Two forms of the GABAA receptor distinguished by anion-exchange chromatography

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.     

A small RNA makes a <Emphasis Type="Italic">Bic</Emphasis> difference

The first highly specific knockouts of a microRNA, miR155, in mice result in multiple defects in adaptive immunity, and also show the feasibility of investigating at least some microRNAs by gene knockout.     

Effects of N-acetylaspartylglutamate (NAAG) peptidase inhibition on release of glutamate and dopamine in prefrontal cortex and nucleus accumbens in phencyclidine model of schizophrenia

The "glutamate" theory of schizophrenia emerged from the observation that phencyclidine (PCP), an open channel antagonist of the NMDA subtype of glutamate receptor, induces schizophrenia-like behaviors in humans. PCP also induces a complex set of behaviors in animal models of this disorder. PCP also increases glutamate and dopamine release in the medial prefrontal cortex and nucleus accumbens, brain regions associated with expression of psychosis. Increased motor activation is among the PCP-induced behaviors that have been widely validated as models for the characterization of new antipsychotic drugs. The peptide transmitter N-acetylaspartylglutamate (NAAG) activates a group II metabotropic receptor, mGluR3. Polymorphisms in this receptor have been associated with schizophrenia. Inhibitors of glutamate carboxypeptidase II, an enzyme that inactivates NAAG following synaptic release, reduce several behaviors induced by PCP in animal models. This research tested the hypothesis that two structurally distinct NAAG peptidase inhibitors, ZJ43 and 2-(phosphonomethyl)pentane-1,5-dioic acid, would elevate levels of synaptically released NAAG and reduce PCP-induced increases in glutamate and dopamine levels in the medial prefrontal cortex and nucleus accumbens. NAAG-like immunoreactivity was found in neurons and presumptive synaptic endings in both regions. These peptidase inhibitors reduced the motor activation effects of PCP while elevating extracellular NAAG levels. They also blocked PCP-induced increases in glutamate but not dopamine or its metabolites. The mGluR2/3 antagonist LY341495 blocked these behavioral and neurochemical effects of the peptidase inhibitors. The data reported here provide a foundation for assessment of the neurochemical mechanism through which NAAG achieves its antipsychotic-like behavioral effects and support the conclusion NAAG peptidase inhibitors warrant further study as a novel antipsychotic therapy aimed at mGluR3.     

The Fractionated Orthology of Bs2 and Rx/Gpa2 Supports Shared Synteny of Disease Resistance in the Solanaceae

Comparative genomics provides a powerful tool for the identification of genes that encode traits shared between crop plants and model organisms. Pathogen resistance conferred by plant R genes of the nucleotide-binding–leucine-rich-repeat (NB–LRR) class is one such trait with great agricultural importance that occupies a critical position in understanding fundamental processes of pathogen detection and coevolution. The proposed rapid rearrangement of R genes in genome evolution would make comparative approaches tenuous. Here, we test the hypothesis that orthology is predictive of R-gene genomic location in the Solanaceae using the pepper R gene Bs2. Homologs of Bs2 were compared in terms of sequence and gene and protein architecture. Comparative mapping demonstrated that Bs2 shared macrosynteny with R genes that best fit criteria determined to be its orthologs. Analysis of the genomic sequence encompassing solanaceous R genes revealed the magnitude of transposon insertions and local duplications that resulted in the expansion of the Bs2 intron to 27 kb and the frequently detected duplications of the 5′-end of R genes. However, these duplications did not impact protein expression or function in transient assays. Taken together, our results support a conservation of synteny for NB–LRR genes and further show that their distribution in the genome has been consistent with global rearrangements.R genes have a central role in plant disease resistance to mediate pathogen detection and response (Martin et al. 2003; Glazebrook 2005). Although R genes are only one of the components required for these responses, they are consistently identified as a critical determinant for qualitative and quantitative resistance (Fluhr 2001; Wisser et al. 2006). The structure, mechanism of action, and evolution of this gene family are still being elucidated and are critical issues for a more efficient deployment of disease resistances in agricultural crops (McDowell and Simon 2006; Takken et al. 2006; Friedman and Baker 2007; van Ooijen et al. 2007).Comparative studies of sequence similarity between plant R proteins and proteins of innate immunity in animals have made important contributions toward understanding R-protein structure, the role of individual protein domains, and the mechanism by which R proteins identify and respond to foreign proteins (Nurnberger et al. 2004; Takken et al. 2006; Rairdan and Moffett 2007). Both share a central nucleotide-binding (NB) site and a region of homology termed the “ARC” domain (collectively referred to as the NB–ARC) (van der Biezen and Jones 1998; Rairdan and Moffett 2007). The plant counterparts have a highly variable leucine-rich-repeat (LRR) domain at the C terminus and, at the N terminus, either a domain with homology to the Toll and interleukin-1 receptors (TIR) or lack this feature, instead possessing a domain that may include a coiled-coil motif. Due to uncertainty regarding the presence of a coiled-coil motif, this class of NB–LRRs is often referred to as non-TIR proteins. The LRR domains are highly variable and tend to be under diversifying selection to adapt to continually changing pathogen proteins (Meyers et al. 1998b; Michelmore and Meyers 1998; Mondragon-Palomino et al. 2002). Other conserved patterns have been identified in the N terminus of non-TIR proteins, most notably, an EDxxD motif that mediates an intramolecular interaction (Rairdan et al. 2008). The interaction with cellular factors is mediated by the N-terminal domains of NB–LRR proteins although domain-swapping experiments between closely related NB–LRR proteins have shown that recognition specificity is determined by the LRR domains (Rairdan and Moffett 2007; van Ooijen et al. 2007).The clustering of R genes has provided both insight into their ability to evolve rapidly and challenges to their identification and cloning. R genes often occur in clusters of tandem duplications that can span several megabases and include a multitude of copies of functional R genes, pseudogenes, and other genes within the clusters (Meyers et al. 1998a; Kuang et al. 2004; Smith et al. 2004). Of the various modes of evolution ascribed to these clusters, sequence exchange between R genes within the cluster by unequal crossing over or illegitimate recombination is especially noteworthy (Michelmore and Meyers 1998; Ellis et al. 2000; Hulbert et al. 2001; McDowell and Simon 2006; Friedman and Baker 2007; Wicker et al. 2007). Under stress conditions, transposon activation, recombination activation, and chromatin modifications related to small RNAs may be induced (Levy et al. 2004; Friedman and Baker 2007; Yi and Richards 2007).Two distinct models for the genomewide arrangement and distribution of NB–LRR genes and these clusters have been proposed. The first predicts rapid rearrangement of R-gene distribution during genome evolution, yielding poor conservation of R-gene locations (Leister et al. 1998; Richly et al. 2002; Meyers et al. 2003). Indeed, in monocots, extensive loss of genomewide R-gene colinearity has been attributed to frequent R-gene duplication and ectopic transposition (Gale and Devos 1998; Paterson et al. 2003). In contrast, the second model supports genomewide conservation of R-gene distribution maintained during speciation. According to this model, most duplication and recombination of R-gene sequences should occur within restricted chromosomal regions, yielding clusters of closely related R-gene sequences. The resulting orthology relationships (homologs related by speciation, not duplication) are complex due to “fractionation” (repeated cycles of duplication, deletion, and recombination) but can, as we have previously shown, be reconstructed (Grube et al. 2000b). Analysis of R genes using the complete Arabidopsis thaliana genome sequence supports this model and accounts for the consensus of NB–LRR sequences (Baumgarten et al. 2003). Resistance to a particular pathogen type is not conserved, and highly similar NB–LRR proteins may confer resistance to very different pathogens (Grube et al. 2000b).Bs2 encodes a non-TIR NB–LRR protein identified in Capsicum chacoense that confers resistance to the bacterium Xanthomonas campestris pv. vesicatoria. This R gene has greatest sequence identity to Rx and Gpa2 in potato, which confer resistance to a virus and nematode, respectively (Bendahmane et al. 1999; Tai et al. 1999b; van der Vossen et al. 2000). Despite the difference in the pathogens recognized by these genes, they are distinguishable from all other known R genes by marked sequence and structural features. In this study, we demonstrate that these three R genes are derived from syntenic regions in solanaceous genomes as predicted by our model of conservation of synteny. In performing these comparisons, we explore conserved amino acid patterns associated with proteins of the non-TIR family and the local genomic context of R genes of the Solanaceae. Finally, advances in the development of the Solanaceae as a system for comparative genomics highlight a role for chromosomal rearrangements in R-gene distribution throughout plant genomes.     

The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.     

Relationship of Salt Marsh Vegetation Zonation to Spatial Patterns in Soil Moisture, Salinity, and Topography

An intertidal San Francisco Bay salt marsh was used to study the spatial relationships between vegetation patterns and hydrologic and edaphic variables. Multiple abiotic variables were represented by six metrics: elevation, distance to major tidal channels and to the nearest channel of any size, edaphic conditions during dry and wet circumstances, and the magnitude of tidally induced changes in soil saturation and salinity. A new approach, quantitative differential electromagnetic induction (Q-DEMI), was developed to obtain the last metric. The approach converts the difference in soil electrical conductivity (ECa) between dry and wet conditions to quantitative maps of tidally induced changes in root zone soil water content and salinity. The result is a spatially exhaustive map of edaphic changes throughout the mapped area of the ecosystem. Spatially distributed data on the six metrics were used to explore two hypotheses: (1) multiple abiotic variables relevant to vegetation zonation each exhibit different, uncorrelated, spatial patterns throughout an intertidal salt marsh; (2) vegetation zones and habitats of individual plant species are uniquely characterized by different combinations of key metrics. The first hypothesis was supported by observed, uncorrelated spatial variability in the metrics. The second hypothesis was supported by binary logistic regression models that identified key vegetation zone and species habitat characteristics from among the six metrics. Based on results from 108 models, the Q-DEMI map of saturation and salinity change was the most useful metric of those tested for distinguishing different vegetation zones and plant species habitats in the salt marsh.     

Driver Ants Invading a Termite Nest: Why Do the Most Catholic Predators of All Seldom Take This Abundant Prey?

Driver ants ( i.e. , epigaeic species in the army ant genus Dorylus , subgenus Anomma ) are among the most extreme polyphagous predators, but termites appear to be conspicuously absent from their prey spectrum and attacks by driver ants on termite nests have not yet been described. Here, we report a Dorylus ( Anomma ) rubellus attack on a colony of the fungus-growing termite Macrotermes subhyalinus that was observed during the dry season in a savannah habitat in Nigeria's Gashaka National Park. It was estimated that several hundred thousand termites (probably more than 2.4 kg dry mass) were retrieved. The apparent rarity of driver ant predation on Macrotermes nests may be explained by different habitat requirements, by the fact that these ants mostly forage aboveground, by efficient termite defense behavior and nest architecture that make entry into the nest difficult, and finally by driver ant worker morphology, which differs remarkably from that of subterranean Dorylus species that regularly invade and destroy termite colonies.     

Comparison of UV inactivation of spores of three encephalitozoon species with that of spores of two DNA repair-deficient Bacillus subtilis biodosimetry strains

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m(2), respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.