Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth of Helicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibitor-insensitive proteinases. Electrophoretic analysis of PI-induced HGP showed differential regulation of proteinase isoforms. Interestingly, HGP activity induced in response to dietary potato PI-II was inhibited by winged bean PIs. The optimized combination of potato PI-II and winged bean PIs identified in the present study and their proposed successive use has potential in developing H. armigera-resistant transgenic plants.     

Ribosomal Pausing and Scanning Arrest as Mechanisms of Translational Regulation from Cap-Distal Iron-Responsive Elements

Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE–IRP-1 complex. In contrast, cap-distal IRE–IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5′ untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE–IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.     

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.     

Suppression and synthetic‐lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin‐binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.     

Nitrous oxide (N<Subscript>2</Subscript>O) and subsequent open-label SSRI treatment of adolescents with depression (NOTAD): study protocol for a randomised controlled trial

Background

The first line of pharmacological treatment for severe depressive disorders in young people is selective serotonin reuptake inhibitors (SSRIs). However, beneficial clinical effects are rarely observed before several weeks into treatment. Nitrous oxide (N₂O) has a long-standing safety record for pain relief and has been used in adults and young people. In adults with severe treatment-resistant depression, a single dose of N₂O had significant antidepressant effects, with maximum antidepressant effects observed 24 h after administration. However, the antidepressant effects of N₂O have never been investigated in adolescents with a confirmed diagnosis of depression in a prospective trial. The aims of this study are to (1) investigate whether a single inhaled N₂O administration leads to antidepressant effects in adolescents with depression at 24 h, (2) determine whether combined N₂O and SSRI administration (commenced after N₂O intervention) provides a clinically significant improvement in mood over and above the benefits from SSRI administration alone, and, (3) investigate whether the effect seen following N₂O administration can be used as a predictor of SSRI treatment response.

Methods/design

In this study, we will use a single-blind, randomised, placebo-controlled design. Patients aged between 12 and 17 years with major depressive disorder will be recruited. This study will consist of two phases: phase A and phase B. During phase A, participants will be randomised to receive either inhaled N₂O or placebo (air) for 1 h. In phase B, participants will receive open-label pharmacological treatment with the SSRI fluoxetine and will be followed over a 12-week period. Participants will undertake mood assessments at 2 and 24 h after N₂O or placebo administration (phase A) and weekly during the 12-week follow up in phase B.

Discussion

We expect an antidepressant effect from a single dose of inhaled N₂O compared with placebo at 24 h after administration. Additionally, we expect that subjects treated with N₂O will also show greater improvements than the placebo group after 6 and 12 weeks into fluoxetine treatment because of potential additive antidepressant effects. Such findings would be of clinical importance because currently children and adolescents often do not experience any symptom alleviation for several weeks following the initiation of SSRIs.

Trial registration

Australian and New Zealand Clinical Trials Registry, ACTRN12616001568404. Registered on 14 November 2016.

     

Plastid signalling to the nucleus and beyond

Communication between the compartments or organelles of cells is essential for plant growth and development. There is an emerging understanding of signals generated within energy-transducing organelles, such as chloroplasts and mitochondria, and the nuclear genes that respond to them, a process known as retrograde signalling. A recent series of unconnected breakthroughs have given scientists a glimpse inside the 'black box' of organellar signalling thanks to the identification of some of the factors involved in generating and propagating signals to the nucleus and, in some instances, systemically throughout photosynthetic tissues. This review will focus on recent developments in our understanding of retrograde and systemic signals generated by organelles, with an emphasis on chloroplasts.     

Proteomics and diagnostics: Let's Get Specific, again

DNA array technology has changed all discussions about proteomics. Whole genome arrays allow unbiased experimentation, and the surprises that flow from those approaches. 'Whole proteome' proteomics is not possible today, and might never be possible unless experiments are guided by careful evaluation of reagent specificity. In this paper we explore some possible ways to increase the content of proteomic analysis.     

Novel insulin thiomer nanoparticles: in vivo evaluation of an oral drug delivery system

It was aim of the study to investigate the in vivo potential of a novel insulin-thiomer complex nanoparticulate delivery system. Insulin loaded nanoparticles were obtained by the formation of hydrogen bonds between poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid)-cysteine (PAA-Cys) or poly(acrylic acid) (PAA), respectively, in the presence of insulin. Dissolution behavior of insulin from tablets as well as nanoparticulate suspensions was evaluated in vitro. Serum insulin concentrations and reduction of blood sugar values were determined after oral administration of nanoparticles formulated as enteric coated tablets and suspensions. Results displayed a low serum insulin concentration and pharmacological efficacy in terms of blood sugar reduction after oral administration of enteric coated tablets. On the contrary, nanoparticulate suspensions led to significant serum insulin concentrations. Furthermore a 2.3-fold improvement of the AUC of insulin could be achieved due to the use of thiolated PAA instead of unmodified PAA. In addition, a blood sugar reduction of 22% was observed. Results demonstrate that this novel complex nanoparticulate formulation is an encouraging new attempt toward the noninvasive delivery of peptide drugs.