Dissociation of the lipid-enzyme complex of hormone-sensitive lipase using high density lipoprotein or apolipoprotein A-I

Hormone-sensitive lipase in homogenates of adipose tissue occurs as a large, lipid-rich complex including several acylhydrolase activities that emerge quantitatively in the void volume on gel filtration chromatography (2% agarose). Incubation with intact human plasma high density lipoprotein or with lipid-free apolipoprotein A-I, however, disrupted the lipid-rich complex almost completely and most of the enzyme activity eluted from a 2% agarose column at about Ve = 2.3 x Vo. This use of the detergent-like properties of apolipoprotein A-I may be of value for dissociation of other lipid-associated or membrane-bound enzymes.     

A modified DNA barcode approach to define trophic interactions between native and exotic pentatomids and their parasitoids

The establishment of invasive Halyomorpha halys (Stål) outside of its native range may impact native species assemblages, including other pentatomids and their scelionid parasitoids. This has generated interest in defining species diversity and host‐parasitoid associations in this system to better understand the impact of invasive alien species on trophic interactions in invaded regions. Information on scelionid–pentatomid associations in natural habitats is lacking, and species‐level identification of these associations can be tenuous using rearing and dissection techniques. Naturally occurring pentatomid eggs were collected in areas where H. halys has established in Canada and were analysed using a modified DNA barcoding approach to define species‐level trophic interactions. Identification was possible for >90% of egg masses. Eleven pentatomid and five scelionid species were identified, and trophic links were established. Approximately 70% of egg masses were parasitized; parasitism and parasitoid species composition were described for each species. Telenomus podisi Ashmead was the dominant parasitoid and was detected in all host species. Trissolcus euschisti Ashmead was detected in several host species, but was significantly more prevalent in Chinavia hilaris (Say) and Brochymena quadripustulata (Fabricius). Trissolcus brochymenae Ashmead and Tr. thyantae Ashmead were recorded sporadically. Parasitism of H. halys was 55%, and this species was significantly less likely to be parasitized than native pentatomids. The scelionid species composition of H. halys consisted of Te. podisi, Tr. euschisti and Tr. thyantae. Although these species cannot develop in fresh H. halys eggs, we demonstrate that parasitoids attempt to exploit this host under field conditions.     

Adaptive variation in beach mice produced by two interacting pigmentation genes

Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.     

Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated

Natural variation in human drug metabolism and target genes can cause pharmacogenetic or interindividual variation in drug sensitivity. We reasoned that natural pharmacogenetic variation in model organisms could be systematically exploited to facilitate the characterization of new small molecules. To test this, we subjected multiple Arabidopsis thaliana accessions to chemical genetic screens and discovered 12 accession-selective hit molecules. As a model for understanding this variation, we characterized natural resistance to hypostatin, a new inhibitor of cell expansion. Map-based cloning identified HYR1, a UDP glycosyltransferase (UGT), as causative for hypostatin resistance. Multiple lines of evidence demonstrate that HYR1 glucosylates hypostatin in vivo to form a bioactive glucoside. Additionally, we delineated a HYR1 substrate motif and used it to identify another molecule modulated by glucosylation. Our results demonstrate that natural variation can be exploited to inform the biology of new small molecules, and that UGT sequence variation affects xenobiotic sensitivity across biological kingdoms.     

ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.     

Human subtelomeric WASH genes encode a new subclass of the WASP family

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences.     

Elevated rates of sister chromatid exchange at chromosome ends

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.     

Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin gamma-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry

Measurement of F(2)-isoprostanes (F(2)-IsoPs) has been independently verified as one of the most reliable approaches to assess oxidative stress in vivo. However, the rapid clearance of F(2)-IsoPs makes the timing of sample collection critical for short-lived oxidative insults. Isoketals (IsoKs) are gamma-ketoaldehydes formed via the IsoP pathway of lipid peroxidation that rapidly react with lysyl residues of proteins to form stable protein adducts. Oxidative stress can also activate cyclooxygenases to produce prostaglandin H(2), which can form two specific isomers of IsoK-levuglandin (LG) D(2) and E(2). Because adducted proteins are not rapidly cleared, IsoK/LG protein adduct levels can serve as a dosimeter of oxidative and inflammatory damage over prolonged periods of time as well as brief episodes of injury. Quantification of IsoK/LG protein adducts begins with liquid-phase extraction to separate proteins from lipid membranes, allowing measurement of both IsoK/LG protein adducts and F(2)-IsoP from the same sample if desired. IsoK/LG-lysyl-lactam adducts are measured by liquid chromatography tandem mass spectrometry after proteolytic digestion of extracted proteins, solid-phase extraction and preparative HPLC.