Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.     

Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomatal guard cells

Components of the polyphosphoinositide signalling pathway have been identified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to release of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca²⁺. 'Isolated' epidermal strips of C. communis (in which all cells other than guard cells have been killed by treatment at low pH) were radiolabelled with myo -[2n-³H]inositol or [³²P]orthophosphate for 17–18 h. The phosphoinositides and inositol phosphates were extracted. Phosphoinositides were deacylated and the head groups resolved by HPLC. The water-soluble products generated by mild periodate cleavage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, PtdIns3 P , PtdIns4 P , PtdIns(3,4) P ₂ and PtdIns(4,5) P ₂. Thex inositol phosphates were resolved by HPLC. Preliminary analysis of HPLC-purified putative inositol phosphate fractions resulted in the identification of each inositol phosphate class, that is, Ins P , Ins P ₂, Ins P ₃, Ins P ₄, Ins P ₅ and Ins_P6. Many of these inositol phosphates occurred in different isomeric forms. The presence of 3-phosphorylated phosphoinositides suggests that they may have a role in signalling in stomatal guard cells.     

microRNAs mature with help from cancer biology

A report of the Keystone Symposium on Molecular and Cellular Biology, 'MicroRNA and Cancer', Keystone, Colorado, USA, 10-15 June 2009.     

Functional anatomy of the Drosophila microRNA-generating enzyme

In Drosophila melanogaster, the multidomain RNase III Dicer-1 (Dcr-1) functions in tandem with the double-stranded (ds)RNA-binding protein Loquacious (Loqs) to catalyze the maturation of microRNAs (miRNAs) from precursor (pre)-miRNAs. Here we dissect the molecular mechanism of pre-miRNA processing by the Dcr-1-Loqs complex. The tandem RNase III (RIII) domains of Dcr-1 form an intramolecular dimer such that one RIII domain cleaves the 3' strand, whereas the other cuts the 5' strand of pre-miRNA. We show that the functional core of Dcr-1 consists of a DUF283 domain, a PAZ domain, and two RIII domains. Dcr-1 preferentially associates with the Loqs-PB splice isoform. Loqs-PB uses the second dsRNA-binding domain to bind pre-miRNA and the third dsRNA-binding domain to interact with Dcr-1. Both domains of Loqs-PB are required for efficient miRNA production by enhancing the affinity of Dcr-1 for pre-miRNA. Thus, our results provide further insights into the functional anatomy of the Drosophila miRNA-generating enzyme.     

Estrogen attenuates postexercise HSP70 expression in skeletal muscle

Exercise has been demonstrated as aphysiological inducer of heat shock protein (HSP)70. Many of theproposed signals of this response exhibit sexual dimorphism. Thus thepresent objectives were to determine whether HSP70 induction afterexercise exhibits gender specificity and to elucidate the mechanismsunderlying such a phenomenon. Postexercise HSP70 induction in skeletalmuscle was greater in male than female rats at the level of protein and mRNA (P = 0.005). Moreover, placebo-treatedovariectomized animals demonstrated a greater HSP70 response toexercise than those treated with estrogen (P = 0.015 and 0.019 for protein and mRNA, respectively). These findings indicatethat the gender-specific HSP70 response to exercise is mediated by thefemale-specific hormone estrogen. Compounds structurally related to17-estradiol, the major endogenous estrogen, but which do notactivate the estrogen receptor, also attenuated HSP70 induction withexercise (P < 0.01), indicating a nongenomic hormonalmechanism. These findings highlight a specific example of thebiological differences between males and females and reiterate thephysiological effects of sex hormones extending beyond their roles inreproductive function.

     

Imaging gene expression using oligonucleotides and peptide nucleic acids

The development of methods for non-invasive, real-time imaging of gene expression would provide powerful tools for biomedical research and medical diagnostics. A broadly applicable strategy for achieving this goal is the use of complementary oligonucleotide probes for recognition of mRNA. The major challenge for molecular imaging is the development of specific and efficient transducers for signaling probe-target interaction. This review summarizes the strengths and limitations of reported molecular approaches for imaging of mRNA expression and discusses the challenges to development of in vivo methods.     

Challenges for RNAi in vivo

Synthetic small interfering RNA (siRNA) has become a valuable tool for investigating gene function in cell culture. This success has led to high expectations for siRNA as a tool for in vivo investigation and as a platform for therapeutic development. siRNA in cell culture owes much of its success to years of development of traditional antisense oligonucleotides, and in vivo applications will also benefit from previous experience in this regard. However, the duplex nature of siRNA presents significant obstacles that will need to be overcome. Here, we discuss the current status of in vivo siRNA technology and describe some of the barriers to widespread application of RNAi-mediated gene silencing in mammals.     

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.     

Metabolism of 3- and 4-phosphorylated phosphatidylinositols in stomatal guard cells of Commelina communis L.

Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants. A non-equilibrium [³²P]P_i labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [³²P]P_i. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP₂) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [³²P]P_i label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P₂ by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type. The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the ³²P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P₂] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P₂, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P₂ was quicker than that of PtdIns(4,5)P₂, and in mass terms PtdIns(3,4)P₂ appeared to predominate over PtdIns(4,5)P₂. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P₂ show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P₂ in addition to those previously indicated for PtdIns(4,5)P₂ in stomatal guard cells.     

Isoproterenol potentiates exercise-induction of Hsp70 in cardiac and skeletal muscle

The response to exercise stress is characterized by an increase in circulating catecholamines and rapid synthesis of the inducible member of the 70 kDa family of heat shock proteins (Hsp70). Cell culture studies indicate that Hsp70 expression is influenced by beta-adrenergic receptor intermediates including cyclic AMP (cAMP) and cAMP dependent protein kinase (PKA). Thus, in the present investigation, the effect of a beta-adrenergic agonist, isoproterenol (ISO; 10 mg/kg) and a beta-adrenergic antagonist, nadolol (NAD; 25 mg/kg), on the in vivo expression of Hsp70 in rodent cardiac and skeletal muscle following moderate (MOD; 17 m/min) and exhaustive (EXH; 30 m/min) exercise was examined. While ISO alone did not induce Hsp70 synthesis, ISO treatment potentiated Hsp70 expression following MOD in the white vastus and heart (395+/-29 and 483+/-29% greater than control respectively, P < 0.05). Furthermore, this effect was reversed with combined beta-adrenergic agonist and antagonist treatment (ISO+NAD) indicating that the isoproterenol induced increase in post-exercise Hsp70 expression was mediated via beta-adrenergic receptor activity. However, there were no differences in Hsp70 levels among treatment groups following EXH. The failure of NAD to attenuate Hsp70 accumulation following EXH suggests that beta-adrenergic receptor activity is not the main signal in the induction of Hsp70 following exercise. Hsp70 induction was dependent on exercise intensity and ISO administration prior to MOD resulted in Hsp70 levels similar to those observed following EXH. The results from the present investigation indicate that beta-adrenergic receptor stimulation does not induce Hsp70 synthesis per se, but may be one factor involved in the complex regulation of the stress response to exercise in vivo.