Chloroplast 2010: a database for large-scale phenotypic screening of Arabidopsis mutants

Large-scale phenotypic screening presents challenges and opportunities not encountered in typical forward or reverse genetics projects. We describe a modular database and laboratory information management system that was implemented in support of the Chloroplast 2010 Project, an Arabidopsis (Arabidopsis thaliana) reverse genetics phenotypic screen of more than 5,000 mutants (http://bioinfo.bch.msu.edu/2010_LIMS; www.plastid.msu.edu). The software and laboratory work environment were designed to minimize operator error and detect systematic process errors. The database uses Ruby on Rails and Flash technologies to present complex quantitative and qualitative data and pedigree information in a flexible user interface. Examples are presented where the database was used to find opportunities for process changes that improved data quality. We also describe the use of the data-analysis tools to discover mutants defective in enzymes of leucine catabolism (heteromeric mitochondrial 3-methylcrotonyl-coenzyme A carboxylase [At1g03090 and At4g34030] and putative hydroxymethylglutaryl-coenzyme A lyase [At2g26800]) based upon a syndrome of pleiotropic seed amino acid phenotypes that resembles previously described isovaleryl coenzyme A dehydrogenase (At3g45300) mutants. In vitro assay results support the computational annotation of At2g26800 as hydroxymethylglutaryl-coenzyme A lyase.     

Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate

Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions of people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg²⁺ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of Escherichia coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA^Met complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.     

Omega-3 fatty acids modulate collagen signaling in human platelets

Dietary intake of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in cardioprotective benefits. However, the cellular and physiological bases for these benefits remain unclear. We hypothesized that EPA and DHA treatments would interfere with collagen-mediated platelet signaling. Thirty healthy volunteers received 28 days of 3.4 g/d EPA+DHA with and without a single dose of aspirin. Clinical hematologic parameters were then measured along with assays of collagen-stimulated platelet activation and protein phosphorylation. Omega-3 therapy led to a small but significant reduction in platelets (6.3%) and red blood cells (1.7%), but did not impair clinical time-to-closure assays. However, collagen-mediated platelet signaling events of integrin activation, α-granule secretion, and phosphatidylserine exposure were all reduced by roughly 50% after omega-3 incorporation, and collagen-induced tyrosine phosphorylation was significantly impaired. The diminished platelet response to collagen may account for some of the cardioprotective benefits provided by DHA and EPA.     

Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes

The funny current, I(f), in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. I(f) is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of I(f) occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of I(f) and that PKA can directly phosphorylate HCN4. In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on I(f) in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated I(f) via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C, or phosphatidylinositol-4,5-bisphosphate. The off-target activation of I(f) by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes.     

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training

We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.     

13C-pyruvate imaging reveals alterations in glycolysis that precede c-Myc-induced tumor formation and regression

Tumor cells have an altered metabolic phenotype characterized by increased glycolysis and diminished oxidative phosphorylation. Despite the suspected importance of glycolysis in tumorigenesis, the temporal relationship between oncogene signaling, in?vivo tumor formation, and glycolytic pathway activity is poorly understood. Moreover, how glycolytic pathways are altered as tumors regress remains unknown. Here, we use a switchable model of Myc-driven liver cancer, along with hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) to visualize glycolysis in de novo tumor formation and regression. LDHA abundance and activity in tumors is tightly correlated to?in?vivo pyruvate conversion to lactate and is rapidly inhibited as tumors begin to regress, as are numerous glycolysis pathway genes. Conversion of pyruvate to alanine predominates in precancerous tissues prior to observable morphologic or histological changes. These results demonstrate that metabolic changes precede tumor formation and regression and are directly linked to the activity of a single oncogene.