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.     

Human anti-prion antibodies block prion peptide fibril formation and neurotoxicity

Prion diseases are a group of rare, fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K-resistant conformer, termed scrapie PrP (PrP(Sc)). Aggregates of PrP(Sc) deposited around neurons lead to neuropathological alterations. Currently, there is no effective treatment for these fatal illnesses; thus, the development of an effective therapy is a priority. PrP peptide-based ELISA assay methods were developed for detection and immunoaffinity chromatography capture was developed for purification of naturally occurring PrP peptide autoantibodies present in human CSF, individual donor serum, and commercial preparations of pooled intravenous immunoglobulin (IVIg). The ratio of anti-PrP autoantibodies (PrP-AA) to total IgG was ～1:1200. The binding epitope of purified PrP-AA was mapped to an N-terminal region comprising the PrP amino acid sequence KTNMK. Purified PrP-AA potently blocked fibril formation by a toxic 21-amino acid fragment of the PrP peptide containing the amino acid alanine to valine substitution corresponding to position 117 of the full-length peptide (A117V). Furthermore, PrP-AA attenuated the neurotoxicity of PrP(A117V) and wild-type peptides in rat cerebellar granule neuron (CGN) cultures. In contrast, IgG preparations depleted of PrP-AA had little effect on PrP fibril formation or PrP neurotoxicity. The specificity of PrP-AA was demonstrated by immunoprecipitating PrP protein in brain tissues of transgenic mice expressing the human PrP(A117V) epitope and Sc237 hamster. Based on these intriguing findings, it is suggested that human PrP-AA may be useful for interfering with the pathogenic effects of pathogenic prion proteins and, thereby has the potential to be an effective means for preventing or attenuating human prion disease progression.     

Influence of Genetic Variation on Plasma Protein Levels in Older Adults Using a Multi-Analyte Panel

Proteins, widely studied as potential biomarkers, play important roles in numerous physiological functions and diseases. Genetic variation may modulate corresponding protein levels and point to the role of these variants in disease pathophysiology. Effects of individual single nucleotide polymorphisms (SNPs) within a gene were analyzed for corresponding plasma protein levels using genome-wide association study (GWAS) genotype data and proteomic panel data with 132 quality-controlled analytes from 521 Caucasian participants in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. Linear regression analysis detected 112 significant (Bonferroni threshold p = 2.44×10⁻⁵) associations between 27 analytes and 112 SNPs. 107 out of these 112 associations were tested in the Indiana Memory and Aging Study (IMAS) cohort for replication and 50 associations were replicated at uncorrected p<0.05 in the same direction of effect as those in the ADNI. We identified multiple novel associations including the association of rs7517126 with plasma complement factor H-related protein 1 (CFHR1) level at p<1.46×10⁻⁶⁰, accounting for 40 percent of total variation of the protein level. We serendipitously found the association of rs6677604 with the same protein at p<9.29×10⁻¹¹². Although these two SNPs were not in the strong linkage disequilibrium, 61 percent of total variation of CFHR1 was accounted for by rs6677604 without additional variation by rs7517126 when both SNPs were tested together. 78 other SNP-protein associations in the ADNI sample exceeded genome-wide significance (5×10⁻⁸). Our results confirmed previously identified gene-protein associations for interleukin-6 receptor, chemokine CC-4, angiotensin-converting enzyme, and angiotensinogen, although the direction of effect was reversed in some cases. This study is among the first analyses of gene-protein product relationships integrating multiplex-panel proteomics and targeted genes extracted from a GWAS array. With intensive searches taking place for proteomic biomarkers for many diseases, the role of genetic variation takes on new importance and should be considered in interpretation of proteomic results.     

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.     

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.     

Quantitative analysis of amyloid-beta peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry.

Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation 相似文献