Discrete signaling regions in the lymphotoxin-beta receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-kappaB pathways

Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR.     

Membrane lymphotoxin is required for the development of different subpopulations of NK T cells

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTalpha and LTbeta, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTalpha-/- and LTbeta-/- mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTalpha-/- and LTbeta-/- mice did not respond to the lipoglycan alpha-galactosylceramide, which is presented by mouse CD1 to Valpha14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and alpha-galactosylceramide reactive and those that are not, were affected by disruption of the LTalpha and LTbeta genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTbeta receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.     

Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.     

Lymphotoxin-alpha-deficient mice can clear a productive infection with murine gammaherpesvirus 68 but fail to develop splenomegaly or lymphocytosis

Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- alpha-deficient (LTalpha(-/-)) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTalpha(-/-) mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTalpha(-/-) and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTalpha(-/-) mice. Furthermore, LTalpha(-/-) mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTalpha(-/-) mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.     

Metabolomic Profiling of the Nectars of Aquilegia pubescens and A. Canadensis

To date, variation in nectar chemistry of flowering plants has not been studied in detail. Such variation exerts considerable influence on pollinator–plant interactions, as well as on flower traits that play important roles in the selection of a plant for visitation by specific pollinators. Over the past 60 years the Aquilegia genus has been used as a key model for speciation studies. In this study, we defined the metabolomic profiles of flower samples of two Aquilegia species, A. Canadensis and A. pubescens. We identified a total of 75 metabolites that were classified into six main categories: organic acids, fatty acids, amino acids, esters, sugars, and unknowns. The mean abundances of 25 of these metabolites were significantly different between the two species, providing insights into interspecies variation in floral chemistry. Using the PlantSEED biochemistry database, we found that the majority of these metabolites are involved in biosynthetic pathways. Finally, we explored the annotated genome of A. coerulea, using the PlantSEED pipeline and reconstructed the metabolic network of Aquilegia. This network, which contains the metabolic pathways involved in generating the observed chemical variation, is now publicly available from the DOE Systems Biology Knowledge Base (KBase; http://kbase.us).     

Shared Segment Analysis and Next-Generation Sequencing Implicates the Retinoic Acid Signaling Pathway in Total Anomalous Pulmonary Venous Return (TAPVR)

Most isolated congenital heart defects are thought to be sporadic and are often ascribed to multifactorial mechanisms with poorly understood genetics. Total Anomalous Pulmonary Venous Return (TAPVR) occurs in 1 in 15,000 live-born infants and occurs either in isolation or as part of a syndrome involving aberrant left-right development. Previously, we reported causative links between TAVPR and the PDGFRA gene. TAPVR has also been linked to the ANKRD1/CARP genes. However, these genes only explain a small fraction of the heritability of the condition. By examination of phased single nucleotide polymorphism genotype data from 5 distantly related TAPVR patients we identified a single 25 cM shared, Identical by Descent genomic segment on the short arm of chromosome 12 shared by 3 of the patients and their obligate-carrier parents. Whole genome sequence (WGS) analysis identified a non-synonymous variant within the shared segment in the retinol binding protein 5 (RBP5) gene. The RBP5 variant is predicted to be deleterious and is overrepresented in the TAPVR population. Gene expression and functional analysis of the zebrafish orthologue, rbp7, supports the notion that RBP5 is a TAPVR susceptibility gene. Additional sequence analysis also uncovered deleterious variants in genes associated with retinoic acid signaling, including NODAL and retinol dehydrogenase 10. These data indicate that genetic variation in the retinoic acid signaling pathway confers, in part, susceptibility to TAPVR.     

Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH₃ debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC₅₀ values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.     

N-alkylated cyclen cobalt(III) complexes of 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol DNA alkylating agent as hypoxia-activated prodrugs

A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).