Genetic diversity in laboratory colonies of western corn rootworm (Coleoptera: Chrysomelidae), including a nondiapause colony

Laboratory-reared western corn rootworms, Diabrotica virgifera virgifera, from colonies maintained at the North Central Agricultural Research Laboratory (NCARL) in Brookings, SD, are used extensively by many researchers in studies of the biology, ecology, behavior, and genetics of this major insect pest. A nondiapause colony developed through artificial selection in the early 1970s is particularly attractive for many studies because its generation time is much shorter than that of typical diapause colonies. However, the nondiapause colony has been in culture for approximately 190 generations without out-crossing. We compared variation at six microsatellite loci among individuals from the NCARL nondiapause colony (approximately 190 generations), main diapause colony (approximately 22 generations), four regional diapause colonies (3-8 generations), and four wild populations. Genetic diversity was very similar among the diapause laboratory colonies and wild populations. However, the nondiapause colony showed approximately 15-39% loss of diversity depending on the measure. Pairwise estimates of F(ST) were very low, revealing little genetic differentiation among laboratory colonies and natural populations. The nondiapause colony showed the greatest genetic differentiation with an average pairwise F(ST) of 0.153. There was little evidence that the laboratory colonies had undergone genetic bottlenecks except for the nondiapause colony. The nondiapause colony has suffered a moderate loss in genetic diversity and is somewhat differentiated from wild populations. This was not unexpected given its history of artificial selection for the nondiapause trait, and the large number of generations in culture. In contrast, the results indicate that the diapause colonies maintained at NCARL are genetically similar to wild populations.     

Identification and characterization of a novel periplasmic polygalacturonic acid binding protein from Yersinia enterolitica

A number of bacteria in the family Enterobacteriaceae harbor the genes comprising well-developed pectinolytic pathways (e.g. Erwinia sp.) or abridged versions of this pathway (e.g. Yersinia sp.). One of the most enigmatic components present in some of these pathways is a small gene that encodes a predicted secreted protein of approximately 160 amino acid residues with unknown function. This protein shows distant amino acid sequence similarity over its entire length to galactose-specific family 32 carbohydrate-binding modules (CBMs). Here we demonstrate the ability of the Yersinia enterocolitica example, here called YeCBM32, to bind polygalacturonic acid containing components of pectin. This binding is selective for highly polymerized galacturonic acid and shows a complex mode of polysaccharide recognition. The high resolution X-ray crystal structure (1.35 A) shows YeCBM32s overall structural similarity to galactose specific CBMs and conserved binding site location but reveals a substantially different binding site topology, which likely reflects its unique polymeric and acidic ligand. The results suggest the possibility of a unique role for YeCBM32 in polygalacturonic acid transport.     

Specific recognition of saturated and 4,5-unsaturated hexuronate sugars by a periplasmic binding protein involved in pectin catabolism

The process of pectin depolymerization by pectate lyases and glycoside hydrolases produced by pectinolytic organisms, particularly the phytopathogens from the genus Erwinia, is reasonably well understood. Indeed each extracellular and intracellular catabolic stage has been identified using either genetic, bioinformatic or biochemical approaches. Nevertheless, the molecular details of many of these stages remain unknown. In particular, the mechanism and ligand binding profiles for the transport of pectin degradation products between cellular compartments remain entirely uninvestigated. Here we present the structure of TogB, a 45.7 kDa periplasmic binding protein from Yersinia enterocolitica. This protein is a component of the TogMNAB ABC transporter involved in the periplasmic transport of oligogalacturonides. In addition to the unliganded complex (at 2.2 A), we have also determined the structures of TogB in complex with digalacturonic acid (at 2.2 A), trigalacturonic acid (at 1.8 A) and 4,5-unsaturated digalacutronic acid (at 2.3 A). The molecular determinants of oligogalacturonide binding include a novel salt-bridge between the non-reducing sugar uronate group, selectivity for the unsaturated ligand, and the overall sugar configuration. Complementing this are UV difference and isothermal titration calorimetry experiments that highlight the thermodynamic basis of ligand specificity. The ligand binding profiles of the TogMNAB transporter complex nicely complement pectate lyase-mediated pectin degradation, which is a significant component of pectin depolymerization reactions.     

Hypermethylation of Fads2 and altered hepatic fatty acid and phospholipid metabolism in mice with hyperhomocysteinemia

Alterations in lipid metabolism may play a role in the vascular pathology associated with hyperhomocysteinemia (HHcy). Homocysteine is linked to lipid metabolism through the methionine cycle and the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine (PE) methyltransferase, which is responsible for the synthesis of 20-40% of liver PC. The goal of the present study was to determine if the reduced methylation capacity in HHcy is associated with alterations in liver phospholipid and fatty acid metabolism. Mice heterozygous for disruption of cystathionine beta-synthase (Cbs+/-) fed a diet to induce HHcy (HH diet) had higher (p<0.001) plasma total homocysteine (30.8+/-4.4 microM, mean+/-S.E.) than C57BL/6 mice (Cbs+/+) fed the HH diet (7.0+/-1.1 microM) or Cbs+/+ mice fed a control diet (2.3+/-0.3 microM). Mild and moderate HHcy was accompanied by lower adenosylmethionine/adenosylhomocysteine ratios (p<0.05), higher PE (p<0.05) and PE/PC ratios (p<0.01), lower PE methyltransferase activity (p<0.001), and higher linoleic acid (p<0.05) and lower arachidonic acid (p<0.05) in PE. Mice with moderate HHcy also had higher linoleic acid and alpha-linolenic acid (p<0.05) and lower arachidonic acid and docosahexaenoic acid (p<0.05) in liver PC. The first step in the desaturation and elongation of linoleic acid and linolenic acid to arachidonic acid and docosahexaenoic acid, respectively, is catalyzed by Delta6-desaturase (encoded by Fads2). We found hypermethylation of the Fads2 promoter (p<0.01), lower Fads2 mRNA (p<0.05), and lower Delta6-desaturase activity (p<0.001) in liver from mice with HHcy. These findings suggest that methylation silencing of liver Fads2 expression and changes in liver fatty acids may contribute to the pathology of HHcy.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.     

FTIR studies of the redox partner interaction in cytochrome P450: The Pdx–P450cam couple

Recently we have developed a new approach to study protein–protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam–Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP–FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in β-sheets and α-helix content, a decrease in the population of random coil/3₁₀-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam–Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx–P450cam complex.     

Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state ³¹P and ²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically ¹³C-labelled Aβ(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely ¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Post-mating disparity between potential and realized immune response in Drosophila melanogaster

Reproductive costs are an essential component of evolutionary theory. For instance, an increase in reproduction is generally coupled with a decrease in immunocompetence shortly after mating. However, recent work in Drosophila melanogaster suggests that the potential to mount an immune response, as measured by the levels of immune gene expression, increases after mating. These data are in contrast to previous studies, which suggest that mating can reduce a fly's ability to survive an actual bacterial challenge (realized immunity). This pattern may be driven by some aspect of mating, independent of resource limitation, which reduces immune function by inhibiting the effective deployment of immune gene products. Though several studies have examined both the potential and the realized immunity after mating, none have examined these immune measures simultaneously. Here, we examined the link between the potential and the realized immunity in a sterile mutant of D. melanogaster. Shortly after mating, we found that female immune gene expression was high, but survival against infection was low. Surprisingly, this pattern was reversed within 24 h. Thus, estimates of immunity based on gene expression do not appear to reflect an actual ability to defend against pathogens in the hours following copulation. We discuss the possible mechanisms that may account for this pattern.     

Preparation of 4-aryl-2-trifluoromethylbenzonitrile derivatives as androgen receptor antagonists for topical suppression of sebum production

A series of substituted 4-aryl-2-trifluoromethylbenzonitrile analogs were evaluated in the human androgen receptor binding and cellular functional assays. Analogs with sufficient in vitro binding and cellular potency (IC(50)<200 nM) were tested in the progesterone receptor binding assay for selectivity and in the Golden Syrian hamster ear model for in vivo efficacy. Within the series, compound 4 e was identified to be the most active analog in vivo (wax ester inhibition=86%).