Molecular mechanisms of insulin receptor substrate protein-mediated modulation of insulin signalling

The insulin receptor substrate (IRS) proteins act as important mediators of insulin action. Their regulation serves to augment the specificity of the insulin signalling cascade. They can be regulated--both positively and negatively--at the level of phosphorylation, and signalling through these proteins can be further modulated through the actions of SOCS (suppressor of cytokine signalling) proteins. Understanding the mechanisms of IRS regulation will provide further insight into the pathophysiology of insulin resistance and type 2 diabetes.     

Menopause in Blackfeet women--a life span perspective

A lifespan perspective, combining quantitative and qualitative approaches, is used to examine factors related to the timing of menopause in Blackfeet women of northern Montana (USA). Cross-sectional survey data demonstrate a median age at menopause using a status quo method of 51.6 years, and a mean age of 47.0 +/- 5.0 years among those women who had already experienced menopause. Age at menopause is inversely associated with age at menarche and having been breastfed, and positively associated with use of contraceptives, household income, and current or recent employment. Household income and age at menarche influence menopause age jointly in multivariate models. These and other patterns are examined in the lives of two women with very divergent ages at menopause. Although these data support an effect of early life influences on shaping reproductive trajectories that culminate in menopause, environmental factors and human agency during adult life may play a modifying role.     

Phosphinic,phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA(C) receptor antagonists

A number of amino acids bioisosterically derived from the specific GABA_A agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA_C ρ₁ receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid (2), was comparable with the standard GABA_C antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA_C versus GABA_A receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid (4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA_C antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA_C antagonist within the group of isonipecotic acid derived amino acids studied.     

The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium

The PAR-1 kinase plays a conserved role in cell polarity in C. elegans, Drosophila and mammals. We have investigated the role of PAR-1 in epithelial polarity by generating null mutant clones in the Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologues, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila, however, as null mutations in the single tau family member in the genome have no effect on the microtubule organisation in the follicle cells. Furthermore, PAR-1 activity stabilises microtubules, as microtubules in mutant cells depolymerise much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localisation of the microtubule plus ends, which are mislocalised to the centre of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organisation of microtubules. Although the direct targets of PAR-1 are unknown, we suggest that it functions by regulating the plus ends, possibly by capping them at the basal cortex.     

The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen

The anteroposterior axis of Drosophila is defined during oogenesis, when the polarisation of the oocyte microtubule cytoskeleton directs the localisation of bicoid and oskar mRNAs to the anterior and posterior poles, respectively. Although maternal-effect lethal and female-sterile screens have identified many mutants that disrupt these processes, these screens could not recover mutations in essential genes. Here we describe a genetic screen in germline clones for mutants that disrupt the localisation of GFP-Staufen in living oocytes, which overcomes this limitation. As Staufen localises to the posterior with oskar mRNA and to the anterior with bicoid mRNA, it acts as a marker for both poles of the oocyte, allowing the identification of mutants that affect the localisation of either mRNA, as well as mutants that disrupt oocyte polarity. Using this approach, we have identified 23 novel complementation groups on chromosome 3R that disrupt anteroposterior axis formation. Analyses of new alleles of spn-E and orb show that both SPN-E and ORB proteins are required to organise the microtubule cytoskeleton at stage 9, and to prevent premature cytoplasmic streaming. Furthermore, yps mutants partially suppress the premature cytoplasmic streaming of orb mutants. As orb, yps and spn-E encode RNA-binding proteins, they may regulate the translation of unidentified RNAs necessary for the polarisation of the microtubule cytoskeleton.     

Sorghum laxiflorum and S. macrospermum, the Australian native species most closely related to the cultivated S. bicolor based on ITS1 and ndhF sequence analysis of 25 Sorghum species

Australian species make up seventeen of the worlds twenty-five recognised species of Sorghum, with the genus separated into five sections: Eu-sorghum, Chaetosorghum, Heterosorghum, Para-sorghum and Stiposorghum. Whereas the genetic relationships within section Eu-sorghum are well known, little is known about the genetic relationships and crossabilities outside the primary genepool. We made a detailed investigation of phylogenetic relationships within Sorghum to identify wild species most closely related to cultivated sorghum (with outgroups Zea mays and Saccharum officinarum). The ribosomal ITS1 gene of ten species and the chloroplast ndhF gene from nineteen species were sequenced. Independent and combined analyses of the ITS1 and ndhF data sets were undertaken. The Eu-sorghum species were resolved into a strongly supported lineage by all three analyses, and included the Australian natives S. laxiflorum and S. macrospermum in the ITS1 and combined analyses. All remaining sorghum species were resolved into a second well-supported lineage in the combined analyses, although some internal relationships within this second lineage remain unresolved. We identified S. laxiflorum and S. macrospermum as the Australian species most closely related to cultivated sorghum. Our data support a reduction in the number of subgeneric sections from five to three: Eu-sorghum (unchanged); a combined Chaetosorghum/Heterosorghum to reflect the very close relationship between these two species; and a combined Para-sorghum/Stiposorghum section, thereby removing the unclear taxonomic and genetic boundaries between these species.     

Interaction of synapsin I with membranes

The synapsins (I, II, and III) comprise a family of peripheral membrane proteins that are involved in both regulation of neurotransmitter release and synaptogenesis. Synapsins are concentrated at presynaptic nerve terminals and are associated with the cytoplasmic surface of synaptic vesicles. Membrane-binding of synapsins involves interaction with both protein and lipid components of synaptic vesicles. Synapsin I binds rapidly and with high affinity to liposomes containing anionic lipids. The binding of bovine synapsin I to liposomes was studied using fluoresceinphosphatidyl-ethanolamine (FPE) to measure membrane electrostatic potential. Synapsin binding to liposomes caused a rapid increase in FPE fluorescence, indicating an increase in positive charge at the membrane surface. Synapsin I binding to monolayers resulted in a substantial increase in monolayer surface pressure. At higher initial surface pressures, the synapsin-induced increase in monolayer surface pressure is dependent on the presence of anionic lipids in the monolayer. Synapsin I also induced rapid aggregation of liposomes, but did not induce leakage of entrapped carboxyfluorescein, while other aggregation-inducing agents promoted extensive leakage. These results are in agreement with the presence of amphipathic stretches of amino acids in synapsin I that exhibit both electrostatic and hydrophobic interactions with membranes, and offer a molecular explanation for the high affinity binding of synapsin I to liposomes and for stabilization of membranes by synapsin I.     

Ubiquitous expression of a gene encoding for uncoupling protein isolated from the thermogenic inflorescence of the dead horse arum Helicodiceros muscivorus

Uncoupling proteins (UCPs) are a family of mitochondrial inner membrane proteins that have been implicated in heat production in mammalian cells. The inflorescences of several members of the arum lily family (Araceae) have also been shown to produce heat during flowering, but the involvement of UCP-mediated heat production in plants is not known. In this work a gene has been isolated termed HmUCPa that encodes for a putative uncoupling protein from Helicodiceros muscivorus, a highly thermogenic arum lily. RT-PCR analysis revealed that the expression of HmUCPa was ubiquitously found, both in thermogenic male florets and appendix, and the non-thermogenic female florets, spathe and club-shaped organs of the spadix. These results suggest that HmUCPa is not primarily involved in organ-specific heat production in H. muscivorus.