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.     

Regeneration of long spinal axons in the rat

Summary To investigate regeneration of long spinal axons, the right lateral column of the rat spinal cord was cut at high cervical, low cervical, midthoracic or lumbar level, and one end of an autologous sciatic nerve segment was grafted to the spinal cord at the site of incision. Three to six months after operation, the origin of axons in the grafts was traced retrogradely with horseradish peroxidase injected into the grafts and, in some cases, anterogradely with radioautography of tritiated amino acids injected into the brainstem. Axons from each of the major lateral spinal tracts arising in the brainstem as well as axons ascending from the lower spinal cord succeeded in growing into low cervical grafts. However, long descending axons rarely regenerated after midthoracic or lumbar injury; axons ascending from lumbar segments of the spinal cord usually failed to enter high cervical grafts. Differences in axonal regrowth at the four segmental levels were not simply attributable to dwindling of axonal number in fibre tracts. Axonal regeneration from Clarke's column or the red nucleus was observed only with lesions causing atrophy of many neurons.There was no obvious example of a fibre tract in the lateral spinal columns from which axons failed to regenerate nor from which axons regenerated exceptionally well. Under the conditions of these experiments, the distance from cell body to injury appeared to be an important determinant of axonal regeneration.     

Conduction of nervous impulses in spinal roots and peripheral nerves of dystrophic mice

Conduction was studied in the sacral ventral roots and ventral tail nerves of dystrophic mice (dy/dy) and phenotypically normal littermates. In myelinated ventral root fibers of normal mice, conduction velocity was uniform with internodal conduction time45 ± 5 μsec (26 °C). In ventral root fibers of dystrophic mice, conduction velocity was decreased and strikingly non-uniform; both saltatory and continuous conduction were observed in different portions of the same nerve fiber. Continuous conduction with velocity <2 m/sec (26 °C) was characteristically observed in mid-root where the axons are bare; conduction was saltatory close to the exit from the spinal canal and near the spinal cord where the axons are myelinated.Maximum conduction velocity in ventral tail nerves was21 ± 3 m/sec for dystrophic mice and31 ± 4 m/sec for littermate controls (37 °C). Internodal lengths were somewhat decreased in the dystrophic peripheral nerves but there was no significant difference in maximum fiber diameters, myelin thickness or nodal morphology between dystrophic and normal nerves.     

Evaluation of [18F]-JNJ-64326067-AAA tau PET tracer in humans

The [¹⁸F]-JNJ-64326067-AAA ([¹⁸F]-JNJ-067) tau tracer was evaluated in healthy older controls (HCs), mild cognitive impairment (MCI), Alzheimer’s disease (AD), and progressive supranuclear palsy (PSP) participants. Seventeen subjects (4 HCs, 5 MCIs, 5 ADs, and 3 PSPs) received a [¹¹C]-PIB amyloid PET scan, and a tau [¹⁸F]-JNJ-067 PET scan 0-90 minutes post-injection. Only MCIs and ADs were amyloid positive. The simplified reference tissue model, Logan graphical analysis distribution volume ratio, and SUVR were evaluated for quantification. The [¹⁸F]-JNJ-067 tau signal relative to the reference region continued to increase to 90 min, indicating the tracer had not reached steady state. There was no significant difference in any bilateral ROIs for MCIs or PSPs relative to HCs; AD participants showed elevated tracer relative to controls in most cortical ROIs (P < 0.05). Only AD participants showed elevated retention in the entorhinal cortex. There was off-target signal in the putamen, pallidum, thalamus, midbrain, superior cerebellar gray, and white matter. [¹⁸F]-JNJ-067 significantly correlated (p < 0.05) with Mini-Mental State Exam in entorhinal cortex and temporal meta regions. There is clear binding of [¹⁸F]-JNJ-067 in AD participants. Lack of binding in HCs, MCIs and PSPs suggests [¹⁸F]-JNJ-067 may not bind to low levels of AD-related tau or 4 R tau.     

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.     

Review of the evidence on the management of blunt renal trauma in pediatric patients

Due to the size and location within the pediatric patient, the kidneys are susceptible to injury from blunt trauma. While it is clear that the goal of management of blunt renal trauma in children is renal preservation, the methods of achieving this goal have not been well established in the current literature. Therefore, we have set out to summarize and clarify the current published information on the management strategies for blunt renal trauma in children. While there is extensive literature available, it consists mostly of retrospective series documenting widely varied management styles. The purpose of this review is to display the current information available and delineate the role for future studies that may allow us to develop consistent management strategies of pediatric patients, who have sustained blunt renal trauma, in a safe and cost-effective manner.     

Prevalences, genotypes, and risk factors for HIV transmission in South America

HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.     

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.