Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.     

Expression in cereal plants of genes that inactivate Fusarium mycotoxins

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an omega enhancer sequence upstream of the start codon.     

Extended TAQing system for large‐scale plant genome reorganization

We previously developed a large‐scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double‐strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5′‐TCGA‐3′ sequences in the genome at higher temperatures (37–42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT‐rich regions. To overcome such problems we developed an extended TAQing (Ex‐TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant‐growing temperatures. We established the Ex‐TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5′‐TTAA‐3′ sequence which is highly abundant in the Arabidopsis genome. A synthetic intron‐spanning MseI gene, which was placed downstream of a heat‐shock‐inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end‐joining at MseI cleavage sites. The Ex‐TAQing system could induce large‐scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex‐TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.     

Competitive incorporation of homologous gene segments of influenza a virus into virions

By using two reporter protein-encoding virus-like RNAs derived from identical viral RNA (vRNA) segments, we assessed their incorporation efficiency into single progeny virions. Most plaques formed by the recombinant viruses that were generated in cells positive for both reporter genes expressed only one or the other protein. These results suggest that two virus-like RNAs encoding different reporter proteins compete for incorporation into virions, and individual influenza virions incorporate single, but not multiple, copies of homologous vRNA segments.     

Redox signaling loops in the unfolded protein response

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology.     

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag"

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.     

Potential Role of Transient Receptor Potential Channel M5 in Sensing Putative Pheromones in Mouse Olfactory Sensory Neurons

Based on pharmacological studies of chemosensory transduction in transient receptor potential channel M5 (TRPM5) knockout mice it was hypothesized that this channel is involved in transduction for a subset of putative pheromones in mouse olfactory sensory neurons (OSNs). Yet, in the same study an electroolfactogram (EOG) in the mouse olfactory epithelium showed no significant difference in the responses to pheromones (and odors) between wild type and TRPM5 knockout mice. Here we show that the number of OSNs expressing TRPM5 is increased by unilateral naris occlusion. Importantly, EOG experiments show that mice lacking TRPM5 show a decreased response in the occluded epithelia to putative pheromones as opposed to wild type mice that show no change upon unilateral naris occlusion. This evidence indicates that under decreased olfactory sensory input TRPM5 plays a role in mediating putative pheromone transduction. Furthermore, we demonstrate that cyclic nucleotide gated channel A2 knockout (CNGA2-KO) mice that show substantially decreased or absent responses to odors and pheromones also have elevated levels of TRPM5 compared to wild type mice. Taken together, our evidence suggests that TRPM5 plays a role in mediating transduction for putative pheromones under conditions of reduced chemosensory input.     

Tópicos y reflexiones sobre la reducción de ingresos hospitalarios: de la evidencia a la práctica

Demographic changes and the economic situation of the recent years have conditioned a turning point in health policies, which have decided to progressively prioritize chronicity care programs. Given that hospital costs were concentrated in attention to patients with chronic diseases, reduction on admissions is now a priority target.