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Components of the mitochondrial electron transport chain have recently gained much interest as potential therapeutic targets. Since mitochondria are essential for the supply of energy that is required for both angiogenic and tumourigenic activity, targeting the mitochondria represents a promising potential therapeutic approach for treating cancer. Here we investigate the established anti-angiogenesis drugs combretastatin A4, thalidomide, OGT 2115 and tranilast that we hypothesise are able to exert a direct anti-cancer effect in the absence of vasculature by targeting the mitochondria. Drug cytotoxicity was measured using the MTT assay. Mitochondrial function was measured in intact isolated mitochondria using polarography, fluorimetry and enzymatic assays to measure mitochondrial oxygen consumption, membrane potential and complex I–IV activities respectively. Combretastatin A4, OGT 2115 and tranilast were both shown to decrease mitochondrial oxygen consumption. OGT 2115 and tranilast decreased mitochondrial membrane potential and reduced complex I activity while combretastatin A4 and thalidomide did not. OGT 2115 inhibited mitochondrial complex II–III activity while combretastatin A4, thalidomide and tranilast did not. Combretastatin A4, thalidomide and OGT 2115 induced bi-phasic concentration-dependent increases and decreases in mitochondrial complex IV activity while tranilast had no evident effect. These data demonstrate that combretastatin A4, thalidomide, OGT 2115 and tranilast are all mitochondrial modulators. OGT 2115 and tranilast are both mitochondrial inhibitors capable of eliciting concentration-dependent reductions in cell viability by decreasing mitochondrial membrane potential and oxygen consumption.

Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant-induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.

The rate of recombination is a key genomic parameter that displays considerable variation among taxa. Species comparisons have demonstrated that the rate of evolution in recombination rate is strongly dependent on the physical scale of measurement. Individual recombination hotspots are poorly conserved among closely related taxa, whereas genomic-scale recombination rate variation bears a strong signature of phylogenetic history. In contrast, the mode and tempo of evolution in recombination rates measured on intermediate physical scales is poorly understood. Here, we conduct a detailed statistical comparison between two whole-genome F2 genetic linkage maps constructed from experimental intercrosses between closely related house mouse subspecies (Mus musculus). Our two maps profile a common wild-derived inbred strain of M. m. domesticus crossed to distinct wild-derived inbred strains representative of two other house mouse subspecies, M. m. castaneus and M. m. musculus. We identify numerous orthologous genomic regions with significant map length differences between these two crosses. Because the genomes of these recently diverged house mice are highly collinear, observed differences in map length (centimorgans) are suggestive of variation in broadscale recombination rate (centimorgans per megabase) within M. musculus. Collectively, these divergent intervals span 19% of the house mouse genome, disproportionately aggregating on the X chromosome. In addition, we uncover strong statistical evidence for a large effect, sex-linked, site-specific modifier of recombination rate segregating within M. musculus. Our findings reveal considerable variation in the megabase-scale recombination landscape among recently diverged taxa and underscore the continued importance of genetic linkage maps in the post-genome era.

Catechol-O-methyltransferase (COMT) is an ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity (Andersen & Skorpen, 2009) and risk for developing psychiatric disorders (Harrison & Tunbridge, 2008). A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation (Nackley et al., 2006). However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis-regulated. A B2 Short Interspersed Element (SINE) was inserted in the 3′UTR of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element demonstrate that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3′ UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.

The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-γ in malaria infection.

The identification of genes influencing sensitivity to stimulants and opioids is important for determining their mechanism of action and may provide fundamental insights into the genetics of drug abuse. We used a panel of C57BL/6J (B6; recipient) x A/J (donor) chromosome substitution strains (CSS) to identify quantitative trait loci (QTL) for both open field activity and sensitivity to the locomotor stimulant response to methamphetamine (MA). Mice were injected with saline (days 1 and 2) and MA (day 3; 2 mg/kg, i.p.). We analyzed the total distance traveled in the open field for thirty minutes following each injection. CSS-8, -11, and -16 showed reduced MA-induced locomotor activity relative to B6 whereas CSS-10 and -12 showed increased MA-induced locomotor activity. Further analysis focused on CSS-11 because it was robustly different from B6 following MA-injection but did not differ in activity following saline injection and because it also showed reduced locomotor activity in response to the mu-opioid receptor agonist fentanyl (0.2 mg/kg, i.p.). Thus, CSS-11 captures a QTL for the response to both psychostimulants and opioids. Using a B6 x CSS-11 F2 intercross, we identified a dominant QTL for the MA response on chromosome 11. We used haplotype association mapping of cis expression QTLs and bioinformatic resources to parse among genes within the 95% confidence interval of the chromosome 11 QTL. Identification of the genes underlying QTLs for psychostimulants and opioid response may provide insights about genetic factors that modulate sensitivity to drugs of abuse.

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d × 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

Mutations in the head and tail domains of the motor protein myosin VIIA (MYO7A) cause deaf-blindness (Usher syndrome Type 1B, USH1B) and non-syndromic deafness (DFNB2, DFNA11). The head domain binds to F-actin and serves as the MYO7A motor domain, but little is known about the function of the tail domain. In a genetic screen, we have identified polka mice, which carry a mutation (c.5742 + 5G>A) that affects splicing of the MYO7A transcript and truncates the MYO7A tail domain at the C-terminal FERM domain. In the inner ear, expression of the truncated MYO7A protein is severely reduced, leading to defects in hair cell development. In retinal pigment epithelial (RPE) cells, the truncated MYO7A protein is expressed at comparative levels to wild-type protein but fails to associate with and transport melanosomes. We conclude that the C-terminal FERM domain of MYO7A is critical for melanosome transport in RPE cells. Our findings also suggest that MYO7A mutations can lead to tissue specific effects on protein levels, which may explain why some mutations in MYO7A lead to deafness without retinal impairment.

No abstract available.

The type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate postsynaptic inhibition. The functional diversity of these receptors comes from the use of a large repertoire of subunits encoded by separate genes, as well as from differences in subunit composition of individual receptors. In mammals, a majority of GABA(A) receptor subunit genes are located in gene clusters that may be important for their regulated expression and function. We have established a high-resolution physical map of the cluster of genes encoding GABA(A) receptor subunits alpha2 (Gabra2), beta1 (Gabrb1), and gamma(1) (Gabrg1) on mouse chromosome 5. Rat cDNA probes and specific sequence probes for all three GABA(A) receptor subunit genes have been used to initiate the construction of a sequence-ready contig of bacterial artificial chromosomes (BACs) encompassing this cluster. In the process of contig construction clones from 129/Sv and C57BL/6J BAC libraries were isolated. The assembled 1.3-Mb contig, consisting of 45 BACs, gives five- to sixfold coverage over the gene cluster and provides an average resolution of one marker every 32 kb. A number of BAC insert ends were sequenced, generating 30 new sequence tag sites (STS) in addition to 6 Gabr gene-based and 3 expressed sequence tag (EST)-based markers. STSs from, and surrounding, the Gabrg1-Gabra2-Gabrb1 gene cluster were mapped in the T31 mouse radiation hybrid panel. The integration of the BAC contig with a map of loci ordered by radiation hybrid mapping suggested the most likely genomic orientation of this cluster on mouse chromosome 5: cen-D5Mit151-Gabrg1-Gabra2-Gabrb1-D5Mit58- tel. This established contig will serve as a template for genomic sequencing and for functional analysis of the GABA(A) gene cluster on mouse chromosome 5 and the corresponding region on human chromosome 4.