrating bilateral OME (BOME) ranged from 12% between 24 to 30 months to 4% between 54 to 60 months of age. The mean proportion of exams revealing bilateral normal ears increased from 77% at 24 to 30 months to 88% at 54 to 60 months of age. Although 60 children had experienced BOME that lasted 4 months or longer in the 6- to 24-month age period, only 8 of these children experienced at least 4 months of continuous BOME between 24 to 60 months. CONCLUSIONS: The proportion of time with BOME decreased progressively with increasing age in this population. Only 8 of 60 children who had experienced more than 4 consecutive months of BOME before 2 years of age continued to manifest persistent effusion or experience recurrences of prolonged BOME after 2 years of age.     

Transforming growth factor beta-1 (TGFB1) and peak bone mass: association between intragenic polymorphisms and quantitative ultrasound of the heel

Background  

Variance of peak bone mass has a substantial genetic component, as has been shown with twin studies examining quantitative measures such as bone mineral density (BMD) and quantitative ultrasound (QUS). Evidence implicating single nucleotide polymorphisms (SNPs) of the transforming growth factor beta-1 (TGFB1) gene is steadily accumulating. However, a comprehensive look at multiple SNPs at this locus for their association with indices of peak bone mass has not been reported.     

The influence of bright light during the daytime upon circadian rhythm of core temperature and its implications for nocturnal sleep

Abstract The purpose of the present study was to investigate how bright light during the daytime could influence circadian rhythms of core temperature and nocturnal sleep. Seven females (age 20 ± 2 years) served as participants. The participants lived in the experimental unit for 4 days and were exposed to either 6000 lx (bright) or 200 lx (dim) light during the daytime. Rectal temperature (T_re) was measured during the experimental period. Subjective alertness was measured by the Kansei-gakuin Sleeping Scale five times a day. The minimum T_re was significantly lower after bright exposure ( P < 0.05). The T_re fell rapidly after bright exposure before they retired ( P < 0.05) and increased more rapidly during bright light after they woke up ( P < 0.05). The morning wakefulness under bright exposure was more active than under dim exposure ( P < 0.05). The melatonin secretion at wake up during bright exposure was significantly lower than during dim exposure ( P < 0.05). Exposure to bright light during daytime lowered the nocturnal level of T_re, its evening fall was faster and the morning rise quicker. This suggests that indoor light during daytime should be bright enough to promote healthy sleep at night.     

Scales for the measurement of attitudes toward blood donation

Attitudes toward blood donation are frequently assumed to vary along a single dimension from unfavorable to favorable. In contrast, theories of attitude structure specify three distinct attitude components: affect, cognition, and behavior. This article describes the development of three new scales for the measurement of affective, cognitive, and behavioral components of attitudes toward blood donation. The scales were developed using the method of equal-appearing intervals and were administered to both donors of blood and nondonors. Correlations among the scales were relatively small and supported the three-component distinction. Affect was more strongly correlated with the number of prior donations than was cognition, which suggested an important role for emotional factors in blood donation. Scores on all three scales showed the attitudes of blood donors to be more favorable than those of nondonors.     

Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions

Autosomal dominant cerebellar ataxia with progressive macular degeneration is caused by a CAG/glutamine repeat expansion in the SCA7 gene/protein. Neuronal intranuclear inclusions were detected in the brain of an early onset SCA7 case with the 1C2 antibody directed against an expanded polyglutamine domain. Nuclear inclusions were most frequent in the inferior olivary complex, a site of severe neuronal loss in SCA7. They were also observed in other brain regions, including the cerebral cortex, not considered to be affected in the disease. Using confocal microscopy we showed that some inclusions were ubiquitinated, but to varying degrees, ranging from <1% in the cerebral cortex to 60% in the inferior olive. In addition, we also observed cytoplasmic staining using the 1C2 antibody, particularly in the supramarginal gyrus, the hippocampus, the thalamus, the lateral geniculate body and the pontine nuclei. These data confirm that the presence of intranuclear inclusions in neurons is a common characteristic of disorders caused by CAG/polyglutamine expansions, but unlike what has been reported for Huntington's disease, SCA1 and SCA3/MJD, in SCA7 the inclusions were not restricted to the sites of severe neuronal loss.      

Increased salt retention and hypertension from non-steroidal agents in the elderly

We studied blood pressure and natriuretic responses to acute salt loading, and the effect of non-steroidal anti-inflammatory agents on these responses, in five healthy normotensive women aged 65 to 71 years. Five women aged 25 to 31 years acted as controls. Intravenous saline loading, with and without prior ingestion of ibuprofen, was 15 ml/kg/h for 3 h. Baseline blood pressures were higher in the elderly. Saline infusion without ibuprofen raised systolic blood pressure (SBP) by about 25 mmHg in the older group only. Ibuprofen increased baseline SBP in the elderly (129 +/- 6 vs. 116 +/- 5 mmHg, p < 0.05). Saline loading after ibuprofen again raised blood pressure by about 25 mmHg in the elderly only. The elderly group showed markedly increased sodium excretion during saline loading, but this was reduced by ibuprofen. Ibuprofen had no effect on SBP or sodium excretion in controls. Ageing appears to increase susceptibility to salt retention and hypertension from non-steroidal anti-inflammatory agents.      

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.