7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N²-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N²-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram− bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10 μg/ml), and were inactive against the Gram− organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.     

Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity

Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.     

Antibody repertoire development in fetal and neonatal piglets. XX. B cell lymphogenesis is absent in the ileal Peyer's patches, their repertoire development is antigen dependent, and they are not required for B cell maintenance

The continuous ileal Peyer's patches (IPP) of sheep are regarded as a type of mammalian bursal equivalent where B cells diversify their repertoire in an Ag-independent fashion. Anatomically and developmentally similar IPP occur in swine. Resection of ～90% of the IPP in piglets at birth did not alter Ig levels in serum and secretions or retard diversification of the Ab repertoire when animals were maintained in isolators and colonized with a defined gut flora. Resection or sham surgery elevated IgG and IgA in serum and in lavage fluid from the gut, lung, and in saliva. No changes in the frequency of IgG-, IgA-, and IgM-containing cells in the spleen and peripheral lymph node were observed. Using an index that quantifies diversification of the VDJ repertoire, no differences were seen in three secondary lymphoid tissues between piglets lacking IPP and colonized controls, whereas both groups displayed >10-fold greater diversification than did late-term fetal piglets or piglets maintained germ-free. Somatic hypermutation was very low in fetal IPP and the IPP of germ-free piglets but increased 3- to 5-fold after colonization. D-J signal joint circles were not recovered in IPP, and V-DJ signal joint circles were 5-fold lower than in bone marrow and similar to those in thymus and spleen. We conclude that the porcine IPP are not a site of B cell lymphogenesis, do not undergo Ag-independent repertoire diversification, and are not primary lymphoid tissue since they are not required for maintenance of Ig levels in serum and secretions.     

Effects of insecticides on behavior of adult Bactericera cockerelli (Hemiptera: Triozidae) and transmission of Candidatus Liberibacter psyllaurous

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes (Solanum tuberosum L.) that can cause yield loss by direct feeding on crop plants and by vectoring a bacterial pathogen, Candidatus Liberibacer psyllaurous. Current pest management practices rely on the use of insecticides to control the potato psyllid to lower disease incidences and increase yields. Although many studies have focused on the mortality that insecticides can cause on potato psyllid populations, little is known regarding the behavioral responses of the potato psyllid to insecticides or whether insecticides can decrease pathogen transmission. Thus, the objectives of this study were to determine the effects of insecticides on adult potato psyllid behaviors, the residual effects of insecticides on potato psyllid behaviors over time, and effects of these insecticides on Ca. L. psyllaurous transmission. Insecticides tested included imidacloprid, kaolin particle film, horticultural spray oil, abamectin, and pymetrozine. All insecticides significantly reduced probing durations and increased the amount of time adult psyllids spent off the leaflets, suggesting that these chemicals may be deterrents to feeding as well as repellents. Nonfeeding behaviors such as tasting, resting, and cleaning showed variable relationships with the different insecticide treatments over time. The insecticides imidacloprid and abamectin significantly lowered transmission of Ca. L. psyllaurous compared with untreated controls. The implications of our results for the selection of insecticides useful for an integrated pest management program for potato psyllid control are discussed.     

Effect of aerosol surface lubricants on the abundance and richness of selected forest insects captured in multiple-funnel and panel traps

Survey and detection programs for native and exotic forest insects frequently rely on traps baited with odorants, which mediate the orientation of target taxa (e.g., the southern pine beetle, Dendroctonusfrontalis Zimmermann) toward a resource (e.g., host material, mates). The influence of trap design on the capture efficiency of baited traps has received far less empirical attention than odorants, despite concerns that intercept traps currently used operationally have poor capture efficiencies for some target taxa (e.g., large woodborers). Several studies have recently demonstrated that treating traps with a surface lubricant to make them "slippery" can increase their capture efficiency; however, previously tested products can be expensive and their application time-consuming. The purpose of this study was to evaluate the effect of alternate, easier to apply aerosol lubricants on trap capture efficiency of selected forest insects. Aerosol formulations of Teflon and silicone lubricants increased both panel and multiple-funnel trap capture efficiencies. Multiple-funnel traps treated with either aerosol lubricant captured significantly more Monochamus spp. and Acanthocinus obsoletus (Olivier) than untreated traps. Similarly, treated panel traps captured significantly more Xylotrechus sagittatus (Germar), Ips calligraphus (Germar), Pissodes nemorensis (Germar), Monochamus spp., A. obsoletus, Thanasimus dubius (F.), and Ibalia leucospoides (Hochenwarth) than untreated traps. This study demonstrates that treating multiple-funnel and panel traps with an aerosol dry film lubricant can increase their capture efficiencies for large woodborers (e.g., Cerambycidae) as well as bark beetles, a weevil, a woodwasp parasitoid and a bark beetle natural enemy (Coleoptera: Cleridae).     

Massive genomic rearrangement acquired in a single catastrophic event during cancer development

Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%-3% of all cancers, across many subtypes, and is present in ～25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer.     

Targeted disruption of the porcine immunoglobulin kappa light chain locus

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (??) light chain constant (C??) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted C?? allele (heterozygous knockout or ±) were bred together to generate C?? homozygous knockout (?/?) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from C?? ?/? pigs were devoid of ??-containing Igs. Furthermore, there was an increase in lambda (??) light chain expression when compared to that of wild-type littermates (C?? +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.