Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu₅ NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu₅ NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu₅ versus DAT.     

Optimal window of caloric restriction onset limits its beneficial impact on T‐cell senescence in primates

We have recently shown in non‐human primates that caloric restriction (CR) initiated during adulthood can delay T‐cell aging and preserve naïve CD8 and CD4 T cells into advanced age. An important question is whether CR can be initiated at any time in life, and whether age at the time of onset would modulate the beneficial effects of CR. In the current study, we evaluated the impact of CR started before puberty or during advanced age on T‐cell senescence and compared it to the effects of CR started in early adulthood. Our data demonstrate that the beneficial effects of adult‐onset CR on T‐cell aging were lost by both early and late CR onset. In fact, some of our results suggest that inappropriate initiation of CR may be harmful to the maintenance of T‐cell function. This suggests that there may be an optimal window during adulthood where CR can delay immune senescence and improve correlates of immunity in primates.     

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).     

Role of prostaglandin E2 in the induction of nonspecific T lymphocyte suppressor activity

Activated human monocytes and concanavalin A (Con A)-activated T lymphocytes are known to suppress T and B lymphocyte proliferation and B cell maturation into immunoglobulin-producing cells. We have now shown that monocyte suppressive activity is predominantly mediated through release of prostaglandin E2 (PGE2), which is active only in the presence of a "short-lived," radiosensitive T lymphocyte subset. PGE2, at high concentration, can activate T suppressor lymphocytes (TS), which display the same characteristics as Con A-activated TS lymphocytes. Moreover, Con A activation of TS lymphocytes was obtained only in the presence of PGE2, as specific anti-PGE2 antiserum or indomethacin prevented TS activation; this suggested a double signal as a prerequisite for activation of the nonspecific TS cell subset. We propose that TS lymphocytes modified by Con A become sensitive to small amounts of PGE2 produced by monocytes that must be present during the Con A-stimulated activation phase of suppressive cells.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.     

Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

Preemption of space can lead to intransitive coexistence of competitors

Intransitive competition has the potential to be a powerful contributor to species coexistence, but there are few proposed biological mechanisms that could create intransitivities in natural communities. Using a three‐species model of competition for space, we demonstrate a mechanism for coexistence that combines a colonization–competition tradeoff between two species with the ability of a third species to preempt space from the other competitors. The combination of differential abilities to colonize, preempt, and overtake space creates a community where no single species can exclude both of its competitors. The dynamics of this kind of community are analogous to rock‐paper‐scissors competition, and the three‐species community can persist even though not all pairs of species can coexist in isolation. In distinction to prior results, this is a mechanism of intransitivity that does not require nonhierarchical local interference competition. We present parameter estimates from a subtidal marine community illustrating how documented competitive traits can lead to preemption‐based intransitivities in natural communities, and we describe methods for an empirical test of the occurrence of this mechanism.     

Enhanced AFLP genome scans detect local adaptation in high-altitude populations of a small rodent (Microtus arvalis)

Adaptation to adverse environmental conditions such as high altitude requires physiological and/or morphological changes. Genome scans provide a means to identify the genetic basis of such adaptations without previous knowledge about the particular genetic variants or traits under selection. In this study, we scanned 3027 amplified fragment length polymorphisms (AFLP) in four populations of the common vole Microtus arvalis for loci associated with local adaptation and high altitude. We investigated voles from two populations at high elevation (~2000 m a.s.l.) representing the upper limit of the altitudinal distribution of the species and two geographically close low-altitude populations (<600 m a.s.l.). Statistical analysis incorporated a new Bayesian F(ST) outlier approach specifically developed for AFLP markers, which considers the intensity of AFLP bands instead of mere presence/absence and allows to derive population-based estimates of allele frequencies and F(IS) values. Computer simulations showed that this approach increases the statistical power of the detection of AFLP markers under selection almost to the power of single nucleotide polymorphism (SNP) data without compromising specificity. Our enhanced genome scan resulted in 20 prime candidate markers for positive selection, which show mostly extremely high allele frequency differences between the low- and high-altitude populations. The comparison of global- and pairwise-enhanced genome scans demonstrated further that very strong selective signatures may also be associated with single populations suggesting the importance of local adaptation in alpine populations of common voles.     

What is the best reference RNA? And other questions regarding the design and analysis of two-color microarray experiments

The reference design is a practical and popular choice for microarray studies using two-color platforms. In the reference design, the reference RNA uses half of all array resources, leading investigators to ask: What is the best reference RNA? We propose a novel method for evaluating reference RNAs and present the results of an experiment that was specially designed to evaluate three common choices of reference RNA. We found no compelling evidence in favor of any particular reference. In particular, a commercial reference showed no advantage in our data. Our experimental design also enabled a new way to test the effectiveness of pre-processing methods for two-color arrays. Our results favor using intensity normalization and foregoing background subtraction. Finally, we evaluate the sensitivity and specificity of data quality filters, and we propose a new filter that can be applied to any experimental design and does not rely on replicate hybridizations.