Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

Ethical and legal dilemmas arising during predictive testing for adult-onset disease: the experience of Huntington disease.

The goal of predictive testing is to modify the risk for currently healthy individuals to develop a genetic disease in the future. Such testing using polymorphic DNA markers has had major application in Huntington disease. The Canadian Collaborative Study of Predictive Testing for Huntington Disease has been guided by major principles of medical ethics, including autonomy, beneficence, confidentiality, and justice. Numerous ethical and legal dilemmas have arisen in this program, challenging these principles and occasionally casting them into conflict. The present report describes these dilemmas and offers our approach to resolving them. These issues will have relevance to predictive-testing programs for other adult-onset disorders.     

EDTA chelation therapy for cardiovascular disease: a systematic review

Background

Experimental studies support an important role for endothelial nitric oxide synthase (eNOS) in the regulation of angiogenesis. In humans, a common polymorphism exists in the eNOS gene that results in the conversion of glutamate to aspartate for codon 298. In vitro and in vivo studies have suggested a decreased NOS activity in patients with the Asp²⁹⁸ variant. We hypothesized that a genetic-mediated decreased eNOS activity may limit collateral development in patients with chronic coronary occlusions.

Methods

We selected 291 consecutive patients who underwent coronary angiography and who had at least one chronic (>15 days) total coronary occlusion. Collateral development was graded angiographically using two different methods: the collateral flow grade and the recipient filling grade. Genomic DNA was extracted from white blood cells and genotyping was performed using previously published techniques.

Results

Collateral development was lower in patients carrying the Asp²⁹⁸ variant than in Glu-Glu homozygotes (collateral flow grade: 2.64 ± 0.08 and 2.89 ± 0.08, respectively, p = 0.04; recipient filling grade: 3.00 ± 0.08 and 3.24 ± 0.07, respectively, p = 0.04). By multivariable analysis, three variables were independently associated with the collateral flow grade: female gender, smoking, and the Asp²⁹⁸ variant (p = 0.03) while the Asp²⁹⁸ variant was the sole variable independently associated with the recipient filling grade (p = 0.03).

Conclusion

Collateral development is lower in patients with the Asp²⁹⁸ variant. This may be explained by the decreased NOS activity in patients with the Asp²⁹⁸ variant. Further studies will have to determine whether increasing eNOS activity in humans is associated with coronary collateral development.     

Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.