MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).     

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.     

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.     

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.