Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry

Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosin's twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.     

Increasing oxytocin receptor expression in the nucleus accumbens of pre-pubertal female prairie voles enhances alloparental responsiveness and partner preference formation as adults

Oxytocin receptors (OXTR) in the nucleus accumbens (NAcc) promote alloparental behavior and partner preference formation in female prairie voles. Within the NAcc there is significant individual variation in OXTR binding and virgin juvenile and adult females with a high density of OXTR in the NAcc display an elevated propensity to engage in alloparental behavior toward novel pups. Over-expression of OXTR in the NAcc of adult female prairie voles using viral vector gene transfer facilitates partner preference formation, but has no effect on alloparental behavior, even though OXTR antagonists infused into the NAcc blocks both behaviors. We therefore hypothesized that long-term increases in OXTR signaling during development may underlie the relationship between adult OXTR density in the NAcc and alloparental behavior. To test this hypothesis, we used viral vector gene transfer to increase OXTR density in the NAcc of prepubertal, 21 day old female prairie voles and tested for both alloparental behavior and partner preference formation as adults. Consistent with a developmental impact of OXTR signaling, adults over-expressing OXTR from weaning display both increased alloparental behavior and partner preference formation. Thus, the relatively acute impact of elevated OXTR signaling in the NAcc on partner preference formation previously reported appears to be dissociable from the effects of longer term, developmentally relevant OXTR signaling necessary for modulating alloparental behavior. These results are consistent with the hypothesis that oxytocin can have both long-term “organizational” effects as well as acute “activational” effects on affiliative behaviors.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Field and vegetable crops as hosts of larval western spotted cucumber beetle (Coleoptera: Chrysomelidae)

The western spotted cucumber beetle, Diabrotica undecimpunctata undecimpunctata Mannerheim, is an important pest of melons (Cucurmis melo L.) in northern California. Recent observations indicate that adults are using alfalfa (Medicago sativa L.) as a feeding host and larvae may be developing on the roots. Greenhouse studies were conducted during the winters of 2009 and 2010 in which larval development was compared on the roots of six field and vegetable crops commonly grown in the southern Sacramento Valley. The growth parameters used to evaluate the hosts were larval and pupal head capsule width, body width, and body length as well as total survival percentage and survival percentage to the third instar. According to larval growth and survivorship in 2009, maize (Zea mays L.) was the best host, followed by alfalfa and tomato (Solanum lycopersicum L.) that were roughly equivalent to one another. Melon was a slightly weaker host than alfalfa and tomato; sunflower (Helianthus annuus L.) was a completely incompatible host and thus dropped from the 2010 study. In 2010, melon was the weakest host for larval development. Maize was the superior host again followed closely by alfalfa that performed slightly better than tomato and lima bean (Phaseolus lunatus L.). Data suggest that larval western spotted cucumber beetles may primarily develop outside of melon fields and the adults emigrate to melons.     

Pathways to meiotic recombination in Arabidopsis thaliana

Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.     

In the heat of the night--alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum

? Philodendron bipinnatifidum inflorescences heat up to 42 °C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). ? Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. ? Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O? isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O?. ? The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O? can aid measurement of respiratory pathway fluxes in dense tissues.