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Eukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. Here, we probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus [1-3]. We show that inactivation of Ned1, the phosphatidic acid phosphatase of the lipin family, by CDK phosphorylation is both necessary and sufficient to promote NE expansion required for "closed" mitosis in S. pombe. In contrast, Ned1 is not regulated during division in S. japonicus, thus limiting membrane availability and necessitating NE breakage. Interspecies gene swaps result in phenotypically normal divisions with the S. japonicus lipin acquiring an S. pombe-like mitotic phosphorylation pattern. Our results provide experimental evidence for the mitotic regulation of phosphatidic acid flux and suggest that the regulatory networks governing lipin activity diverged in evolution to give rise to strikingly dissimilar mitotic programs.
Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.
BACKGROUND - At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.
RESULTS - A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.
CONCLUSIONS - We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.
The structure, in particular symmetry, geometry and morphology of organic chelates coordinated with the lanthanide ions are analyzed in the present review. This is the first part of a complete presentation of a theoretical description of the properties of systems, which are widely used in technology, but most of all, in molecular biology and medicine. The discussion is focused on the symmetry and geometry of the cages, since these features play a dominant role in the spectroscopic activity of the lanthanides caged by organic chelates. At the same time, the spectroscopic properties require more formal presentation in the language of Racah algebra, and deserve a separate analysis. In addition to the parent systems of DOTA, DOTP, EDTMP and CDTMP presented here, their modifications by various antennas are analyzed. The conclusions that have a strong impact upon the theory of the energy transfer and the sensitized luminescence of these systems are based on the results of numerical density functional theory calculations.
Striatal medium spiny neurons (MSNs) receive glutamatergic afferents from the cerebral cortex and dopaminergic inputs from the substantia nigra (SN). Striatal dopamine loss decreases the number of MSN dendritic spines. This loss of spines has been suggested to reflect the removal of tonic dopamine inhibitory control over corticostriatal glutamatergic drive, with increased glutamate release culminating in MSN spine loss. We tested this hypothesis in two ways. We first determined in vivo if decortication reverses or prevents dopamine depletion-induced spine loss by placing motor cortex lesions 4 weeks after, or at the time of, 6-hydroxydopamine lesions of the SN. Animals were sacrificed 4 weeks after cortical lesions. Motor cortex lesions significantly reversed the loss of MSN spines elicited by dopamine denervation; a similar effect was observed in the prevention experiment. We then determined if modulating glutamate release in organotypic cocultures prevented spine loss. Treatment of the cultures with the mGluR2/3 agonist LY379268 to suppress corticostriatal glutamate release completely blocked spine loss in dopamine-denervated cultures. These studies provide the first evidence to show that MSN spine loss associated with parkinsonism can be reversed and point to suppression of corticostriatal glutamate release as a means of slowing progression in Parkinson's disease.
Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (approximately 300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000-30,000 cells.
Pharmacological inhibition or genetic deletion of cyclooxygenase (COX)-2, but not COX-1, has been shown to increase susceptibility to kainic acid (KA)-induced excitotoxicity. However, it is unclear if susceptibility to excitotoxins that act through other neurotransmitter receptors is altered by COX-2 inhibition. To further understand the involvement of COX-2 in regulating susceptibility to excitotoxicity, we investigated the effect of COX-2 deletion on excitotoxicity induced by peripheral injection of N-methyl-d-aspartate (NMDA, a specific agonist of the NMDA receptors) or lindane (a GABA(A) receptor antagonist). COX-2(-/-) mice injected intraperitoneally with NMDA (50-100mg/kg) exhibited significantly increased median seizure intensity when compared to COX-2(+/+) mice. Further, COX-2(-/-) mice exposed to NMDA showed neuronal damage, detected by Fluoro Jade B (FJB) staining, in the CA3 region of the hippocampus. There was no FJB staining nor any significant difference in median or maximal seizure intensity in COX-2(+/+) and COX-2(-/-) mice exposed to lindane. LC-MS/MS analysis of brain prostaglandin profile in COX-2(-/-) mice demonstrated a significant increase in PGF(2alpha), TXB(2), PGE(2) and PGD(2) expression 1h after administration of an excitotoxic dose of KA, but not of NMDA. Our findings demonstrate that COX-2 regulates susceptibility to KA and NMDA excitotoxicity, which directly activate glutamatergic neurotransmission, but not to lindane, which indirectly alters glutamatergic neurotransmission. Furthermore, increased levels of prostaglandins after seizures are associated with consistent manifestation of neuronal damage.
Seizure-induced damage elicits a loss of hippocampal neurons mediated to a great extent by the p75 neurotrophin receptor (NTR). Proneurotrophins, which are potent apoptosis-inducing ligands for p75(NTR), were increased in the hippocampus, particularly in astrocytes, by pilocarpine-induced seizures; and infusion of anti-pro-NGF dramatically attenuated neuronal loss after seizures. The p75(NTR) is expressed in many different cell types in the nervous system, and can mediate a variety of different cellular functions by recruiting specific intracellular binding proteins to activate distinct signaling pathways. In this study, we demonstrate that neurotrophin receptor-interacting factor (NRIF) mediates apoptotic signaling via p75(NTR) in hippocampal neurons in vitro and in vivo. After seizure-induced injury, NRIF(-/-) mice showed an increase in p75(NTR) expression in the hippocampus; however, these neurons failed to undergo apoptosis in contrast to wild-type mice. Treatment of cultured hippocampal neurons with proneurotrophins induced association of NRIF with p75(NTR) and subsequent translocation of NRIF to the nucleus, which was dependent on cleavage of the receptor. Neurons lacking NRIF were resistant to p75(NTR)-mediated apoptosis in vitro and in vivo. In addition, we demonstrate some mechanistic differences in p75(NTR) signaling in hippocampal neurons compared with other cell types. Overall, these studies demonstrate the requirement for NRIF to signal p75(NTR)-mediated apoptosis of hippocampal neurons and that blocking pro-NGF can inhibit neuronal loss after seizures.
The mosquito Anopheles gambiae is the principal Afrotropical vector for human malaria. A central component of its vectorial capacity is the ability to maintain sufficient populations of adults. During both adult and preadult (larval) stages, the mosquitoes depend on the ability to recognize and respond to chemical cues that mediate feeding and survival. In this study, we used a behavioral assay to identify a range of odorant-specific responses of An. gambiae larvae that are dependent on the integrity of the larval antennae. Parallel molecular analyses have identified a subset of the An. gambiae odorant receptors (AgOrs) that are localized to discrete neurons within the larval antennae and facilitate odor-evoked responses in Xenopus oocytes that are consistent with the larval behavioral spectrum. These studies shed light on chemosensory-driven behaviors and represent molecular and cellular characterization of olfactory processes in mosquito larvae. These advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in larval-stage mosquitoes to reduce the catastrophic effects of malaria and other diseases.
Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.