Determination of the pharmacokinetics and oral bioavailability of salicylamine, a potent γ-ketoaldehyde scavenger, by LC/MS/MS

Levels of reactive γ-ketoaldehydes derived from arachidonate increase in diseases associated with inflammation and oxidative injury. To assess the biological importance of these γ-ketoaldehydes, we previously identified salicylamine as an effective γ-ketoaldehyde scavenger in vitro and in cells. To determine if salicylamine could be administered in vivo, we developed an LC/MS/MS assay to measure salicylamine in plasma and tissues. In mice, half-life (t1/2) was 62 minutes. Drinking water supplementation (1-10 g/L) generated tissue concentrations (10-500 μM) within the range previously shown to inhibit γ-ketoaldehydes in cells. Therefore, oral administration of salicylamine can be used to assess the contribution of γ-ketoaldehydes in animal models of disease. © 2010 by the authors licensee Molecular Diversity Preservation International, Basel, Switzerland

SNPs in Multi-Species Conserved Sequences (MCS) as useful markers in association studies: a practical approach

<p>Abstract</p> <p>Background</p> <p>Although genes play a key role in many complex diseases, the specific genes involved in most complex diseases remain largely unidentified. Their discovery will hinge on the identification of key sequence variants that are conclusively associated with disease. While much attention has been focused on variants in protein-coding DNA, variants in noncoding regions may also play many important roles in complex disease by altering gene regulation. Since the vast majority of noncoding genomic sequence is of unknown function, this increases the challenge of identifying "functional" variants that cause disease. However, evolutionary conservation can be used as a guide to indicate regions of noncoding or coding DNA that are likely to have biological function, and thus may be more likely to harbor SNP variants with functional consequences. To help bias marker selection in favor of such variants, we devised a process that prioritizes annotated SNPs for genotyping studies based on their location within Multi-species Conserved Sequences (MCSs) and used this process to select SNPs in a region of linkage to a complex disease. This allowed us to evaluate the utility of the chosen SNPs for further association studies. Previously, a region of chromosome 1q43 was linked to Multiple Sclerosis (MS) in a genome-wide screen. We chose annotated SNPs in the region based on location within MCSs (termed MCS-SNPs). We then obtained genotypes for 478 MCS-SNPs in 989 individuals from MS families.</p> <p>Results</p> <p>Analysis of our MCS-SNP genotypes from the 1q43 region and comparison to HapMap data confirmed that annotated SNPs in MCS regions are frequently polymorphic and show subtle signatures of selective pressure, consistent with previous reports of genome-wide variation in conserved regions. We also present an online tool that allows MCS data to be directly exported to the UCSC genome browser so that MCS-SNPs can be easily identified within genomic regions of interest.</p> <p>Conclusion</p> <p>Our results showed that MCS can easily be used to prioritize markers for follow-up and candidate gene association studies. We believe that this novel approach demonstrates a paradigm for expediting the search for genes contributing to complex diseases.</p

Mitochondrial DNA Polymorphism A4917G Is Independently Associated with Age-Related Macular Degeneration

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20–3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms

Using genetic variation and environmental risk factor data to identify individuals at high risk for age-related macular degeneration

A major goal of personalized medicine is to pre-symptomatically identify individuals at high risk for disease using knowledge of each individual's particular genetic profile and constellation of environmental risk factors. With the identification of several well-replicated risk factors for age-related macular degeneration (AMD), the leading cause of legal blindness in older adults, this previously unreachable goal is beginning to seem less elusive. However, recently developed algorithms have either been much less accurate than expected, given the strong effects of the identified risk factors, or have not been applied to independent datasets, leaving unknown how well they would perform in the population at large. We sought to increase accuracy by using novel modeling strategies, including multifactor dimensionality reduction (MDR) and grammatical evolution of neural networks (GENN), in addition to the traditional logistic regression approach. Furthermore, we rigorously designed and tested our models in three distinct datasets: a Vanderbilt-Miami (VM) clinic-based case-control dataset, a VM family dataset, and the population-based Age-related Maculopathy Ancillary (ARMA) Study cohort. Using a consensus approach to combine the results from logistic regression and GENN models, our algorithm was successful in differentiating between high- and low-risk groups (sensitivity 77.0%, specificity 74.1%). In the ARMA cohort, the positive and negative predictive values were 63.3% and 70.7%, respectively. We expect that future efforts to refine this algorithm by increasing the sample size available for model building, including novel susceptibility factors as they are discovered, and by calibrating the model for diverse populations will improve accuracy

Primate-specific evolution of noncoding element insertion into PLA2G4C and human preterm birth

Background The onset of birth in humans, like other apes, differs from non-primate mammals in its endocrine physiology. We hypothesize that higher primate-specific gene evolution may lead to these differences and target genes involved in human preterm birth, an area of global health significance. Methods We performed a comparative genomics screen of highly conserved noncoding elements and identified PLA2G4C, a phospholipase A isoform involved in prostaglandin biosynthesis as human accelerated. To examine whether this gene demonstrating primate-specific evolution was associated with birth timing, we genotyped and analyzed 8 common single nucleotide polymorphisms (SNPs) in PLA2G4C in US Hispanic (n = 73 preterm, 292 control), US White (n = 147 preterm, 157 control) and US Black (n = 79 preterm, 166 control) mothers. Results Detailed structural and phylogenic analysis of PLA2G4C suggested a short genomic element within the gene duplicated from a paralogous highly conserved element on chromosome 1 specifically in primates. SNPs rs8110925 and rs2307276 in US Hispanics and rs11564620 in US Whites were significant after correcting for multiple tests (p < 0.006). Additionally, rs11564620 (Thr360Pro) was associated with increased metabolite levels of the prostaglandin thromboxane in healthy individuals (p = 0.02), suggesting this variant may affect PLA2G4C activity. Conclusions Our findings suggest that variation in PLA2G4C may influence preterm birth risk by increasing levels of prostaglandins, which are known to regulate labor.Children’s Discovery InstituteMarch of Dimes Birth Defects FoundationNational Institute of General Medical Sciences (U.S.) (grant T32 GM081739)Washington University (Saint Louis, Mo.) (Mr. and Mrs. Spencer T. Olin Fellowship for Women in Graduate Study)Sigrid Jusélius FoundationSigne and Anne Gyllenberg FoundationAcademy of FinlandVanderbilt University (Turner-Hazinski grant award

Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies