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Sample records for drosophila require pka

  1. The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila.

    Science.gov (United States)

    Cassar, Marlène; Sunderhaus, Elizabeth; Wentzell, Jill S; Kuntz, Sara; Strauss, Roland; Kretzschmar, Doris

    2018-02-06

    Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. We now show that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.

  2. A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons.

    Science.gov (United States)

    Vagnoni, Alessio; Bullock, Simon L

    2018-04-23

    Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles [1, 2]. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms [3-6], and has been proposed to contribute to the broader decline in neuronal function that occurs during aging [3, 5-7]. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. Here we provide evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, our results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. Our study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA).

    Science.gov (United States)

    Copf, Tijana

    2014-09-15

    Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons we test in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. We find that genetic manipulations of the PKA activity levels affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outposts and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative we find that PKA interacts genetically with the early endosomal pathway. We test if the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. We find that Drosophila cytoplasmic dynein components are direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. Our data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Early-onset sleep defects in Drosophila models of Huntington's disease reflect alterations of PKA/CREB signaling

    Science.gov (United States)

    Gonzales, Erin D.; Tanenhaus, Anne K.; Zhang, Jiabin; Chaffee, Ryan P.; Yin, Jerry C.P.

    2016-01-01

    Huntington's disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. Here, we report Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in our HD models. We were curious whether aberrations in PKA/CREB signaling were responsible for our early-onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, our data suggest that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention. PMID:26604145

  5. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    Science.gov (United States)

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  6. Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling.

    Science.gov (United States)

    Koschinski, Andreas; Zaccolo, Manuela

    2017-10-26

    cAMP is a ubiquitous second messenger responsible for the cellular effects of multiple hormones and neurotransmitters via activation of its main effector, protein kinase A (PKA). Multiple studies have shown that the basal concentration of cAMP in several cell types is about 1 μM. This value is well above the reported concentration of cAMP required to half-maximally activate PKA, which measures in the 100-300 nM range. Several hypotheses have been suggested to explain this apparent discrepancy including inaccurate measurements of intracellular free cAMP, inaccurate measurement of the apparent activation constant of PKA or shielding of PKA from bulk cytosolic cAMP via localization of the enzyme to microdomains with lower basal cAMP concentration. However, direct experimental evidence in support of any of these models is limited and a firm conclusion is missing. In this study we use multiple FRET-based reporters for the detection of cAMP and PKA activity in intact cells and we establish that the sensitivity of PKA to cAMP is almost twenty times lower when measured in cell than when measured in vitro. Our findings have important implications for the understanding of compartmentalized cAMP signalling.

  7. Context generalization in Drosophila visual learning requires the mushroom bodies

    Science.gov (United States)

    Liu, Li; Wolf, Reinhard; Ernst, Roman; Heisenberg, Martin

    1999-08-01

    The world is permanently changing. Laboratory experiments on learning and memory normally minimize this feature of reality, keeping all conditions except the conditioned and unconditioned stimuli as constant as possible. In the real world, however, animals need to extract from the universe of sensory signals the actual predictors of salient events by separating them from non-predictive stimuli (context). In principle, this can be achieved ifonly those sensory inputs that resemble the reinforcer in theirtemporal structure are taken as predictors. Here we study visual learning in the fly Drosophila melanogaster, using a flight simulator,, and show that memory retrieval is, indeed, partially context-independent. Moreover, we show that the mushroom bodies, which are required for olfactory but not visual or tactile learning, effectively support context generalization. In visual learning in Drosophila, it appears that a facilitating effect of context cues for memory retrieval is the default state, whereas making recall context-independent requires additional processing.

  8. Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

    Science.gov (United States)

    Battistone, M A; Da Ros, V G; Salicioni, A M; Navarrete, F A; Krapf, D; Visconti, P E; Cuasnicú, P S

    2013-09-01

    were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

  9. Drosophila Myc is required for normal DREF gene expression

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Seto, Hirokazu; Yamaguchi, Masamitsu

    2008-01-01

    The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm 4 /Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm 2 /dm 2 homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression

  10. Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

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    Rafael Falcón-Moya

    2018-06-01

    Full Text Available We elucidated the mechanisms underlying the kainate receptor (KAR-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs at synapses between axon terminals of parallel fibers (PF and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC. KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.

  11. Mushroom body glycolysis is required for olfactory memory in Drosophila.

    Science.gov (United States)

    Wu, Chia-Lin; Chang, Ching-Ching; Wu, Jie-Kai; Chiang, Meng-Hsuan; Yang, Chu-Huai; Chiang, Hsueh-Cheng

    2018-04-01

    Glucose catabolism, also known as glycolysis, is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules. The glycolysis process generates adenosine triphosphate as a byproduct. In this study, we investigated whether glycolysis plays a role in maintaining neuronal functions in the Drosophila mushroom bodies (MBs), which are generally accepted to be an olfactory learning and memory center. Our data showed that individual knockdown of glycolytic enzymes in the MBs, including hexokinase (HexA), phosphofructokinase (Pfk), or pyruvate kinase (PyK), disrupts olfactory memory. Whole-mount brain immunostaining indicated that pyruvate kinase is strongly expressed in the MB αβ, α'β', and γ neuron subsets. We conclude that HexA, Pfk, and PyK are required in each MB neuron subset for olfactory memory formation. Our data therefore indicates that glucose catabolism in the MBs is important for olfactory memory formation in Drosophila. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

    Science.gov (United States)

    Katchman, Alexander; Yang, Lin; Zakharov, Sergey I; Kushner, Jared; Abrams, Jeffrey; Chen, Bi-Xing; Liu, Guoxia; Pitt, Geoffrey S; Colecraft, Henry M; Marx, Steven O

    2017-08-22

    Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2.

  13. Functional requirements driving the gene duplication in 12 Drosophila species.

    Science.gov (United States)

    Zhong, Yan; Jia, Yanxiao; Gao, Yang; Tian, Dacheng; Yang, Sihai; Zhang, Xiaohui

    2013-08-15

    Gene duplication supplies the raw materials for novel gene functions and many gene families arisen from duplication experience adaptive evolution. Most studies of young duplicates have focused on mammals, especially humans, whereas reports describing their genome-wide evolutionary patterns across the closely related Drosophila species are rare. The sequenced 12 Drosophila genomes provide the opportunity to address this issue. In our study, 3,647 young duplicate gene families were identified across the 12 Drosophila species and three types of expansions, species-specific, lineage-specific and complex expansions, were detected in these gene families. Our data showed that the species-specific young duplicate genes predominated (86.6%) over the other two types. Interestingly, many independent species-specific expansions in the same gene family have been observed in many species, even including 11 or 12 Drosophila species. Our data also showed that the functional bias observed in these young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses. This study reveals the evolutionary patterns of young duplicates across 12 Drosophila species on a genomic scale. Our results suggest that convergent evolution acts on young duplicate genes after the species differentiation and adaptive evolution may play an important role in duplicate genes for adaption to ecological factors and environmental changes in Drosophila.

  14. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

    OpenAIRE

    Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

    1994-01-01

    Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the ...

  15. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Chris Snowdon

    Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  16. AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

    Science.gov (United States)

    Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W; Heinemann, Udo; Klussmann, Enno

    2016-07-01

    A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  17. G(o) Activation Is Required for Both Appetitive and Aversive Memory Acquisition in "Drosophila"

    Science.gov (United States)

    Madalan, Adrian; Yang, Xiao; Ferris, Jacob; Zhang, Shixing; Roman, Gregg

    2012-01-01

    Heterotrimeric G(o) is an abundant brain protein required for negatively reinforced short-term associative olfactory memory in "Drosophila". G(o) is the only known substrate of the S1 subunit of pertussis toxin (PTX) in fly, and acute expression of PTX within the mushroom body neurons (MB) induces a reversible deficit in associative olfactory…

  18. Matrix metalloproteinase 2 is required for ovulation and corpus luteum formation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Lylah D Deady

    2015-02-01

    Full Text Available Ovulation is critical for successful reproduction and correlates with ovarian cancer risk, yet genetic studies of ovulation have been limited. It has long been thought that the mechanism controlling ovulation is highly divergent due to speciation and fast evolution. Using genetic tools available in Drosophila, we now report that ovulation in Drosophila strongly resembles mammalian ovulation at both the cellular and molecular levels. Just one of up to 32 mature follicles per ovary pair loses posterior follicle cells ("trimming" and protrudes into the oviduct, showing that a selection process prefigures ovulation. Follicle cells that remain after egg release form a "corpus luteum (CL" at the end of the ovariole, develop yellowish pigmentation, and express genes encoding steroid hormone biosynthetic enzymes that are required for full fertility. Finally, matrix metalloproteinase 2 (Mmp2, a type of protease thought to facilitate mammalian ovulation, is expressed in mature follicle and CL cells. Mmp2 activity is genetically required for trimming, ovulation and CL formation. Our studies provide new insights into the regulation of Drosophila ovulation and establish Drosophila as a model for genetically investigating ovulation in diverse organisms, including mammals.

  19. PKA spectrum file

    Energy Technology Data Exchange (ETDEWEB)

    Kawai, M. [Toshiba Corp., Kawasaki, Kanagawa (Japan). Nuclear Engineering Lab.

    1997-03-01

    In the Japanese Nuclear Data Committee, the PKA/KERMA file containing PKA spectra, KERMA factors and DPA cross sections in the energy range between 10{sup -5} eV and 50 MeV is being prepared from the evaluated nuclear data. The processing code ESPERANT was developed to calculate quantities of PKA, KERMA and DPA from evaluated nuclear data for medium and heavy elements by using the effective single particle emission approximation (ESPEA). For light elements, the PKA spectra are evaluated by the SCINFUL/DDX and EXIFON codes, simultaneously with other neutron cross sections. The DPA cross sections due to charged particle emitted from light elements are evaluated for high neutron energy above 20 MeV. (author)

  20. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca(2+)/PKA crosstalk.

    Science.gov (United States)

    Schott, Micah B; Gonowolo, Faith; Maliske, Benjamin; Grove, Bryon

    2016-04-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca(2+)]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium "switch" that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca(2+) and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca(2+)/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca(2+) and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  1. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

    Science.gov (United States)

    Schott, Micah; Gonowolo, Faith; Maliske, Ben; Grove, Bryon

    2016-01-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca2+]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium “switch” that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca2+ and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca2+/PKA crosstalk was not observed in cells expressing a gravin mutant that resists calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  2. The Drosophila small GTPase Rac2 is required for normal feeding and mating behaviour.

    Science.gov (United States)

    Goergen, Philip; Kasagiannis, Anna; Schiöth, Helgi B; Williams, Michael J

    2014-03-01

    All multicellular organisms require the ability to regulate bodily processes in order to maintain a stable condition, which necessitates fluctuations in internal metabolics, as well as modifications of outward behaviour. Understanding the genetics behind this modulation is important as a general model for the metabolic modification of behaviour. This study demonstrates that the activity of the small GTPase Rac2 is required in Drosophila for the proper regulation of lipid storage and feeding behaviour, as well as aggression and mating behaviours. Rac2 mutant males and females are susceptible to starvation and contain considerably less lipids than controls. Furthermore, Rac2 mutants also have disrupted feeding behaviour, eating fewer but larger meals than controls. Intriguingly, Rac2 mutant males rarely initiate aggressive behaviour and display significantly increased levels of courtship behaviour towards other males and mated females. From these results we conclude that Rac2 has a central role in regulating the Drosophila homeostatic system.

  3. Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates.

    Science.gov (United States)

    Tillo, Shane E; Xiong, Wei-Hong; Takahashi, Maho; Miao, Sheng; Andrade, Adriana L; Fortin, Dale A; Yang, Guang; Qin, Maozhen; Smoody, Barbara F; Stork, Philip J S; Zhong, Haining

    2017-04-18

    Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. A sleep state in Drosophila larvae required for neural stem cell proliferation

    Science.gov (United States)

    Szuperak, Milan; Churgin, Matthew A; Borja, Austin J; Raizen, David M; Fang-Yen, Christopher

    2018-01-01

    Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation. PMID:29424688

  5. Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

    DEFF Research Database (Denmark)

    Deng, Wu-Min; Schneider, Martina; Frock, Richard

    2003-01-01

    The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan......, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell......, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics....

  6. Multiple Arginine Residues Are Methylated in Drosophila Mre11 and Required for Survival Following Ionizing Radiation.

    Science.gov (United States)

    Yuan, Qing; Tian, Ran; Zhao, Haiying; Li, Lijuan; Bi, Xiaolin

    2018-05-31

    Mre11 is a key player for DNA double strand break repair. Previous studies have shown that mammalian Mre11 is methylated at multiple arginines in its C-terminal Glycine-Arginine-Rich motif (GAR) by protein arginine methyltransferase PRMT1. Here, we found that the Drosophila Mre11 is methylated at arginines 559, 563, 565, and 569 in the GAR motif by DART1, the Drosophila homolog of PRMT1. Mre11 interacts with DART1 in S2 cells, and this interaction does not require the GAR motif. Arginines methylated Mre11 localizes exclusively in the nucleus as soluble nuclear protein or chromatin-binding protein. To study the in vivo functions of methylation, we generated the single Arg-Ala and all Arginines mutated flies. We found these mutants were sensitive to ionizing radiation. Furthermore, Arg-Ala mutated flies had no irradiation induced G2/M checkpoint defect in wing disc and eye disc. Thus, we provided evidence that arginines in Drosophila Mre11 are methylated by DART1 methytransferase and flies loss of arginine methylation are sensitive to irradiation. Copyright © 2018 Yuan et al.

  7. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    Science.gov (United States)

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  8. Bedu-Addo, PKA

    African Journals Online (AJOL)

    Bedu-Addo, PKA. Vol 32, No 1-2 (2013) - Articles Work-related Stress Among Ghanaian Bankers: Implications For Counselling. Abstract. ISSN: 0189-0263. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's Partners · Terms and Conditions of ...

  9. SNMP is a signaling component required for pheromone sensitivity in Drosophila.

    Science.gov (United States)

    Jin, Xin; Ha, Tal Soo; Smith, Dean P

    2008-08-05

    The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, we have identified a third component required for cVA reception: sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. We show that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, we show that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling.

  10. hemingway is required for sperm flagella assembly and ciliary motility in Drosophila.

    Science.gov (United States)

    Soulavie, Fabien; Piepenbrock, David; Thomas, Joëlle; Vieillard, Jennifer; Duteyrat, Jean-Luc; Cortier, Elisabeth; Laurençon, Anne; Göpfert, Martin C; Durand, Bénédicte

    2014-04-01

    Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left-right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604-amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.

  11. PKA and Apicomplexan Parasite Diseases.

    Science.gov (United States)

    Haidar, M; Ramdani, G; Kennedy, E J; Langsley, G

    2017-04-01

    The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum -infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata -infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma. © Georg Thieme Verlag KG Stuttgart · New York.

  12. Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

    Science.gov (United States)

    Garrity, P A; Rao, Y; Salecker, I; McGlade, J; Pawson, T; Zipursky, S L

    1996-05-31

    Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

  13. Insulin signaling is acutely required for long-term memory in Drosophila.

    Science.gov (United States)

    Chambers, Daniel B; Androschuk, Alaura; Rosenfelt, Cory; Langer, Steven; Harding, Mark; Bolduc, Francois V

    2015-01-01

    Memory formation has been shown recently to be dependent on energy status in Drosophila. A well-established energy sensor is the insulin signaling (InS) pathway. Previous studies in various animal models including human have revealed the role of insulin levels in short-term memory but its role in long-term memory remains less clear. We therefore investigated genetically the spatial and temporal role of InS using the olfactory learning and long-term memory model in Drosophila. We found that InS is involved in both learning and memory. InS in the mushroom body is required for learning and long-term memory whereas long-term memory specifically is impaired after InS signaling disruption in the ellipsoid body, where it regulates the level of p70s6k, a downstream target of InS and a marker of protein synthesis. Finally, we show also that InS is acutely required for long-term memory formation in adult flies.

  14. A presynaptic role for PKA in synaptic tagging and memory.

    Science.gov (United States)

    Park, Alan Jung; Havekes, Robbert; Choi, Jennifer Hk; Luczak, Vince; Nie, Ting; Huang, Ted; Abel, Ted

    2014-10-01

    Protein kinase A (PKA) and other signaling molecules are spatially restricted within neurons by A-kinase anchoring proteins (AKAPs). Although studies on compartmentalized PKA signaling have focused on postsynaptic mechanisms, presynaptically anchored PKA may contribute to synaptic plasticity and memory because PKA also regulates presynaptic transmitter release. Here, we examine this issue using genetic and pharmacological application of Ht31, a PKA anchoring disrupting peptide. At the hippocampal Schaffer collateral CA3-CA1 synapse, Ht31 treatment elicits a rapid decay of synaptic responses to repetitive stimuli, indicating a fast depletion of the readily releasable pool of synaptic vesicles. The interaction between PKA and proteins involved in producing this pool of synaptic vesicles is supported by biochemical assays showing that synaptic vesicle protein 2 (SV2), Rim1, and SNAP25 are components of a complex that interacts with cAMP. Moreover, acute treatment with Ht31 reduces the levels of SV2. Finally, experiments with transgenic mouse lines, which express Ht31 in excitatory neurons at the Schaffer collateral CA3-CA1 synapse, highlight a requirement for presynaptically anchored PKA in pathway-specific synaptic tagging and long-term contextual fear memory. These results suggest that a presynaptically compartmentalized PKA is critical for synaptic plasticity and memory by regulating the readily releasable pool of synaptic vesicles. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Drosophila Atlastin in motor neurons is required for locomotion and presynaptic function.

    Science.gov (United States)

    De Gregorio, Cristian; Delgado, Ricardo; Ibacache, Andrés; Sierralta, Jimena; Couve, Andrés

    2017-10-15

    Hereditary spastic paraplegias (HSPs) are characterized by spasticity and weakness of the lower limbs, resulting from length-dependent axonopathy of the corticospinal tracts. In humans, the HSP-related atlastin genes ATL1 - ATL3 catalyze homotypic membrane fusion of endoplasmic reticulum (ER) tubules. How defects in neuronal Atlastin contribute to axonal degeneration has not been explained satisfactorily. Using Drosophila , we demonstrate that downregulation or overexpression of Atlastin in motor neurons results in decreased crawling speed and contraction frequency in larvae, while adult flies show progressive decline in climbing ability. Broad expression in the nervous system is required to rescue the atlastin -null Drosophila mutant ( atl 2 ) phenotype. Importantly, both spontaneous release and the reserve pool of synaptic vesicles are affected. Additionally, axonal secretory organelles are abnormally distributed, whereas presynaptic proteins diminish at terminals and accumulate in distal axons, possibly in lysosomes. Our findings suggest that trafficking defects produced by Atlastin dysfunction in motor neurons result in redistribution of presynaptic components and aberrant mobilization of synaptic vesicles, stressing the importance of ER-shaping proteins and the susceptibility of motor neurons to their mutations or depletion. © 2017. Published by The Company of Biologists Ltd.

  16. Liquid facets-related (lqfR is required for egg chamber morphogenesis during Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Peter A Leventis

    Full Text Available Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR is an Epsin N-terminal homology (ENTH domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR. LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis.

  17. Orthodenticle is required for the development of olfactory projection neurons and local interneurons in Drosophila

    Directory of Open Access Journals (Sweden)

    Sonia Sen

    2014-07-01

    Full Text Available The accurate wiring of nervous systems involves precise control over cellular processes like cell division, cell fate specification, and targeting of neurons. The nervous system of Drosophila melanogaster is an excellent model to understand these processes. Drosophila neurons are generated by stem cell like precursors called neuroblasts that are formed and specified in a highly stereotypical manner along the neuroectoderm. This stereotypy has been attributed, in part, to the expression and function of transcription factors that act as intrinsic cell fate determinants in the neuroblasts and their progeny during embryogenesis. Here we focus on the lateral neuroblast lineage, ALl1, of the antennal lobe and show that the transcription factor-encoding cephalic gap gene orthodenticle is required in this lineage during postembryonic brain development. We use immunolabelling to demonstrate that Otd is expressed in the neuroblast of this lineage during postembryonic larval stages. Subsequently, we use MARCM clonal mutational methods to show that the majority of the postembryonic neuronal progeny in the ALl1 lineage undergoes apoptosis in the absence of orthodenticle. Moreover, we demonstrate that the neurons that survive in the orthodenticle loss-of-function condition display severe targeting defects in both the proximal (dendritic and distal (axonal neurites. These findings indicate that the cephalic gap gene orthodenticle acts as an important intrinsic determinant in the ALl1 neuroblast lineage and, hence, could be a member of a putative combinatorial code involved in specifying the fate and identity of cells in this lineage.

  18. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

    Science.gov (United States)

    Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

    2005-08-15

    Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

  19. The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila.

    Directory of Open Access Journals (Sweden)

    Rahul Pandey

    Full Text Available During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC. We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

  20. The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila.

    Science.gov (United States)

    Pandey, Rahul; Blanco, Jorge; Udolph, Gerald

    2011-01-01

    During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

  1. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer L Bandura

    Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  2. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Science.gov (United States)

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  3. Drosophila Longevity Assurance Conferred by Reduced Insulin Receptor Substrate Chico Partially Requires d4eBP.

    Directory of Open Access Journals (Sweden)

    Hua Bai

    Full Text Available Mutations of the insulin/IGF signaling (IIS pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit, suggesting there are additional outputs of IIS to mediate aging. One candidate is target of rapamycin complex 1 (TORC1. Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. Here we use genetic epistasis analyses to test whether longevity assurance mutants of chico, the Drosophila insulin receptor substrate homolog, require Drosophila d4eBP to slow aging. In chico heterozygotes, which are robustly long-lived, d4eBP is required but not sufficient to slow aging. Remarkably, d4eBP is not required or sufficient for chico homozygotes to extend longevity. Likewise, chico heterozygote females partially require d4eBP to preserve age-dependent locomotion, and both chico genotypes require d4eBP to improve stress-resistance. Reproduction and most measures of growth affected by either chico genotype are always independent of d4eBP. In females, chico heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP. Altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP.

  4. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Shin-Hae; Park, Joung-Sun [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of); Kim, Young-Shin [Research Institute of Genetic Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Chung, Hae-Young [Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-03-10

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  5. Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

    International Nuclear Information System (INIS)

    Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin; Chung, Hae-Young; Yoo, Mi-Ae

    2012-01-01

    Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: ► Mmp1 is expressed in the adult midgut. ► Mmp1 is involved in the regulation of ISC proliferation activity. ► Mmp1-related ISC proliferation is associated with EGFR signaling. ► Mmp1 in the gut is required for the intestinal homeostasis and longevity.

  6. Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

    Directory of Open Access Journals (Sweden)

    Aditya Sen

    2015-06-01

    Full Text Available Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1 and Parkin (Park, either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu, a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

  7. The Gemin associates of survival motor neuron are required for motor function in Drosophila.

    Science.gov (United States)

    Borg, Rebecca; Cauchi, Ruben J

    2013-01-01

    Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3(ΔN) mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3(ΔN) overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3(ΔN), we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3(ΔN)-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3(ΔN). Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.

  8. E-cadherin is required for centrosome and spindle orientation in Drosophila male germline stem cells.

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    Mayu Inaba

    2010-08-01

    Full Text Available Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs are attached to niche component cells (i.e., the hub via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.

  9. Drosophila divalent metal ion transporter Malvolio is required in dopaminergic neurons for feeding decisions.

    Science.gov (United States)

    Søvik, E; LaMora, A; Seehra, G; Barron, A B; Duncan, J G; Ben-Shahar, Y

    2017-06-01

    Members of the natural resistance-associated macrophage protein (NRAMP) family are evolutionarily conserved metal ion transporters that play an essential role in regulating intracellular divalent cation homeostasis in both prokaryotes and eukaryotes. Malvolio (Mvl), the sole NRAMP family member in insects, plays a role in food choice behaviors in Drosophila and other species. However, the specific physiological and cellular processes that require the action of Mvl for appropriate feeding decisions remain elusive. Here, we show that normal food choice requires Mvl function specifically in the dopaminergic system, and can be rescued by supplementing food with manganese. Collectively, our data indicate that the action of the Mvl transporter affects food choice behavior via the regulation of dopaminergic innervation of the mushroom bodies, a principle brain region associated with decision-making in insects. Our studies suggest that the homeostatic regulation of the intraneuronal levels of divalent cations plays an important role in the development and function of the dopaminergic system and associated behaviors. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  10. Neurotransmitter Transporter-Like: a male germline-specific SLC6 transporter required for Drosophila spermiogenesis.

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    Nabanita Chatterjee

    2011-01-01

    Full Text Available The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl, is expressed only in the male germline. Mobilization of a transposon inserted near the 3' end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism.

  11. Requirement for nuclear calcium signaling in Drosophila long-term memory.

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    Weislogel, Jan-Marek; Bengtson, C Peter; Müller, Michaela K; Hörtzsch, Jan N; Bujard, Martina; Schuster, Christoph M; Bading, Hilmar

    2013-05-07

    Calcium is used throughout evolution as an intracellular signal transducer. In the mammalian central nervous system, calcium mediates the dialogue between the synapse and the nucleus that is required for transcription-dependent persistent neuronal adaptations. A role for nuclear calcium signaling in similar processes in the invertebrate brain has yet to be investigated. Here, we show by in vivo calcium imaging of adult brain neurons of the fruit fly Drosophila melanogaster, that electrical foot shocks used in olfactory avoidance conditioning evoked transient increases in cytosolic and nuclear calcium concentrations in neurons. These calcium signals were detected in Kenyon cells of the flies' mushroom bodies, which are sites of learning and memory related to smell. Acute blockade of nuclear calcium signaling during conditioning selectively and reversibly abolished the formation of long-term olfactory avoidance memory, whereas short-term, middle-term, or anesthesia-resistant olfactory memory remained unaffected. Thus, nuclear calcium signaling is required in flies for the progression of memories from labile to transcription-dependent long-lasting forms. These results identify nuclear calcium as an evolutionarily conserved signal needed in both invertebrate and vertebrate brains for transcription-dependent memory consolidation.

  12. Elongator complex is required for long-term olfactory memory formation in Drosophila.

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    Yu, Dinghui; Tan, Ying; Chakraborty, Molee; Tomchik, Seth; Davis, Ronald L

    2018-04-01

    The evolutionarily conserved Elongator Complex associates with RNA polymerase II for transcriptional elongation. Elp3 is the catalytic subunit, contains histone acetyltransferase activity, and is associated with neurodegeneration in humans. Elp1 is a scaffolding subunit and when mutated causes familial dysautonomia. Here, we show that elp3 and elp1 are required for aversive long-term olfactory memory in Drosophila RNAi knockdown of elp3 in adult mushroom bodies impairs long-term memory (LTM) without affecting earlier forms of memory. RNAi knockdown with coexpression of elp3 cDNA reverses the impairment. Similarly, RNAi knockdown of elp1 impairs LTM and coexpression of elp1 cDNA reverses this phenotype. The LTM deficit in elp3 and elp1 knockdown flies is accompanied by the abolishment of a LTM trace, which is registered as increased calcium influx in response to the CS+ odor in the α-branch of mushroom body neurons. Coexpression of elp1 or elp3 cDNA rescues the memory trace in parallel with LTM. These data show that the Elongator complex is required in adult mushroom body neurons for long-term behavioral memory and the associated long-term memory trace. © 2018 Yu et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Feedback regulation between autophagy and PKA.

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  14. Drosophila Nociceptive Sensitization Requires BMP Signaling via the Canonical SMAD Pathway.

    Science.gov (United States)

    Follansbee, Taylor L; Gjelsvik, Kayla J; Brann, Courtney L; McParland, Aidan L; Longhurst, Colin A; Galko, Michael J; Ganter, Geoffrey K

    2017-08-30

    Nociceptive sensitization is a common feature in chronic pain, but its basic cellular mechanisms are only partially understood. The present study used the Drosophila melanogaster model system and a candidate gene approach to identify novel components required for modulation of an injury-induced nociceptive sensitization pathway presumably downstream of Hedgehog. This study demonstrates that RNAi silencing of a member of the Bone Morphogenetic Protein (BMP) signaling pathway, Decapentaplegic (Dpp), specifically in the Class IV multidendritic nociceptive neuron, significantly attenuated ultraviolet injury-induced sensitization. Furthermore, overexpression of Dpp in Class IV neurons was sufficient to induce thermal hypersensitivity in the absence of injury. The requirement of various BMP receptors and members of the SMAD signal transduction pathway in nociceptive sensitization was also demonstrated. The effects of BMP signaling were shown to be largely specific to the sensitization pathway and not associated with changes in nociception in the absence of injury or with changes in dendritic morphology. Thus, the results demonstrate that Dpp and its pathway play a crucial and novel role in nociceptive sensitization. Because the BMP family is so strongly conserved between vertebrates and invertebrates, it seems likely that the components analyzed in this study represent potential therapeutic targets for the treatment of chronic pain in humans. SIGNIFICANCE STATEMENT This report provides a genetic analysis of primary nociceptive neuron mechanisms that promote sensitization in response to injury. Drosophila melanogaster larvae whose primary nociceptive neurons were reduced in levels of specific components of the BMP signaling pathway, were injured and then tested for nocifensive responses to a normally subnoxious stimulus. Results suggest that nociceptive neurons use the BMP2/4 ligand, along with identified receptors and intracellular transducers to transition to a

  15. The Drosophila DmGluRA is required for social interaction and memory

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    Brian P. Schoenfeld

    2013-05-01

    Full Text Available Metabotropic glutamate receptors (mGluRs have well established roles in cognition andsocial behavior in mammals. Whether or not these roles have been conserved throughoutevolution from invertebrate species is less clear. Mammals have 8 mGluRs whereasDrosophila have a single DmGluRA, which has both Gi and Gq coupled signalingactivity. We have utilized Drosophila to examine the role of DmGluRA in social behaviorand various phases of memory. We have found that flies that are homozygous orheterozygous for loss of function mutations of DmGluRA have impaired social behaviorin male Drosophila. Futhermore, flies that are homozygous or heterozygous for loss offunction mutations of DmGluRA have impaired learning during training, immediate recallmemory, short-term memory and long-term memory as young adults. This workdemonstrates a role for metabotropic glutamate receptor activity in both social behaviorand memory in Drosophila.

  16. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain.

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    Jan J Vonk

    Full Text Available Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis.

  17. Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.

    Science.gov (United States)

    Lucas, Eliana P; Raff, Jordan W

    2007-08-27

    Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.

  18. DNA replication machinery is required for development in Drosophila.

    Science.gov (United States)

    Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

    2018-01-01

     In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

  19. Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration.

    Science.gov (United States)

    Takemura, Masahiko; Nakato, Hiroshi

    2017-01-15

    Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. Here, we show that a specific modification of heparan sulfate is crucial for regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular heparan sulfate endosulfatase Sulf1 resulted in increased ISC division during normal homeostasis, which was caused by upregulation of mitogenic signaling including the JAK-STAT, EGFR and Hedgehog pathways. Using a regeneration model, we found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. We propose that post-transcriptional regulation of mitogen signaling by heparan sulfate structural modifications provides a new regulatory step for precise temporal control of stem cell activity during regeneration. © 2017. Published by The Company of Biologists Ltd.

  20. Drosophila convoluted/dALS is an essential gene required for tracheal tube morphogenesis and apical matrix organization.

    Science.gov (United States)

    Swanson, Lianna E; Yu, Marcus; Nelson, Kevin S; Laprise, Patrick; Tepass, Ulrich; Beitel, Greg J

    2009-04-01

    Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.

  1. Mitochondrial PKA mediates sperm motility.

    Science.gov (United States)

    Mizrahi, Rashel; Breitbart, Haim

    2014-12-01

    Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics. Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy. Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria. Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60kDa proteins does not occur in the absence of bicarbonate. Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

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    Assia Hijazi

    Full Text Available BACKGROUND: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. METHODOLOGY/PRINCIPAL FINDINGS: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. CONCLUSION/SIGNIFICANCE: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

  3. The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

    Science.gov (United States)

    Hijazi, Assia; Haenlin, Marc; Waltzer, Lucas; Roch, Fernando

    2011-03-15

    Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

  4. SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

    Science.gov (United States)

    Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

    2010-02-08

    Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

  5. Transcriptional Adaptor ADA3 of Drosophila melanogaster Is Required for Histone Modification, Position Effect Variegation, and Transcription▿ †

    OpenAIRE

    Grau, Benjamin; Popescu, Cristina; Torroja, Laura; Ortuño-Sahagún, Daniel; Boros, Imre; Ferrús, Alberto

    2007-01-01

    The Drosophila melanogaster gene diskette (also known as dik or dAda3) encodes a protein 29% identical to human ADA3, a subunit of GCN5-containing histone acetyltransferase (HAT) complexes. The fly dADA3 is a major contributor to oogenesis, and it is also required for somatic cell viability. dADA3 localizes to chromosomes, and it is significantly reduced in dGcn5 and dAda2a, but not in dAda2b, mutant backgrounds. In dAda3 mutants, acetylation at histone H3 K9 and K14, but not K18, and at hist...

  6. PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

    Science.gov (United States)

    Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2018-05-03

    β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

  7. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.

    Science.gov (United States)

    Clark, Ira E; Dodson, Mark W; Jiang, Changan; Cao, Joseph H; Huh, Jun R; Seol, Jae Hong; Yoo, Soon Ji; Hay, Bruce A; Guo, Ming

    2006-06-29

    Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease

  8. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    Science.gov (United States)

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  9. Localization and activation of the Drosophila protease easter require the ER-resident saposin-like protein seele.

    Science.gov (United States)

    Stein, David; Charatsi, Iphigenie; Cho, Yong Suk; Zhang, Zhenyu; Nguyen, Jesse; DeLotto, Robert; Luschnig, Stefan; Moussian, Bernard

    2010-11-09

    Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. PKA distributions: Contributions from transmutation products and from radioactive decay

    Directory of Open Access Journals (Sweden)

    M.R. Gilbert

    2016-12-01

    Full Text Available The neutrons generated in fusion plasmas interact with materials via nuclear reactions. The resulting transmutations and atomic displacements have life-limiting consequences for fusion reactor components. A detailed understanding of the production, evolution and material consequences of the damage created by cascades of atomic displacements requires, as a vital primary input, a complete description of the energy-spectrum of initial (prompt atomic displacement events (the primary knock on atoms or PKAs produced by direct neutron nuclear interactions. There is also the possibility that the radionuclides produced under transmutation will create further PKAs as they decay, and so the rate of these must also be quantified. This paper presents the latest results from the analysis of PKA spectra under neutron irradiation, focussing particularly on the variation in PKA distributions due to changes in composition under transmutation, but also on the PKA contributions from radioactive decay of materials that become activated under irradiation.

  11. The Visual Orientation Memory of "Drosophila" Requires Foraging (PKG) Upstream of Ignorant (RSK2) in Ring Neurons of the Central Complex

    Science.gov (United States)

    Kuntz, Sara; Poeck, Burkhard; Sokolowski, Marla B.; Strauss, Roland

    2012-01-01

    Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking "Drosophila" flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. Here we show that this type of working memory requires the cGMP-dependent protein…

  12. GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila.

    Science.gov (United States)

    Gowda, Swetha B M; Paranjpe, Pushkar D; Reddy, O Venkateswara; Thiagarajan, Devasena; Palliyil, Sudhir; Reichert, Heinrich; VijayRaghavan, K

    2018-02-27

    Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. Here we take advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, we have developed an algorithm for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, we use genetic reagents for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila Our findings indicate that targeted down-regulation of the GABA A receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila . Copyright © 2018 the Author(s). Published by PNAS.

  13. PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

    Science.gov (United States)

    Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

    2016-11-15

    Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  15. The Heat Shock Protein 26 Gene is Required for Ethanol Tolerance in Drosophila

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    Awoyemi A. Awofala

    2011-01-01

    Full Text Available Stress plays an important role in drug- and addiction-related behaviours. However, the mechanisms underlying these behavioural responses are still poorly understood. In the light of recent reports that show consistent regulation of many genes encoding stress proteins including heat shock proteins following ethanol exposure in Drosophila , it was hypothesised that transition to alcohol dependence may involve the dysregulation of the circuits that mediate behavioural responses to stressors. Thus, behavioural genetic methodologies were used to investigate the role of the Drosophila hsp26 gene, a small heat shock protein coding gene which is induced in response to various stresses, in the development of rapid tolerance to ethanol sedation. Rapid tolerance was quantified as the percentage difference in the mean sedation times between the second and first ethanol exposure. Two independently isolated P-element mutations near the hsp26 gene eliminated the capacity for tolerance. In addition, RNAi-mediated functional knockdown of hsp26 expression in the glial cells and the whole nervous system also caused a defect in tolerance development. The rapid tolerance phenotype of the hsp26 mutants was rescued by the expression of the wild-type hsp26 gene in the nervous system. None of these manipulations of the hsp26 gene caused changes in the rate of ethanol absorption. Hsp26 genes are evolutionary conserved, thus the role of hsp26 in ethanol tolerance may present a new direction for research into alcohol dependency.

  16. Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

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    Hongyu Zhang

    2015-09-01

    Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

  17. Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila.

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    Valbona Hoxha

    Full Text Available Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood-brain barrier (bbb to interact with the sex-specific brain circuits that control courtship is unknown. Here we show that male identity of the blood-brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. We show that the bbb-specific G-protein coupled receptor moody and bbb-specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior.

  18. Drosophila Clock Is Required in Brain Pacemaker Neurons to Prevent Premature Locomotor Aging Independently of Its Circadian Function.

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    Alexandra Vaccaro

    2017-01-01

    Full Text Available Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. In many organisms, oxidative stress and aging negatively impact the circadian system and sleep. Conversely, loss of the clock decreases resistance to oxidative stress, and may reduce lifespan and speed up brain aging and neurodegeneration. Here we examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. We found that lifespan was similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0 and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing than cyc0 and tim0, or than control flies under constant light. Reactive oxygen species accumulated more with age in ClkAR mutant brains, but this did not appear to contribute to the accelerated locomotor decline of the mutant. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1 clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. Our results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.

  19. A presynaptic role for PKA in synaptic tagging and memory

    NARCIS (Netherlands)

    Park, Alan Jung; Havekes, Robbert; Choi, Jennifer H K; Luczak, Vincent; Nie, Ting; Huang, Ted; Abel, Ted

    2014-01-01

    Protein kinase A (PKA) and other signaling molecules are spatially restricted within neurons by A-kinase anchoring proteins (AKAPs). Although studies on compartmentalized PKA signaling have focused on postsynaptic mechanisms, presynaptically anchored PKA may contribute to synaptic plasticity and

  20. Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila.

    Science.gov (United States)

    Park, Joung-Sun; Na, Hyun-Jin; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Yoo, Mi-Ae

    2015-05-01

    The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study was focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adultDrosophila midgut. We explored the role of ATM and ATR, utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, γ-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, our data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.

  1. A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections.

    Directory of Open Access Journals (Sweden)

    Janelle S Ayres

    2008-12-01

    Full Text Available Organisms evolve two routes to surviving infections-they can resist pathogen growth (resistance and they can endure the pathogenesis of infection (tolerance. The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.

  2. Saccharomyces cerevisiae mitochondria are required for optimal attractiveness to Drosophila melanogaster

    Science.gov (United States)

    Schiabor, Kelly M.; Quan, Allison S.; Eisen, Michael B.

    2014-01-01

    While screening a large collection of wild and laboratory yeast strains for their ability to attract Drosophila melanogaster adults, we noticed a large difference in fly preference for two nearly isogenic strains of Saccharomyces cerevisiae, BY4741 and BY4742. Using standard genetic analyses, we tracked the preference difference to the lack of mitochondria in the BY4742 strain used in the initial experiment. We used gas chromatography coupled with mass spectroscopy to examine the volatile compounds produced by BY4741 and the mitochondria-deficient BY4742, and found that they differed significantly. We observed that several ethyl esters are present at much higher levels in strains with mitochondria, even in fermentative conditions. We found that nitrogen levels in the substrate affect the production of these compounds, and that they are produced at the highest level by strains with mitochondria when fermenting natural fruit substrates. Collectively these observations demonstrate that core metabolic processes mediate the interaction between yeasts and insect vectors, and highlight the importance mitochondrial functions in yeast ecology. PMID:25462617

  3. Absolute requirement of cholesterol binding for Hedgehog gradient formation in Drosophila

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    Antoine Ducuing

    2013-05-01

    How morphogen gradients are shaped is a major question in developmental biology, but remains poorly understood. Hedgehog (Hh is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to Hh N-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.

  4. Xbp1-Independent Ire1 Signaling Is Required for Photoreceptor Differentiation and Rhabdomere Morphogenesis in Drosophila

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    Dina S. Coelho

    2013-11-01

    Full Text Available The unfolded protein response (UPR is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD, including for the fatty acid transport protein (fatp. Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

  5. Precise temporal regulation of roughest is required for correct salivary gland autophagic cell death in Drosophila.

    Science.gov (United States)

    Simon, Claudio R; Moda, Livia M R; Octacilio-Silva, Shirlei; Anhezini, Lucas; Machado-Gitai, Luciana C H; Ramos, Ricardo Guelerman P

    2009-07-01

    The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst(D) mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. 2009 Wiley-Liss, Inc.

  6. The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

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    Morishita, Kazushge; Anh Suong, Dang Ngoc; Yoshida, Hideki; Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp

    2017-05-15

    Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway. - Highlights: • Spg mainly localizes in the air sac primordium in wing imaginal discs. • Spg plays a critical role in air sac primordium development. • Spg positively regulates the ERK signal cascade.

  7. Polarization of the epithelial layer and apical localization of integrins are required for engulfment of apoptotic cells in the Drosophila ovary

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    Tracy L. Meehan

    2015-12-01

    Full Text Available Inefficient clearance of dead cells or debris by epithelial cells can lead to or exacerbate debilitating conditions such as retinitis pigmentosa, macular degeneration, chronic obstructive pulmonary disease and asthma. Despite the importance of engulfment by epithelial cells, little is known about the molecular changes that are required within these cells. The misregulation of integrins has previously been associated with disease states, suggesting that a better understanding of the regulation of receptor trafficking could be key to treating diseases caused by defects in phagocytosis. Here, we demonstrate that the integrin heterodimer αPS3/βPS becomes apically enriched and is required for engulfment by the epithelial follicle cells of the Drosophila ovary. We found that integrin heterodimer localization and function is largely directed by the α-subunit. Moreover, proper cell polarity promotes asymmetric integrin enrichment, suggesting that αPS3/βPS trafficking occurs in a polarized fashion. We show that several genes previously known for their roles in trafficking and cell migration are also required for engulfment. Moreover, as in mammals, the same α-integrin subunit is required by professional and non-professional phagocytes and migrating cells in Drosophila. Our findings suggest that migrating and engulfing cells use common machinery, and demonstrate a crucial role for integrin function and polarized trafficking of integrin subunits during engulfment. This study also establishes the epithelial follicle cells of the Drosophila ovary as a powerful model for understanding the molecular changes required for engulfment by a polarized epithelium.

  8. Notch is required in adult Drosophila sensory neurons for morphological and functional plasticity of the olfactory circuit.

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    Simon Kidd

    2015-05-01

    Full Text Available Olfactory receptor neurons (ORNs convey odor information to the central brain, but like other sensory neurons were thought to play a passive role in memory formation and storage. Here we show that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila ORNs for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. Specifically, we show that Notch activity in ORNs is necessary for the odor specific increase in the volume of glomeruli that occurs as a consequence of prolonged odor exposure. Calcium imaging experiments indicate that Notch in ORNs is also required for the chronic odor induced changes in the physiology of ORNs and the ensuing changes in the physiological response of their second order projection neurons (PNs. We further show that Notch in ORNs acts by both canonical cleavage-dependent and non-canonical cleavage-independent pathways. The Notch ligand Delta (Dl in PNs switches the balance between the pathways. These data define a circuit whereby, in conjunction with odor, N activity in the periphery regulates the activity of neurons in the central brain and Dl in the central brain regulates N activity in the periphery. Our work highlights the importance of experience dependent plasticity at the first olfactory synapse.

  9. Dynein-dependent transport of nanos RNA in Drosophila sensory neurons requires Rumpelstiltskin and the germ plasm organizer Oskar.

    Science.gov (United States)

    Xu, Xin; Brechbiel, Jillian L; Gavis, Elizabeth R

    2013-09-11

    Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

  10. Inhibition of PKA anchoring to A-kinase anchoring proteins impairs consolidation and facilitates extinction of contextual fear memories

    NARCIS (Netherlands)

    Nijholt, Ingrid M.; Ostroveanu, Anghelus; Scheper, Wouter A.; Penke, Botond; Luiten, Paul G. M.; Van der Zee, Eddy A.; Eisel, Ulrich L. M.

    Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial

  11. Ionization Constants pKa of Cardiolipin

    OpenAIRE

    Olofsson, Gerd; Sparr, Emma

    2013-01-01

    Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5-9.5. Still, there are several examples of experimental ob...

  12. rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

    Science.gov (United States)

    Gates, Julie; Lam, Geanette; Ortiz, José A; Losson, Régine; Thummel, Carl S

    2004-01-01

    Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more

  13. p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development.

    Science.gov (United States)

    Contreras, Esteban G; Sierralta, Jimena; Glavic, Alvaro

    2018-01-01

    Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, we hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. Here, we studied the function of p53 during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. We showed that p53 loss of function reduced animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, we showed that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. Using genetic approaches, we demonstrate that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals.

  14. Hormonal Signaling Cascade during an Early-Adult Critical Period Required for Courtship Memory Retention in Drosophila.

    Science.gov (United States)

    Lee, Sang Soo; Ding, Yike; Karapetians, Natalie; Rivera-Perez, Crisalejandra; Noriega, Fernando Gabriel; Adams, Michael E

    2017-09-25

    Formation and expression of memories are critical for context-dependent decision making. In Drosophila, a courting male rejected by a mated female subsequently courts less avidly when paired with a virgin female, a behavioral modification attributed to "courtship memory." Here we show the critical role of hormonal state for maintenance of courtship memory. Ecdysis-triggering hormone (ETH) is essential for courtship memory through regulation of juvenile hormone (JH) levels in adult males. Reduction of JH levels via silencing of ETH signaling genes impairs short-term courtship memory, a phenotype rescuable by the JH analog methoprene. JH-deficit-induced memory impairment involves rapid decay rather than failure of memory acquisition. A critical period governs memory performance during the first 3 days of adulthood. Using sex-peptide-expressing "pseudo-mated" trainers, we find that robust courtship memory elicited in the absence of aversive chemical mating cues also is dependent on ETH-JH signaling. Finally, we find that JH acts through dopaminergic neurons and conclude that an ETH-JH-dopamine signaling cascade is required during a critical period for promotion of social-context-dependent memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Short- and long-term memory in Drosophila require cAMP signaling in distinct neuron types.

    Science.gov (United States)

    Blum, Allison L; Li, Wanhe; Cressy, Mike; Dubnau, Josh

    2009-08-25

    A common feature of memory and its underlying synaptic plasticity is that each can be dissected into short-lived forms involving modification or trafficking of existing proteins and long-term forms that require new gene expression. An underlying assumption of this cellular view of memory consolidation is that these different mechanisms occur within a single neuron. At the neuroanatomical level, however, different temporal stages of memory can engage distinct neural circuits, a notion that has not been conceptually integrated with the cellular view. Here, we investigated this issue in the context of aversive Pavlovian olfactory memory in Drosophila. Previous studies have demonstrated a central role for cAMP signaling in the mushroom body (MB). The Ca(2+)-responsive adenylyl cyclase RUTABAGA is believed to be a coincidence detector in gamma neurons, one of the three principle classes of MB Kenyon cells. We were able to separately restore short-term or long-term memory to a rutabaga mutant with expression of rutabaga in different subsets of MB neurons. Our findings suggest a model in which the learning experience initiates two parallel associations: a short-lived trace in MB gamma neurons, and a long-lived trace in alpha/beta neurons.

  16. HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

    Science.gov (United States)

    Li, Yan; Padmanabha, Divya; Gentile, Luciana B.; Dumur, Catherine I.; Beckstead, Robert B.; Baker, Keith D.

    2013-01-01

    Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. PMID:23382692

  17. Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune response in a Drosophila melanogaster oral infection model

    Science.gov (United States)

    Limmer, Stefanie; Haller, Samantha; Drenkard, Eliana; Lee, Janice; Yu, Shen; Kocks, Christine; Ausubel, Frederick M.; Ferrandon, Dominique

    2011-01-01

    An in-depth mechanistic understanding of microbial infection necessitates a molecular dissection of host–pathogen relationships. Both Drosophila melanogaster and Pseudomonas aeruginosa have been intensively studied. Here, we analyze the infection of D. melanogaster by P. aeruginosa by using mutants in both host and pathogen. We show that orally ingested P. aeruginosa crosses the intestinal barrier and then proliferates in the hemolymph, thereby causing the infected flies to die of bacteremia. Host defenses against ingested P. aeruginosa included an immune deficiency (IMD) response in the intestinal epithelium, systemic Toll and IMD pathway responses, and a cellular immune response controlling bacteria in the hemocoel. Although the observed cellular and intestinal immune responses appeared to act throughout the course of the infection, there was a late onset of the systemic IMD and Toll responses. In this oral infection model, P. aeruginosa PA14 did not require its type III secretion system or other well-studied virulence factors such as the two-component response regulator GacA or the protease AprA for virulence. In contrast, the quorum-sensing transcription factor RhlR, but surprisingly not LasR, played a key role in counteracting the cellular immune response against PA14, possibly at an early stage when only a few bacteria are present in the hemocoel. These results illustrate the power of studying infection from the dual perspective of host and pathogen by revealing that RhlR plays a more complex role during pathogenesis than previously appreciated. PMID:21987808

  18. Differential dpa calculations with SPECTRA-PKA

    Science.gov (United States)

    Gilbert, M. R.; Sublet, J.-Ch.

    2018-06-01

    The processing code SPECTRA-PKA produces energy spectra of primary atomic recoil events (or primary knock-on atoms, PKAs) for any material composition exposed to an irradiation spectrum. Such evaluations are vital inputs for simulations aimed at understanding the evolution of damage in irradiated material, which is generated in cascade displacement events initiated by PKAs. These PKA spectra present the full complexity of the input (to SPECTRA-PKA) nuclear data-library evaluations of recoil events. However, the commonly used displacements per atom (dpa) measure, which is an integral measure over all possible recoil events of the displacement damage dose, is still widely used and has many useful applications - as both a comparative and correlative quantity. This paper describes the methodology employed that allows the SPECTRA-PKA code to evaluate dpa rates using the energy-dependent recoil (PKA) cross section data used for the PKA distributions. This avoids the need for integral displacement kerma cross sections and also provides new insight into the relative importance of different reaction channels (and associated different daughter residual and emitted particles) to the total integrated dpa damage dose. Results are presented for Fe, Ni, W, and SS316. Fusion dpa rates are compared to those in fission, highlighting the increased contribution to damage creation in the former from high-energy threshold reactions.

  19. Ionization constants pKa of cardiolipin.

    Directory of Open Access Journals (Sweden)

    Gerd Olofsson

    Full Text Available Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5-9.5. Still, there are several examples of experimental observations from cardiolipin-containing systems that do not fit with this dissociation behavior. Therefore, we have carried out pH-titration and titration calorimetric experiments on two synthetic cardiolipins, 1,1',2,2'-tetradecanoyl cardiolipin, CL (C14:0, and 1,1',2,2'-tetraoctadecenoyl cardiolipin, CL (C18:1. Our results show that both behave as strong dibasic acids with pKa1 about the same as the first pKa of phosphoric acid, 2.15, and pKa2 about one unit larger. The characterization of the acidic properties of cardiolipin is crucial for the understanding of the molecular organization in self-assembled systems that contain cardiolipin, and for their biological function.

  20. Ionization constants pKa of cardiolipin.

    Science.gov (United States)

    Olofsson, Gerd; Sparr, Emma

    2013-01-01

    Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5-9.5. Still, there are several examples of experimental observations from cardiolipin-containing systems that do not fit with this dissociation behavior. Therefore, we have carried out pH-titration and titration calorimetric experiments on two synthetic cardiolipins, 1,1',2,2'-tetradecanoyl cardiolipin, CL (C14:0), and 1,1',2,2'-tetraoctadecenoyl cardiolipin, CL (C18:1). Our results show that both behave as strong dibasic acids with pKa1 about the same as the first pKa of phosphoric acid, 2.15, and pKa2 about one unit larger. The characterization of the acidic properties of cardiolipin is crucial for the understanding of the molecular organization in self-assembled systems that contain cardiolipin, and for their biological function.

  1. Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres.

    Science.gov (United States)

    Blattner, Ariane C; Chaurasia, Soumya; McKee, Bruce D; Lehner, Christian F

    2016-04-01

    Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase.

  2. Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

    Science.gov (United States)

    Delgado, María Graciela; Oliva, Carlos; López, Estefanía; Ibacache, Andrés; Galaz, Alex; Delgado, Ricardo; Barros, L Felipe; Sierralta, Jimena

    2018-01-19

    The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

  3. Subcellular Location of PKA Controls Striatal Plasticity: Stochastic Simulations in Spiny Dendrites

    Science.gov (United States)

    Oliveira, Rodrigo F.; Kim, MyungSook; Blackwell, Kim T.

    2012-01-01

    Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity. PMID:22346744

  4. Computing pKa Values in Different Solvents by Electrostatic Transformation.

    Science.gov (United States)

    Rossini, Emanuele; Netz, Roland R; Knapp, Ernst-Walter

    2016-07-12

    We introduce a method that requires only moderate computational effort to compute pKa values of small molecules in different solvents with an average accuracy of better than 0.7 pH units. With a known pKa value in one solvent, the electrostatic transform method computes the pKa value in any other solvent if the proton solvation energy is known in both considered solvents. To apply the electrostatic transform method to a molecule, the electrostatic solvation energies of the protonated and deprotonated molecular species are computed in the two considered solvents using a dielectric continuum to describe the solvent. This is demonstrated for 30 molecules belonging to 10 different molecular families by considering 77 measured pKa values in 4 different solvents: water, acetonitrile, dimethyl sulfoxide, and methanol. The electrostatic transform method can be applied to any other solvent if the proton solvation energy is known. It is exclusively based on physicochemical principles, not using any empirical fetch factors or explicit solvent molecules, to obtain agreement with measured pKa values and is therefore ready to be generalized to other solute molecules and solvents. From the computed pKa values, we obtained relative proton solvation energies, which agree very well with the proton solvation energies computed recently by ab initio methods, and used these energies in the present study.

  5. Drosophila larvae food intake cessation following exposure to Erwinia contaminated media requires odor perception, Trpa1 channel and evf virulence factor.

    Science.gov (United States)

    Keita, Seydou; Masuzzo, Ambra; Royet, Julien; Kurz, C Leopold

    2017-05-01

    When exposed to microorganisms, animals use several protective strategies. On one hand, as elegantly exemplified in Drosophila melanogaster, the innate immune system recognizes microbial compounds and triggers an antimicrobial response. On the other hand, behaviors preventing an extensive contact with the microbes and thus reducing the risk of infection have been described. However, these reactions ranging from microbes aversion to intestinal transit increase or food intake decrease have been rarely defined at the molecular level. In this study, we set up an experimental system that allowed us to rapidly identify and quantify food intake decreases in Drosophila larvae exposed to media contaminated with bacteria. Specifically, we report a robust dose-dependent food intake decrease following exposure to the bacteria Erwinia carotovora carotovora strain Ecc15. We demonstrate that this response does not require Imd innate immune pathway, but rather the olfactory neuronal circuitry, the Trpa1 receptor and the evf virulence factor. Finally, we show that Ecc15 induce the same behavior in the invasive pest insect Drosophila suzukii. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Discovery of Allostery in PKA Signaling.

    Science.gov (United States)

    Zhang, Ping; Kornev, Alexandr P; Wu, Jian; Taylor, Susan S

    2015-06-01

    cAMP-dependent protein kinase (PKA) was the second protein kinase to be discovered and the PKA catalytic (C) subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate much of biology in eukaryotic cells and they are now also a major therapeutic target. Although PKA was discovered nearly 50 years ago and the subsequent discovery of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme followed quickly. Thus in PKA we see the convergence of two major signaling mechanisms - protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and the structure of the isolated regulatory (R) and C-subunits have been extremely informative. Yet it is the R 2 C 2 holoenzyme that predominates in cells, and one can only appreciate the allosteric features of PKA signaling by seeing the full length protein. The symmetry and the quaternary constraints that one R:C hetero-dimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C hetero-dimer.

  7. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila

    NARCIS (Netherlands)

    Song, Ho-Juhn; Billeter, Jean-Christophe; Reynaud, Enrique; Carlo, Troy; Spana, Eric P; Perrimon, Norbert; Goodwin, Stephen F; Baker, Bruce S; Taylor, Barbara J

    2002-01-01

    The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less

  8. Drosophila Ana1 is required for centrosome assembly and centriole elongation.

    Science.gov (United States)

    Saurya, Saroj; Roque, Hélio; Novak, Zsofia A; Wainman, Alan; Aydogan, Mustafa G; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano; Raff, Jordan W

    2016-07-01

    Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. © 2016. Published by The Company of Biologists Ltd.

  9. DelPhiPKa web server: predicting pKa of proteins, RNAs and DNAs.

    Science.gov (United States)

    Wang, Lin; Zhang, Min; Alexov, Emil

    2016-02-15

    A new pKa prediction web server is released, which implements DelPhi Gaussian dielectric function to calculate electrostatic potentials generated by charges of biomolecules. Topology parameters are extended to include atomic information of nucleotides of RNA and DNA, which extends the capability of pKa calculations beyond proteins. The web server allows the end-user to protonate the biomolecule at particular pH based on calculated pKa values and provides the downloadable file in PQR format. Several tests are performed to benchmark the accuracy and speed of the protocol. The web server follows a client-server architecture built on PHP and HTML and utilizes DelPhiPKa program. The computation is performed on the Palmetto supercomputer cluster and results/download links are given back to the end-user via http protocol. The web server takes advantage of MPI parallel implementation in DelPhiPKa and can run a single job on up to 24 CPUs. The DelPhiPKa web server is available at http://compbio.clemson.edu/pka_webserver. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila.

    Directory of Open Access Journals (Sweden)

    Michelle T Juarez

    2011-12-01

    Full Text Available The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. We have developed a variety of fluorescent reporters that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. We also find that four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-ß-cyclodextrin, and hydrogen peroxide are sufficient to globally activate epidermal wound response genes in Drosophila embryos. We explore the epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals. Our results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating

  11. Shal/K(v4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

    Directory of Open Access Journals (Sweden)

    Yong Ping

    2011-01-01

    Full Text Available Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K(+ current (I(A, encoded by the highly conserved Shal/K(v4 gene, in neuronal firing patterns and repetitive behaviors. While I(A is present in nearly all neurons across species, elimination of I(A has been complicated in mammals because of multiple genes underlying I(A, and/or electrical remodeling that occurs in response to affecting one gene.In Drosophila, the single Shal/K(v4 gene encodes the predominant I(A current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v4, we show that I(A is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v4 neurons display multiple defects during prolonged stimuli. DNK(v4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K(v4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K(v4 function. Further, knock-out of Shal/K(v4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Based on our results, Shal/K(v4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K(v4 channels play a key role in repetitively firing neurons during prolonged

  12. A glial variant of the vesicular monoamine transporter is required to store histamine in the Drosophila visual system.

    Directory of Open Access Journals (Sweden)

    Rafael Romero-Calderón

    2008-11-01

    Full Text Available Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems.

  13. Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

    OpenAIRE

    Oikemus, Sarah R.; McGinnis, Nadine; Queiroz-Machado, Joana; Tukachinsky, Hanna; Takada, Saeko; Sunkel, Claudio E.; Brodsky, Michael H.

    2004-01-01

    Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We dem...

  14. Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect.

    Science.gov (United States)

    Oikemus, Sarah R; McGinnis, Nadine; Queiroz-Machado, Joana; Tukachinsky, Hanna; Takada, Saeko; Sunkel, Claudio E; Brodsky, Michael H

    2004-08-01

    Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres.

  15. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

    Science.gov (United States)

    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant. © 2015 John Wiley & Sons Ltd.

  16. Organophosphate-Induced Changes in the PKA Regulatory Function of Swiss Cheese/NTE Lead to Behavioral Deficits and Neurodegeneration

    Science.gov (United States)

    Kretzschmar, Doris

    2014-01-01

    Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing

  17. PKA/KIN-1 mediates innate immune responses to bacterial pathogens in Caenorhabditis elegans.

    Science.gov (United States)

    Xiao, Yi; Liu, Fang; Zhao, Pei-Ji; Zou, Cheng-Gang; Zhang, Ke-Qin

    2017-11-01

    The genetically tractable organism Caenorhabditis elegans is a powerful model animal for the study of host innate immunity. Although the intestine and the epidermis of C. elegans that is in contact with pathogens are likely to function as sites for the immune function, recent studies indicate that the nervous system could control innate immunity in C. elegans. In this report, we demonstrated that protein kinase A (PKA)/KIN-1 in the neurons contributes to resistance against Salmonella enterica infection in C. elegans. Microarray analysis revealed that PKA/KIN-1 regulates the expression of a set of antimicrobial effectors in the non-neuron tissues, which are required for innate immune responses to S. enterica. Furthermore, PKA/KIN-1 regulated the expression of lysosomal genes during S. enterica infection. Our results suggest that the lysosomal signaling molecules are involved in autophagy by controlling autophagic flux, rather than formation of autophagosomes. As autophagy is crucial for host defense against S. enterica infection in a metazoan, the lysosomal pathway also acts as a downstream effector of the PKA/KIN-1 signaling for innate immunity. Our data indicate that the PKA pathway contributes to innate immunity in C. elegans by signaling from the nervous system to periphery tissues to protect the host against pathogens.

  18. Drosophila-Cdh1 (Rap/Fzr) a regulatory subunit of APC/C is required for synaptic morphology, synaptic transmission and locomotion.

    Science.gov (United States)

    Wise, Alexandria; Schatoff, Emma; Flores, Julian; Hua, Shao-Ying; Ueda, Atsushi; Wu, Chun-Fang; Venkatesh, Tadmiri

    2013-11-01

    The assembly of functional synapses requires the orchestration of the synthesis and degradation of a multitude of proteins. Protein degradation and modification by the conserved ubiquitination pathway has emerged as a key cellular regulatory mechanism during nervous system development and function (Kwabe and Brose, 2011). The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase complex primarily characterized for its role in the regulation of mitosis (Peters, 2002). In recent years, a role for APC/C in nervous system development and function has been rapidly emerging (Stegmuller and Bonni, 2005; Li et al., 2008). In the mammalian central nervous system the activator subunit, APC/C-Cdh1, has been shown to be a regulator of axon growth and dendrite morphogenesis (Konishi et al., 2004). In the Drosophila peripheral nervous system (PNS), APC2, a ligase subunit of the APC/C complex has been shown to regulate synaptic bouton size and activity (van Roessel et al., 2004). To investigate the role of APC/C-Cdh1 at the synapse we examined loss-of-function mutants of Rap/Fzr (Retina aberrant in pattern/Fizzy related), a Drosophila homolog of the mammalian Cdh1 during the development of the larval neuromuscular junction in Drosophila. Our cell biological, ultrastructural, electrophysiological, and behavioral data showed that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function as well as locomotion defects. Data presented here show changes in size and morphology of synaptic boutons, and, muscle tissue organization. Electrophysiological experiments show that loss-of-function mutants exhibit increased frequency of spontaneous miniature synaptic potentials, indicating a higher rate of spontaneous synaptic vesicle fusion events. In addition, larval locomotion and peristaltic movement were also impaired. These findings suggest a role for Drosophila APC/C-Cdh1 mediated ubiquitination in regulating synaptic morphology

  19. Drosophila C-terminal binding protein, dCtBP is required for sensory organ prepattern and sharpens proneural transcriptional activity of the GATA factor Pnr.

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    Biryukova, Inna; Heitzler, Pascal

    2008-11-01

    The peripheral nervous system is required for animals to detect and to relay environmental stimuli to central nervous system for the information processing. In Drosophila, the precise spatial and temporal expression of two proneural genes achaete (ac) and scute (sc), is necessary for development of the sensory organs. Here we present an evidence that the transcription co-repressor, dCtBP acts as a negative regulator of sensory organ prepattern. Loss of dCtBP function mutant exhibits ectopic sensory organs, while overexpression of dCtBP results in a dramatic loss of sensory organs. These phenotypes are correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. Mammalian CtBP-1 was identified via interaction with the consensus motif PXDLSX(K/R) of adenovirus E1A oncoprotein. We demonstrated that dCtBP binds directly to PLDLS motif of Drosophila Friend of GATA-1 protein, U-shaped and sharpens the adult sensory organ development. Moreover, we found that dCtBP mediates multivalent interaction with the GATA transcriptional activator Pannier and acts as a direct co-repressor of the Pannier-mediated activation of proneural genes. We demonstrated that Pannier genetically interacts with dCtBP-interacting protein HDAC1, suggesting that the dCtBP-dependent regulation of Pannier activity could utilize a repressive mechanism involving alteration of local chromatine structure.

  20. Two Components of Aversive Memory in Drosophila, Anesthesia-Sensitive and Anesthesia-Resistant Memory, Require Distinct Domains Within the Rgk1 Small GTPase.

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    Murakami, Satoshi; Minami-Ohtsubo, Maki; Nakato, Ryuichiro; Shirahige, Katsuhiko; Tabata, Tetsuya

    2017-05-31

    Multiple components have been identified that exhibit different stabilities for aversive olfactory memory in Drosophila These components have been defined by behavioral and genetic studies and genes specifically required for a specific component have also been identified. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies, which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. We propose that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact. SIGNIFICANCE STATEMENT Memory consists of multiple components. Drosophila olfactory memory serves as a fundamental model with which to investigate the mechanisms that underlie memory formation and has provided genetic and molecular means to identify the components of memory, namely short-term, intermediate-term, and long-term memory, depending on how long the memory lasts. Intermediate memory is further divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We have identified a small GTPase in Drosophila , Rgk1, which plays a pivotal role in the regulation of olfactory memory stability. Rgk1 is required for both ASM and ARM. Moreover, N

  1. Drosophila brakeless interacts with atrophin and is required for tailless-mediated transcriptional repression in early embryos.

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    Haecker, Achim; Qi, Dai; Lilja, Tobias; Moussian, Bernard; Andrioli, Luiz Paulo; Luschnig, Stefan; Mannervik, Mattias

    2007-06-01

    Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. We isolated mutations in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, we noted an expanded expression pattern of the Krüppel (Kr) and knirps (kni) genes. We found that Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. We show that both Drosophila and human Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. We discuss the possibility that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA).

  2. The Putative HORMA Domain Protein Atg101 Dimerizes and Is Required for Starvation-Induced and Selective Autophagy in Drosophila

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    Krisztina Hegedűs

    2014-01-01

    Full Text Available The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.

  3. The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

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    Yan, Rihui; McKee, Bruce D

    2013-01-01

    Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome

  4. TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

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    Abhishek Ghosh

    2014-10-01

    Full Text Available The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

  5. Endoplasmic reticulum (ER) stress and cAMP/PKA pathway mediated Zn-induced hepatic lipolysis.

    Science.gov (United States)

    Song, Yu-Feng; Hogstrand, Christer; Wei, Chuan-Chuan; Wu, Kun; Pan, Ya-Xiong; Luo, Zhi

    2017-09-01

    The present study was performed to determine the effect of Zn exposure influencing endoplasmic reticulum (ER) stress, explore the underlying molecular mechanism of Zn-induced hepatic lipolysis in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco. We found that waterborne Zn exposure evoked ER stress and unfolded protein response (UPR), and activated cAMP/PKA pathway, and up-regulated hepatic lipolysis. The increase in ER stress and lipolysis were associated with activation of cAMP/PKA signaling pathway. Zn also induced an increase in intracellular Ca 2+ level, which could be partially prevented by dantrolene (RyR receptor inhibitor) and 2-APB (IP3 receptor inhibitor), demonstrating that the disturbed Ca 2+ homeostasis in ER contributed to ER stress and dysregulation of lipolysis. Inhibition of ER stress by PBA attenuated UPR, inhibited the activation of cAMP/PKA pathway and resulted in down-regulation of lipolysis. Inhibition of protein kinase RNA-activated-like ER kinase (PERK) by GSK2656157 and inositol-requiring enzyme (IRE) by STF-083010 differentially influenced Zn-induced changes of lipid metabolism, indicating that PERK and IRE pathways played different regulatory roles in Zn-induced lipolysis. Inhibition of PKA by H89 blocked the Zn-induced activation of cAMP/PKA pathway with a concomitant inhibition of ER stress-mediated lipolysis. Taken together, our findings highlight the importance of the ER stress-cAMP/PKA axis in Zn-induced lipolysis, which provides new insights into Zn toxicology in fish and probably in other vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Two Pairs of Mushroom Body Efferent Neurons Are Required for Appetitive Long-Term Memory Retrieval in Drosophila

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    Pierre-Yves Plaçais

    2013-11-01

    Full Text Available One of the challenges facing memory research is to combine network- and cellular-level descriptions of memory encoding. In this context, Drosophila offers the opportunity to decipher, down to single-cell resolution, memory-relevant circuits in connection with the mushroom bodies (MBs, prominent structures for olfactory learning and memory. Although the MB-afferent circuits involved in appetitive learning were recently described, the circuits underlying appetitive memory retrieval remain unknown. We identified two pairs of cholinergic neurons efferent from the MB α vertical lobes, named MB-V3, that are necessary for the retrieval of appetitive long-term memory (LTM. Furthermore, LTM retrieval was correlated to an enhanced response to the rewarded odor in these neurons. Strikingly, though, silencing the MB-V3 neurons did not affect short-term memory (STM retrieval. This finding supports a scheme of parallel appetitive STM and LTM processing.

  7. The nociception genes painless and Piezo are required for the cellular immune response of Drosophila larvae to wasp parasitization.

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    Tokusumi, Yumiko; Tokusumi, Tsuyoshi; Schulz, Robert A

    2017-05-13

    In vertebrates, interaction between the nervous system and immune system is important to protect a challenged host from stress inputs from external sources. In this study, we demonstrate that sensory neurons are involved in the cellular immune response elicited by wasp infestation of Drosophila larvae. Multidendritic class IV neurons sense contacts from external stimuli and induce avoidance behaviors for host defense. Our findings show that inactivation of these sensory neurons impairs the cellular response against wasp parasitization. We also demonstrate that the nociception genes encoding the mechanosensory receptors Painless and Piezo, both expressed in class IV neurons, are essential for the normal cellular immune response to parasite challenge. Copyright © 2017. Published by Elsevier Inc.

  8. Global regulatory roles of the cAMP/PKA pathway revealed by phenotypic, transcriptomic and phosphoproteomic analyses in a null mutant of the PKA catalytic subunit in Candida albicans.

    Science.gov (United States)

    Cao, Chengjun; Wu, Mei; Bing, Jian; Tao, Li; Ding, Xuefen; Liu, Xiaoyun; Huang, Guanghua

    2017-07-01

    The conserved cAMP-dependent protein kinase (PKA) plays critical roles in the regulation of morphological transitions and virulence in the human fungal pathogen Candida albicans. It has long been thought that the PKA catalytic subunit is essential for cell viability in this fungus. Paradoxically, the single adenylyl cyclase-encoding gene, CYR1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. Here, a double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1, t2t1), which encode two isoforms of the PKA catalytic subunit was successfully generated, suggesting that this subunit is not essential for cell viability. Inactivation of the PKA catalytic subunit blocked filamentation and dramatically attenuated white-to-opaque switching, but promoted sexual mating. Comparative transcriptomic analyses demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared with the WT strain, the general transcriptional activity and metabolism were significantly decreased in both the t2t1 and cyr1/cyr1 mutants. Using combined phosphoproteomic and bioinformatic analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. The study sheds new insights into the global regulatory features of the cAMP/PKA pathway in C. albicans. © 2017 John Wiley & Sons Ltd.

  9. Altered gene regulation and synaptic morphology in Drosophila learning and memory mutants

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    Guan, Zhuo; Buhl, Lauren K.; Quinn, William G.; Littleton, J. Troy

    2011-01-01

    Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. Here, we demonstrate that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, we assayed synaptic connectivity and performed an expression analysis to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed ∼375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila. PMID:21422168

  10. An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

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    Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu; Hu, Jennifer F.; Ramroop, Johnny; Kellner, Stefanie M.; Benton, Matthew A.; Govind, Shubha; Dedon, Peter C.; Sternglanz, Rolf; Lai, Eric C.

    2015-01-01

    N6-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease. PMID:26516084

  11. The Drosophila nerfin-1 mRNA requires multiple microRNAs to regulate its spatial and temporal translation dynamics in the developing nervous system.

    Science.gov (United States)

    Kuzin, Alexander; Kundu, Mukta; Brody, Thomas; Odenwald, Ward F

    2007-10-01

    The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter-controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, we have studied their ability to block or limit the expression of reporter protein in nerfin-1-expressing cells during embryonic development. Our results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression.

  12. Dendritic diameter influences the rate and magnitude of hippocampal cAMP and PKA transients during β-adrenergic receptor activation.

    Science.gov (United States)

    Luczak, Vincent; Blackwell, Kim T; Abel, Ted; Girault, Jean-Antoine; Gervasi, Nicolas

    2017-02-01

    In the hippocampus, cyclic-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) form a critical signaling cascade required for long-lasting synaptic plasticity, learning and memory. Plasticity and memory are known to occur following pathway-specific changes in synaptic strength that are thought to result from spatially and temporally coordinated intracellular signaling events. To better understand how cAMP and PKA dynamically operate within the structural complexity of hippocampal neurons, we used live two-photon imaging and genetically-encoded fluorescent biosensors to monitor cAMP levels or PKA activity in CA1 neurons of acute hippocampal slices. Stimulation of β-adrenergic receptors (isoproterenol) or combined activation of adenylyl cyclase (forskolin) and inhibition of phosphodiesterase (IBMX) produced cAMP transients with greater amplitude and rapid on-rates in intermediate and distal dendrites compared to somata and proximal dendrites. In contrast, isoproterenol produced greater PKA activity in somata and proximal dendrites compared to intermediate and distal dendrites, and the on-rate of PKA activity did not differ between compartments. Computational models show that our observed compartmental difference in cAMP can be reproduced by a uniform distribution of PDE4 and a variable density of adenylyl cyclase that scales with compartment size to compensate for changes in surface to volume ratios. However, reproducing our observed compartmental difference in PKA activity required enrichment of protein phosphatase in small compartments; neither reduced PKA subunits nor increased PKA substrates were sufficient. Together, our imaging and computational results show that compartment diameter interacts with rate-limiting components like adenylyl cyclase, phosphodiesterase and protein phosphatase to shape the spatial and temporal components of cAMP and PKA signaling in CA1 neurons and suggests that small neuronal compartments are most sensitive to c

  13. Requirement of the Combination of Mushroom Body ? Lobe and a/ß Lobes for the Retrieval of Both Aversive and Appetitive Early Memories in "Drosophila"

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    Xie, Zhiyong; Huang, Cheng; Ci, Bo; Lianzhang, Wang; Zhong, Yi

    2013-01-01

    Extensive studies of "Drosophila" mushroom body in formation and retrieval of olfactory memories allow us to delineate the functional logic for memory storage and retrieval. Currently, there is a questionable disassociation of circuits for memory storage and retrieval during "Drosophila" olfactory memory processing. Formation…

  14. E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

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    Erokhin, Maksim; Parshikov, Alexander; Georgiev, Pavel; Chetverina, Darya

    2010-06-01

    Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

  15. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

    Science.gov (United States)

    Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

    2016-08-01

    Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation. © FASEB.

  16. The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

    Science.gov (United States)

    Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

    2014-07-01

    Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

  17. The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

    Directory of Open Access Journals (Sweden)

    Karen Linnemannstöns

    2014-07-01

    Full Text Available Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7 was identified as a Wnt co-receptor required for control of planar cell polarity (PCP in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964, which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

  18. Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development

    Directory of Open Access Journals (Sweden)

    Péter Lőrincz

    2014-01-01

    Full Text Available Atg6 (Beclin 1 in mammals is a core component of the Vps34 PI3K (III complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation.

  19. Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2.

    Science.gov (United States)

    Bendzunas, N George; Dörfler, Sabrina; Autenrieth, Karolin; Bertinetti, Daniela; Machal, Erik M F; Kennedy, Eileen J; Herberg, Friedrich W

    2018-03-15

    Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Machine Learning Analysis Identifies Drosophila Grunge/Atrophin as an Important Learning and Memory Gene Required for Memory Retention and Social Learning.

    Science.gov (United States)

    Kacsoh, Balint Z; Greene, Casey S; Bosco, Giovanni

    2017-11-06

    High-throughput experiments are becoming increasingly common, and scientists must balance hypothesis-driven experiments with genome-wide data acquisition. We sought to predict novel genes involved in Drosophila learning and long-term memory from existing public high-throughput data. We performed an analysis using PILGRM, which analyzes public gene expression compendia using machine learning. We evaluated the top prediction alongside genes involved in learning and memory in IMP, an interface for functional relationship networks. We identified Grunge/Atrophin ( Gug/Atro ), a transcriptional repressor, histone deacetylase, as our top candidate. We find, through multiple, distinct assays, that Gug has an active role as a modulator of memory retention in the fly and its function is required in the adult mushroom body. Depletion of Gug specifically in neurons of the adult mushroom body, after cell division and neuronal development is complete, suggests that Gug function is important for memory retention through regulation of neuronal activity, and not by altering neurodevelopment. Our study provides a previously uncharacterized role for Gug as a possible regulator of neuronal plasticity at the interface of memory retention and memory extinction. Copyright © 2017 Kacsoh et al.

  1. The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

    Science.gov (United States)

    Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V.; Karpen, Gary H.

    2012-01-01

    CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote. PMID:23300382

  2. The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

    Science.gov (United States)

    Basquin, Denis; Spierer, Anne; Begeot, Flora; Koryakov, Dmitry E; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

  3. The Drosophila Su(var)3–7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing

    Science.gov (United States)

    Begeot, Flora; Koryakov, Dmitry E.; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing. PMID:24820312

  4. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  5. PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

    International Nuclear Information System (INIS)

    Ohtsuka, Masanari; Fukumitsu, Hidefumi; Furukawa, Shoei

    2008-01-01

    Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway

  6. Anchored PKA as a gatekeeper for gap junctions.

    Science.gov (United States)

    Pidoux, Guillaume; Taskén, Kjetil

    2015-01-01

    Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types.

  7. Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability.

    Directory of Open Access Journals (Sweden)

    Hilla Weidberg

    2016-06-01

    Full Text Available Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA and target of rapamycin complex I (TORC1 signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.

  8. The Determination of "Apparent" pKa's. Part II: An Experiment Using Very Weak Acids (pKa's > 11.4).

    Science.gov (United States)

    Cawley, John J.

    1995-01-01

    Presents an experiment designed to show students that the Henderson-Hasselbalch equation will fail when they use this particular one-half titration technique for acids with large pKa's. Involves determining the apparent pKa for such acids and using that to calculate the true pKa. (JRH)

  9. Developing hybrid approaches to predict pKa values of ionizable groups

    Science.gov (United States)

    Witham, Shawn; Talley, Kemper; Wang, Lin; Zhang, Zhe; Sarkar, Subhra; Gao, Daquan; Yang, Wei

    2011-01-01

    Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure. On another hand, if the change of the charge state is accompanied by a structural reorganization of the target protein, then the relevant conformational changes have to be taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups, which ionization is expected to cause conformational changes, termed “problematic” residues, then applies a special protocol on them, while the rest of the pKa’s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for “problematic” groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the pKa of the “problematic” residues and then the results are averaged. PMID:21744395

  10. Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways

    Directory of Open Access Journals (Sweden)

    Wei-Chien Hung

    2016-05-01

    Full Text Available Summary: Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments. : Hung et al. demonstrate that a Piezo1-dependent intracellular calcium increase negatively regulates protein kinase A (PKA as cells transit from unconfined to confined spaces. The Piezo1/PKA and myosin II signaling modules constitute two confinement-sensing mechanisms. This study provides a paradigm by which signaling enables cells to sense and adapt to different microenvironments.

  11. On the development of protein pKa calculation algorithms

    Science.gov (United States)

    Carstensen, Tommy; Farrell, Damien; Huang, Yong; Baker, Nathan A.; Nielsen, Jens Erik

    2011-01-01

    Protein pKa calculation methods are developed partly to provide fast non-experimental estimates of the ionization constants of protein side chains. However, the most significant reason for developing such methods is that a good pKa calculation method is presumed to provide an accurate physical model of protein electrostatics, which can be applied in methods for drug design, protein design and other structure-based energy calculation methods. We explore the validity of this presumption by simulating the development of a pKa calculation method using artificial experimental data derived from a human-defined physical reality. We examine the ability of an RMSD-guided development protocol to retrieve the correct (artificial) physical reality and find that a rugged optimization landscape and a huge parameter space prevent the identification of the correct physical reality. We examine the importance of the training set in developing pKa calculation methods and investigate the effect of experimental noise on our ability to identify the correct physical reality, and find that both effects have a significant and detrimental impact on the physical reality of the optimal model identified. Our findings are of relevance to all structure-based methods for protein energy calculations and simulation, and have large implications for all types of current pKa calculation methods. Our analysis furthermore suggests that careful and extensive validation on many types of experimental data can go some way in making current models more realistic. PMID:21744393

  12. Predicting pKa for proteins using COSMO-RS

    DEFF Research Database (Denmark)

    Andersson, Martin Peter; Jensen, Jan Halborg; Stipp, Susan Louise Svane

    2013-01-01

    We have used the COSMO-RS implicit solvation method to calculate the equilibrium constants, pKa, for deprotonation of the acidic residues of the ovomucoid inhibitor protein, OMTKY3. The root mean square error for comparison with experimental data is only 0.5 pH units and the maximum error 0.8 p......H units. The results show that the accuracy of pKa prediction using COSMO-RS is as good for large biomolecules as it is for smaller inorganic and organic acids and that the method compares very well to previous pKa predictions of the OMTKY3 protein using Quantum Mechanics/Molecular Mechanics. Our approach...

  13. Computer simulation of cascade damage in iron: PKA mass effects

    International Nuclear Information System (INIS)

    Calder, A.; Bacon, D.J.; Barashev, A.; Osetsky, Y.

    2007-01-01

    Full text of publication follows: Results are presented from an extensive series of computer simulations of the damage created by displacement cascades in alpha-iron. The objective has been to determine for the first time the effect of the mass of the primary knock-on atom (PKA) on defect number, defect clustering and cluster morphology. Cascades with PKA energy in the range 5 to 20 keV have been simulated by molecular dynamics for temperature up to 600 K using an interatomic potential for iron for which the energy difference between the dumbbell interstitial and the crowdion is close to the value from ab initio calculation (Ackland et al., J. Phys.: Condens. Matter 2004). At least 30 cascades have been simulated for each condition in order to generate reasonable statistics. The influence of PKA species on damage has been investigated in two ways. In one, the PKA atom was treated as an Fe atom as far as its interaction with other atoms was concerned, but its atomic weight (in amu) was either 12 (C), 56 (Fe) or 209 (Bi). Pairs of Bi PKAs have also been used to mimic heavy molecular ion irradiation. In the other approach, the short-range pair part of the interatomic potential was changed from Fe-Fe to that for Bi-Fe, either with or without a change of PKA mass, in order to study the influence of high-energy collisions on the cascade outcome. It is found that PKA mass is more influential than the interatomic potential between the PKA and Fe atoms. At low cascade energy (5-10 keV), increasing PKA mass leads to a decrease in number of interstitials and vacancies. At high energy (20 keV), the main effect of increasing mass is to increase the probability of creation of interstitial and vacancy clusters in the form of 1/2 and dislocation loops. The simulation results are consistent with experimental TEM observations of damage in irradiated iron. (authors)

  14. Schistosoma mansoni c-AMP-dependent Protein Kinase (PKA): A Potential New Drug Target

    Science.gov (United States)

    2009-12-07

    subunits from other eukaryotic organisms (Aplysia californica, S. japonicum, Caenorhabditis 143 elegans Mus musculus, Onchocerca volvulus , and Homo...Caenorhabditis elegans PKA-R (J05220); OvR, Onchocerca volvulus PKA-R (AY159364). 156 157 Figure 19: RNAi of Sm04765 in...PKA cancer chemotherapeutics [12]. Interestingly, the PKA-R subunit homologue in Onchocerca volvulus , causative agent of river blindness, is being

  15. cAMP-Dependent Protein Kinase A (PKA)-Mediated c-Myc Degradation Is Dependent on the Relative Proportion of PKA-I and PKA-II Isozymes.

    Science.gov (United States)

    Liu, Qingyuan; Nguyen, Eric; Døskeland, Stein; Ségal-Bendirdjian, Évelyne

    2015-09-01

    The transcription factor c-Myc regulates numerous target genes that are important for multiple cellular processes such as cell growth and differentiation. It is commonly deregulated in leukemia. Acute promyelocytic leukemia (APL) is characterized by a blockade of granulocytic differentiation at the promyelocyte stage. Despite the great success of all-trans retinoic acid (ATRA)-based therapy, which results in a clinical remission by inducing promyelocyte maturation, a significant number of patients relapse due to the development of ATRA resistance. A significant role has been ascribed to the cAMP/cAMP-dependent protein kinase A (PKA) signaling pathway in retinoid treatment since PKA activation is able to restore differentiation in some ATRA-resistant cells and eradicate leukemia-initiating cells in vivo. In this study, using NB4 APL cell variants resistant to ATRA-induced differentiation, we reveal distinct functional roles of the two PKA isozymes, PKA type I (PKA-I) and PKA-type II (PKA-II), on the steady-state level of c-Myc protein, providing a likely mechanism by which cAMP-elevating agents can restore differentiation in ATRA maturation-resistant APL cells. Therefore, both the inhibition of c-Myc activity and the PKA-I/PKA-II ratio should be taken into account if cAMP-based therapy is considered in the clinical management of APL. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Humidity Sensing in Drosophila.

    Science.gov (United States)

    Enjin, Anders; Zaharieva, Emanuela E; Frank, Dominic D; Mansourian, Suzan; Suh, Greg S B; Gallio, Marco; Stensmyr, Marcus C

    2016-05-23

    Environmental humidity influences the fitness and geographic distribution of all animals [1]. Insects in particular use humidity cues to navigate the environment, and previous work suggests the existence of specific sensory mechanisms to detect favorable humidity ranges [2-5]. Yet, the molecular and cellular basis of humidity sensing (hygrosensation) remains poorly understood. Here we describe genes and neurons necessary for hygrosensation in the vinegar fly Drosophila melanogaster. We find that members of the Drosophila genus display species-specific humidity preferences related to conditions in their native habitats. Using a simple behavioral assay, we find that the ionotropic receptors IR40a, IR93a, and IR25a are all required for humidity preference in D. melanogaster. Yet, whereas IR40a is selectively required for hygrosensory responses, IR93a and IR25a mediate both humidity and temperature preference. Consistent with this, the expression of IR93a and IR25a includes thermosensory neurons of the arista. In contrast, IR40a is excluded from the arista but is expressed (and required) in specialized neurons innervating pore-less sensilla of the sacculus, a unique invagination of the third antennal segment. Indeed, calcium imaging showed that IR40a neurons directly respond to changes in humidity, and IR40a knockdown or IR93a mutation reduced their responses to stimuli. Taken together, our results suggest that the preference for a specific humidity range depends on specialized sacculus neurons, and that the processing of environmental humidity can happen largely in parallel to that of temperature. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Assigning the pKa's of Polyprotic Acids.

    Science.gov (United States)

    Bodner, George M.

    1986-01-01

    Discusses (1) polyproptic acids for which the difference between K-a's is large; (2) the Henderson-Hasselbach equation; (3) polyprotic acids for which the difference between K-a's is small; (4) analysis of microscopic dissociation constants for cysteine; and (5) analysis of pK-a data. (JN)

  18. A Proteomics Investigation of Anchored PKA-RI Signaling

    NARCIS (Netherlands)

    Kovanich, D.

    2013-01-01

    Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring

  19. Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

    Directory of Open Access Journals (Sweden)

    Ronald A Merrill

    2011-04-01

    Full Text Available Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM targeted form of the protein kinase A (PKA catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1 as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1, inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.

  20. [Physiopathology of cAMP/PKA signaling in neurons].

    Science.gov (United States)

    Castro, Liliana; Yapo, Cedric; Vincent, Pierre

    2016-01-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, synaptic transmission, regulation of excitability or long term changes in the nucleus. Genetically-encoded optical biosensors for cAMP or PKA considerably improved our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progresses made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the subcellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus and axon. Combining this imaging approach with pharmacology or genetical models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly help understand the mechanism of action of current drugs as well as help in devising novel therapeutic strategies for neuropsychiatric diseases. © Société de Biologie, 2017.

  1. O-GlcNAcylation modulates PKA-CREB signaling in a manner specific to PKA catalytic subunit isoforms.

    Science.gov (United States)

    Jin, Nana; Ma, Denglei; Gu, Jianlan; Shi, Jianhua; Xu, Xiaotao; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei; Chu, Dandan

    2018-02-26

    O-GlcNAcylation is a post-translational modification of proteins. Protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling plays critical roles in multiple biological processes. Isoforms α and β of PKA catalytic subunit (PKAc) and CREB are modified by O-GlcNAcylation. In the present study, we determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAcα-CREB signaling, but enhanced PKAcβ-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. This study suggests a role of O-GlcNAcylation in PKA-CREB signaling by affecting phosphorylation of CREB in a PKAc isoform-specific manner. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

    Science.gov (United States)

    Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

    2018-04-15

    T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization

  3. Normal Female Germ Cell Differentiation Requires the Female X Chromosome to Autosome Ratio and Expression of Sex-Lethal in DROSOPHILA MELANOGASTER

    OpenAIRE

    Schüpbach, Trudi

    1985-01-01

    In somatic cells of Drosophila, the ratio of X chromosomes to autosomes (X:A ratio) determines sex and dosage compensation. The present paper addresses the question of whether germ cells also use the X:A ratio for sex determination and dosage compensation. Triploid female embryos were generated which, through the loss of an unstable ring-X chromosome, contained some germ cells of 2X;3A constitution in their ovaries. Such germ cells were shown to differentiate along one of two alternative pat...

  4. A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

    Science.gov (United States)

    Guergnon, Julien; Dessauge, Frederic; Traincard, François; Cayla, Xavier; Rebollo, Angelita; Bost, Pierre Etienne; Langsley, Gordon; Garcia, Alphonse

    2006-08-01

    T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

  5. A Drosophila wing spot test

    International Nuclear Information System (INIS)

    Ayaki, Toshikazu; Yoshikawa, Isao; Niikawa, Norio; Hoshi, Masaharu.

    1986-01-01

    A Drosophila wing spot test system was used to investigate the effects of low doses of X-rays, gamma rays, and both 2.3 and 14.1 MeV neutrons on somatic chromosome mutation (SCM) induction. The incidence of SCM was significantly increased with any type of radiation, with evident linear dose-response relationship within the range of 3 to 20 cGy. It was estimated that relative biological effectiveness value for SCM induction of 2.3 MeV neutrons to X-rays and gamma rays is much higher than that of 14.1 MeV neutrons to those photons (2.4 vs 8.0). The Drosophila wing spot test system seems to become a promising in vivo experimental method for higher animals in terms of the lack of necessity for a marvelously large number of materials required in conventional test system. (Namekawa, K.)

  6. Limited taste discrimination in Drosophila.

    Science.gov (United States)

    Masek, Pavel; Scott, Kristin

    2010-08-17

    In the gustatory systems of mammals and flies, different populations of sensory cells recognize different taste modalities, such that there are cells that respond selectively to sugars and others to bitter compounds. This organization readily allows animals to distinguish compounds of different modalities but may limit the ability to distinguish compounds within one taste modality. Here, we developed a behavioral paradigm in Drosophila melanogaster to evaluate directly the tastes that a fly distinguishes. These studies reveal that flies do not discriminate among different sugars, or among different bitter compounds, based on chemical identity. Instead, flies show a limited ability to distinguish compounds within a modality based on intensity or palatability. Taste associative learning, similar to olfactory learning, requires the mushroom bodies, suggesting fundamental similarities in brain mechanisms underlying behavioral plasticity. Overall, these studies provide insight into the discriminative capacity of the Drosophila gustatory system and the modulation of taste behavior.

  7. 'Peer pressure' in larval Drosophila?

    Science.gov (United States)

    Niewalda, Thomas; Jeske, Ines; Michels, Birgit; Gerber, Bertram

    2014-06-06

    Understanding social behaviour requires a study case that is simple enough to be tractable, yet complex enough to remain interesting. Do larval Drosophila meet these requirements? In a broad sense, this question can refer to effects of the mere presence of other larvae on the behaviour of a target individual. Here we focused in a more strict sense on 'peer pressure', that is on the question of whether the behaviour of a target individual larva is affected by what a surrounding group of larvae is doing. We found that innate olfactory preference of a target individual was neither affected (i) by the level of innate olfactory preference in the surrounding group nor (ii) by the expression of learned olfactory preference in the group. Likewise, learned olfactory preference of a target individual was neither affected (iii) by the level of innate olfactory preference of the surrounding group nor (iv) by the learned olfactory preference the group was expressing. We conclude that larval Drosophila thus do not take note of specifically what surrounding larvae are doing. This implies that in a strict sense, and to the extent tested, there is no social interaction between larvae. These results validate widely used en mass approaches to the behaviour of larval Drosophila. © 2014. Published by The Company of Biologists Ltd.

  8. Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

    Science.gov (United States)

    Röck, Ruth; Mayrhofer, Johanna E; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

  9. Determination of pK(a) of felodipine using UV-Visible spectroscopy.

    Science.gov (United States)

    Pandey, M M; Jaipal, A; Kumar, A; Malik, R; Charde, S Y

    2013-11-01

    In the present study, for the first time, experimental pKa value of felodipine is reported. Dissociation constant, pKa, is one of the very important physicochemical properties of drugs. It is of paramount significance from the perspective of pharmaceutical analysis and dosage form design. The method used for the pKa determination of felodipine was essentially a UV-Visible spectrophotometric method. The spectrophotometric method for the pKa determination was opted by acknowledging the established fact that spectrophotometric determination of pKa produces most precise values. The pKa of felodipine was found to be 5.07. Furthermore, the ruggedness of the determined value is also validated in this study in order to produce exact pKa of the felodipine. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase*

    Science.gov (United States)

    Di Nardo, Giovanna; Breitner, Maximilian; Bandino, Andrea; Ghosh, Debashis; Jennings, Gareth K.; Hackett, John C.; Gilardi, Gianfranco

    2015-01-01

    Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp309 in substrate binding and catalysis. These studies predicted an elevated pKa for Asp309 and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp309 with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp309 based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pKa of 8.2 observed for androstenedione binding to the side chain of Asp309. To our knowledge, this work represents the first experimental assignment of a pKa value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures. PMID:25425647

  11. Molecular evolution of a-kinase anchoring protein (AKAP-7: implications in comparative PKA compartmentalization

    Directory of Open Access Journals (Sweden)

    Johnson Keven R

    2012-07-01

    Full Text Available Abstract Background A-Kinase Anchoring Proteins (AKAPs are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA, directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18 are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. Results Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels that promote the production of AKAP7γ instead of AKAP7δ. Conclusions This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

  12. Quantification of Drosophila Grooming Behavior.

    Science.gov (United States)

    Barradale, Francesca; Sinha, Kairav; Lebestky, Tim

    2017-07-19

    Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

  13. Displacement cross sections and PKA spectra: tables and applications

    International Nuclear Information System (INIS)

    Doran, D.G.; Graves, N.J.

    1976-12-01

    Damage energy cross sections to 20 MeV are given for aluminum, vanadium, chromium, iron, nickel, copper, zirconium, niobium, molybdenum, tantalum, tungsten, lead, and 18Cr10Ni stainless steel. They are based on ENDF/B-IV nuclear data and the Lindhard energy partition model. Primary knockon atom (PKA) spectra are given for aluminum, iron, niobium, tantalum, and lead for neutron energies up to 15 MeV at approximately one-quarter lethargy intervals. The contributions of various reactions to both the displacement cross sections (taken to be proportional to the damage energy cross sections) and the PKA spectra are presented graphically. Spectral-averaged values of the displacement cross sections are given for several spectra, including approximate maps for the Experimental Breeder Reactor-II (EBR-II) and several positions in the Fast Test Reactor (FTR). Flux values are included to permit estimation of displacement rates. Graphs show integral PKA spectra for the five metals listed above for neutron spectra corresponding to locations in the EBR-II, the High Flux Isotope Reactor (HFIR), and a conceptual fusion reactor (UWMAK-I). Detailed calculations are given only for cases not previously documented. Uncertainty estimates are included

  14. Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation.

    Science.gov (United States)

    Vimal, Divya; Kumar, Saurabh; Pandey, Ashutosh; Sharma, Divya; Saini, Sanjay; Gupta, Snigdha; Ravi Ram, Kristipati; Chowdhuri, Debapratim Kar

    2018-03-01

    Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1 e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1 e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1 e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1 e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Systems-level identification of PKA-dependent signaling in epithelial cells.

    Science.gov (United States)

    Isobe, Kiyoshi; Jung, Hyun Jun; Yang, Chin-Rang; Claxton, J'Neka; Sandoval, Pablo; Burg, Maurice B; Raghuram, Viswanathan; Knepper, Mark A

    2017-10-17

    G protein stimulatory α-subunit (G αs )-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G αs -coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

  16. Review: Bilirubin pKa studies; new models and theories indicate high pKa values in water, dimethylformamide and DMSO

    Directory of Open Access Journals (Sweden)

    Ostrow J

    2010-03-01

    Full Text Available Abstract Background Correct aqueous pKa values of unconjugated bilirubin (UCB, a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB. Results These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide. Conclusions No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.

  17. Rationalization of the pKa values of alcohols and thiols using atomic charge descriptors and its application to the prediction of amino acid pKa's.

    Science.gov (United States)

    Ugur, Ilke; Marion, Antoine; Parant, Stéphane; Jensen, Jan H; Monard, Gerald

    2014-08-25

    In a first step toward the development of an efficient and accurate protocol to estimate amino acids' pKa's in proteins, we present in this work how to reproduce the pKa's of alcohol and thiol based residues (namely tyrosine, serine, and cysteine) in aqueous solution from the knowledge of the experimental pKa's of phenols, alcohols, and thiols. Our protocol is based on the linear relationship between computed atomic charges of the anionic form of the molecules (being either phenolates, alkoxides, or thiolates) and their respective experimental pKa values. It is tested with different environment approaches (gas phase or continuum solvent-based approaches), with five distinct atomic charge models (Mulliken, Löwdin, NPA, Merz-Kollman, and CHelpG), and with nine different DFT functionals combined with 16 different basis sets. Moreover, the capability of semiempirical methods (AM1, RM1, PM3, and PM6) to also predict pKa's of thiols, phenols, and alcohols is analyzed. From our benchmarks, the best combination to reproduce experimental pKa's is to compute NPA atomic charge using the CPCM model at the B3LYP/3-21G and M062X/6-311G levels for alcohols (R(2) = 0.995) and thiols (R(2) = 0.986), respectively. The applicability of the suggested protocol is tested with tyrosine and cysteine amino acids, and precise pKa predictions are obtained. The stability of the amino acid pKa's with respect to geometrical changes is also tested by MM-MD and DFT-MD calculations. Considering its strong accuracy and its high computational efficiency, these pKa prediction calculations using atomic charges indicate a promising method for predicting amino acids' pKa in a protein environment.

  18. Metabolomic Studies in Drosophila.

    Science.gov (United States)

    Cox, James E; Thummel, Carl S; Tennessen, Jason M

    2017-07-01

    Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila , often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research. Copyright © 2017 by the Genetics Society of America.

  19. Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

    Science.gov (United States)

    Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

    2018-03-23

    cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

  20. Drosophila Melanogaster as an Experimental Organism.

    Science.gov (United States)

    Rubin, Gerald M.

    1988-01-01

    Discusses the role of the fruit fly in genetics research requiring a multidisciplinary approach. Describes embryological and genetic methods used in the experimental analysis of this organism. Outlines the use of Drosophila in the study of the development and function of the nervous system. (RT)

  1. PINK1 regulates mitochondrial trafficking in dendrites of cortical neurons through mitochondrial PKA.

    Science.gov (United States)

    Das Banerjee, Tania; Dagda, Raul Y; Dagda, Marisela; Chu, Charleen T; Rice, Monica; Vazquez-Mayorga, Emmanuel; Dagda, Ruben K

    2017-08-01

    Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding-deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit β of PKA (PKA/RIIβ) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIβ and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity. © 2017 International Society for Neurochemistry.

  2. PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling

    Science.gov (United States)

    Yang, Jason H.; Polanowska-Grabowska, Renata K.; Smith, Jeffrey S.; Shields, Charles W.; Saucerman, Jeffrey J.

    2014-01-01

    β-adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca2+ handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50 = 10.60±0.68 min; EC50 = 89.00 nmol/L) than in the cytosol (t50 = 3.71±0.25 min; EC50 = 1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca2+ and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50 = 1.84 nmol/L) over cell hypertrophy (EC50 = 85.88 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses. PMID:24225179

  3. Potentiometric pKa Determination of Piroxicam and Tenoxicam in Acetonitrile-Water Binary Mixtures

    OpenAIRE

    Çubuk Demiralay, Ebru; Yılmaz, Hülya

    2012-01-01

    Abstract: Ionization constant (pKa) is one among the parameter to be estimated with accuracy, irrespective of solubility constraints. In the present study, acid-base behaviour of the piroxicam and tenoxicam was studied. By using the potentiometric method, pKa values of piroxicam and tenoxicam have been determined in different percentage of acetonitrile-water binary mixtures (acetonitrile content between 30 and 45% in volume). Aqueous pKa values of these compounds were calculated by mole fract...

  4. GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

    Science.gov (United States)

    Loh, Joon-Khim; Lin, Ching-Chih; Yang, Ming-Chang; Chou, Chia-Hua; Chen, Wan-Shia; Hong, Ming-Chang; Cho, Chung-Lung; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Chang, Chung-Hsing; Tseng, Chao-Neng; Wang, Chi-Huei; Lieu, Ann-Shung; Howng, Shen-Long; Hong, Yi-Ren

    2015-08-01

    GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Highly Perturbed pKa Values in the Unfolded State of Hen Egg White Lysozyme

    OpenAIRE

    Bradley, John; O'Meara, Fergal; Farrell, Damien; Nielsen, Jens Erik

    2012-01-01

    The majority of pKa values in protein unfolded states are close to the amino acid model pKa values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pKa values and the pH dependence of protein stability for this enzyme. The availability of accurate pKa values for the folded state of HEWL and separa...

  6. Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway.

    Science.gov (United States)

    Jiang, Huili; Zhang, Xuhui; Wang, Yu; Zhang, Huimin; Li, Jing; Yang, Xinjing; Zhao, Bingcong; Zhang, Chuntao; Yu, Miao; Xu, Mingmin; Yu, Qiuyun; Liang, Xingchen; Li, Xiang; Shi, Peng; Bao, Tuya

    2017-01-01

    Protein kinase A (PKA)/cAMP response element-binding (CREB) protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA- α and p-CREB was significantly downregulated by chronic unpredicted mild stress (CUMS) procedures. Importantly, acupuncture reversed the downregulation of PKA- α and p-CREB. The expression of PKA- α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA- α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA- α . There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB.

  7. Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

    Science.gov (United States)

    Imamura, Haruna; Wagih, Omar; Niinae, Tomoya; Sugiyama, Naoyuki; Beltrao, Pedro; Ishihama, Yasushi

    2017-04-07

    Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

  8. cAMP-dependent Protein Kinase (PKA) Signaling Is Impaired in the Diabetic Heart.

    Science.gov (United States)

    Bockus, Lee B; Humphries, Kenneth M

    2015-12-04

    Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced β-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. cAMP-dependent Protein Kinase (PKA) Signaling Is Impaired in the Diabetic Heart*

    Science.gov (United States)

    Bockus, Lee B.; Humphries, Kenneth M.

    2015-01-01

    Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced β-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function. PMID:26468277

  10. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor: EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING.

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G H; Fukuhara, Shigetomo; Taylor, Susan S; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-03-18

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The Drosophila Translational Control Element (TCE) is required for high-level transcription of many genes that are specifically expressed in testes.

    Science.gov (United States)

    Katzenberger, Rebeccah J; Rach, Elizabeth A; Anderson, Ashley K; Ohler, Uwe; Wassarman, David A

    2012-01-01

    To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE). The TCE functions in the 5' untranslated region of Mst(3)CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp) genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID) subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and understanding the

  12. upSET, the Drosophila homologue of SET3, Is Required for Viability and the Proper Balance of Active and Repressive Chromatin Marks

    Directory of Open Access Journals (Sweden)

    Kyle A. McElroy

    2017-02-01

    Full Text Available Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5. Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET. We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.

  13. Organization and evolution of Drosophila terminin: similarities and differences between Drosophila and human telomeres

    Directory of Open Access Journals (Sweden)

    Grazia Daniela Raffa

    2013-05-01

    Full Text Available Drosophila lacks telomerase and fly telomeres are elongated by occasional transposition of three specialized retroelements. Drosophila telomeres do not terminate with GC-rich repeats and are assembled independently of the sequence of chromosome ends. Recent work has shown that Drosophila telomeres are capped by the terminin complex, which includes the fast-evolving proteins HOAP, HipHop, Moi and Ver. These proteins are not conserves outside Drosophilidae and localize and function exclusively at telomeres, protecting them from fusion events. Other proteins required to prevent end-to-end fusion in flies include HP1, Eff/UbcD1, ATM, the components of the Mre11-Rad50-Nbs (MRN complex, and the Woc transcription factor. These proteins do not share the terminin properties; they are evolutionarily conserved non-fast-evolving proteins that do not accumulate only telomeres and do not serve telomere-specific functions. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner. This hypothesis suggests that terminin is the functional analog of the shelterin complex that protects human telomeres. The non-terminin proteins are instead likely to correspond to ancestral telomere-associated proteins that did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. Thus, it appears that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. We believe that Drosophila telomeres offer excellent opportunities for investigations on human telomere biology. The identification of additional Drosophila genes encoding non-terminin proteins involved in telomere protection might lead to the discovery of novel components of human telomeres.

  14. AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Neeta Bala Tannan

    2018-01-01

    Full Text Available AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.

  15. AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster.

    Science.gov (United States)

    Bala Tannan, Neeta; Collu, Giovanna; Humphries, Ashley C; Serysheva, Ekatherina; Weber, Ursula; Mlodzik, Marek

    2018-01-01

    AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.

  16. Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

    Science.gov (United States)

    Barbagallo, Federica; Xu, Bing; Reddy, Gopireddy R; West, Toni; Wang, Qingtong; Fu, Qin; Li, Minghui; Shi, Qian; Ginsburg, Kenneth S; Ferrier, William; Isidori, Andrea M; Naro, Fabio; Patel, Hemal H; Bossuyt, Julie; Bers, Donald; Xiang, Yang K

    2016-09-30

    In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation. © 2016 American Heart Association, Inc.

  17. Molecular Dynamics Pinpoint the Global Fluorine Effect in Balanoid Binding to PKCε and PKA.

    Science.gov (United States)

    Hardianto, Ari; Liu, Fei; Ranganathan, Shoba

    2018-02-26

    (-)-Balanol is an adenosine triphosphate mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is known as a tumor promoter, PKC isozymes can be tumor promoters or suppressors. In particular, PKCε is frequently involved in tumorigenesis and a potential target for anticancer drugs. We recently reported that stereospecific fluorination of balanol yielded a balanoid with enhanced selectivity for PKCε over other PKC isozymes and PKA, although the global fluorine effect behind the selectivity enhancement is not fully understood. Interestingly, in contrast to PKA, PKCε is more sensitive to this fluorine effect. Here we investigate the global fluorine effect on the different binding responses of PKCε and PKA to balanoids using molecular dynamics (MD) simulations. For the first time to the best of our knowledge, we found that a structurally equivalent residue in each kinase, Thr184 in PKA and Ala549 in PKCε, is essential for the different binding responses. Furthermore, the study revealed that the invariant Lys, Lys73 in PKA and Lys437 in PKCε, already known to have a crucial role in the catalytic activity of kinases, serves as the main anchor for balanol binding. Overall, while Thr184 in PKA attenuates the effect of fluorination, Ala549 permits remote response of PKCε to fluorine substitution, with implications for rational design of future balanol-based PKCε inhibitors.

  18. The importance of the PKA-energy spectrum for radiation damage simulation

    International Nuclear Information System (INIS)

    Dierckx, R.

    1987-01-01

    Primary damage phenomena as a function of the PKA-energy are simulated with the MARLOWE code. The PKA's studied have energies up to 2 MeV. The displacement cascades are divided into subcascades, the characteristics of which are determined. (orig.)

  19. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics.

    Science.gov (United States)

    Qiang, Zhimin; Adams, Craig

    2004-07-01

    This work determined the acid dissociation constants (pKa) of 26 common human and veterinary antibiotics by potentiometric titration. Selected antibiotics consisted of sulfonamides, macrolides, tetracyclines, fluoroquinolones, and other miscellaneous antibiotics. After validation of analysis methods using phosphoric acid as a model compound, a second-derivative (delta2pH/deltaV2) method was primarily applied to determining pKa's from titration curves for most antibiotics due to its convenience and accuracy. For tetracyclines, however, a least-square non-linear regression method was developed to determine their pKa's because the second-derivative method cannot well distinguish the pKa,2 and pKa,3 of tetracyclines. Results indicate that the pKa values are approximately 2 and 5-7.5 for sulfonamides; 7.5-9 for macrolides; 3-4, 7-8 and 9-10 for tetracyclines; 3-4, 6, 7.5-9 and 10-11 for fluoroquinolones; while compound-specific for other miscellaneous antibiotics. The moieties corresponding to specific pKa's were identified based on chemical structures of antibiotics. In addition, the pKa's available in literature determined by various techniques are compiled in comparison with the values of this work. These results are expected to essentially facilitate the research on occurrence, fate and effects, analysis methods development, and control of antibiotics in various treatment operations.

  20. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza; Jung, Sung Yun; Liu, Dou; Su, Bing; Qin, Jun

    2012-01-01

    capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit

  1. The Drosophila Translational Control Element (TCE is required for high-level transcription of many genes that are specifically expressed in testes.

    Directory of Open Access Journals (Sweden)

    Rebeccah J Katzenberger

    Full Text Available To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE. The TCE functions in the 5' untranslated region of Mst(3CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and

  2. pKa predictions for proteins, RNAs, and DNAs with the Gaussian dielectric function using DelPhi pKa.

    Science.gov (United States)

    Wang, Lin; Li, Lin; Alexov, Emil

    2015-12-01

    We developed a Poisson-Boltzmann based approach to calculate the pKa values of protein ionizable residues (Glu, Asp, His, Lys and Arg), nucleotides of RNA and single stranded DNA. Two novel features were utilized: the dielectric properties of the macromolecules and water phase were modeled via the smooth Gaussian-based dielectric function in DelPhi and the corresponding electrostatic energies were calculated without defining the molecular surface. We tested the algorithm by calculating pKa values for more than 300 residues from 32 proteins from the PPD dataset and achieved an overall RMSD of 0.77. Particularly, the RMSD of 0.55 was achieved for surface residues, while the RMSD of 1.1 for buried residues. The approach was also found capable of capturing the large pKa shifts of various single point mutations in staphylococcal nuclease (SNase) from pKa-cooperative dataset, resulting in an overall RMSD of 1.6 for this set of pKa's. Investigations showed that predictions for most of buried mutant residues of SNase could be improved by using higher dielectric constant values. Furthermore, an option to generate different hydrogen positions also improves pKa predictions for buried carboxyl residues. Finally, the pKa calculations on two RNAs demonstrated the capability of this approach for other types of biomolecules. © 2015 Wiley Periodicals, Inc.

  3. The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA

    Directory of Open Access Journals (Sweden)

    Herberg Friedrich W

    2011-08-01

    Full Text Available Abstract Background The two variants of the α-form of the catalytic (C subunit of protein kinase A (PKA, designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. Results We show that Cα2 interacts with the two major forms of the regulatory subunit (R of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR, we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. Conclusion We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable.

  4. Hearing regulates Drosophila aggression.

    Science.gov (United States)

    Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C; Heinrich, Ralf; Callaerts, Patrick

    2017-02-21

    Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

  5. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum.

    Science.gov (United States)

    Zhu, Wenjun; Zhou, Man; Xiong, Zeyang; Peng, Fang; Wei, Wei

    2017-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant- C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2 , that encode the catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase A (PKA). To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum , we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase ( ChAC ) gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H 2 O 2 . In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum . Moreover, we failed to generate Δ ChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum .

  6. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    2017-07-01

    Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant–C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2, that encode the catalytic subunits of cyclic AMP (cAMP-dependent protein kinase A (PKA. To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum, we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase (ChAC gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H2O2. In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum. Moreover, we failed to generate ΔChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum.

  7. Acute vs chronic exposure to high fat diet leads to distinct regulation of PKA.

    Science.gov (United States)

    London, Edra; Nesterova, Maria; Stratakis, Constantine A

    2017-07-01

    The cAMP-dependent protein kinase (PKA) is an essential regulator of lipid and glucose metabolism that plays a critical role in energy homeostasis. The impact of diet on PKA signaling has not been defined, although perturbations in individual PKA subunits are associated with changes in adiposity, physical activity and energy intake in mice and humans. We hypothesized that a high fat diet (HFD) would elicit peripheral and central alterations in the PKA system that would differ depending on length of exposure to HFD; these differences could protect against or promote diet-induced obesity (DIO). 12-week-old C57Bl/6J mice were randomly assigned to a regular diet or HFD and weighed weekly throughout the feeding studies (4 days, 14 weeks; respectively), and during killing. PKA activity and subunit expression were measured in liver, gonadal adipose tissue (AT) and brain. Acute HFD-feeding suppressed basal hepatic PKA activity. In contrast, hepatic and hypothalamic PKA activities were significantly increased after chronic HFD-feeding. Changes in AT were more subtle, and overall, altered PKA regulation in response to chronic HFD exposure was more profound in female mice. The suppression of hepatic PKA activity after 4 day HFD-feeding was indicative of a protective peripheral effect against obesity in the context of overnutrition. In response to chronic HFD-feeding, and with the development of DIO, dysregulated hepatic and hypothalamic PKA signaling was a signature of obesity that is likely to promote further metabolic dysfunction in mice. © 2017 Society for Endocrinology.

  8. Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations.

    Science.gov (United States)

    Mena-Ulecia, Karel; Gonzalez-Norambuena, Fabian; Vergara-Jaque, Ariela; Poblete, Horacio; Tiznado, William; Caballero, Julio

    2018-02-05

    Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site. In this work, differential affinities of PKA for the peptide substrate Kemptide (LRRASLG) and mutants that substitute the arginine residues by the unnatural peptide homoarginine were evaluated through molecular dynamics (MD) and free energy perturbation (FEP) calculations. The FEP study for the homoarginine mutants required previous elaboration of a CHARMM "arginine to homoarginine" (R2B) hybrid topology file which is available in this manuscript as Supporting Information. Mutants substituting the arginine residues by alanine, lysine, and histidine were also considered in the comparison by using the same protocol. FEP calculations allowed estimating the free energy changes from the free PKA to PKA-substrate complex (ΔΔG E→ES ) when Kemptide structure was mutated. Both ΔΔG S→ES values for homoarginine mutants were predicted with a difference below 1 kcal/mol. In addition, FEP correctly predicted that all the studied mutations decrease the catalytic efficiency of Kemptide for PKA. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  9. Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells.

    Science.gov (United States)

    Yaniv, Yael; Ganesan, Ambhighainath; Yang, Dongmei; Ziman, Bruce D; Lyashkov, Alexey E; Levchenko, Andre; Zhang, Jin; Lakatta, Edward G

    2015-09-01

    cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca(2+)-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. BMAA neurotoxicity in Drosophila.

    Science.gov (United States)

    Zhou, Xianchong; Escala, Wilfredo; Papapetropoulos, Spyridon; Bradley, Walter G; Zhai, R Grace

    2009-01-01

    We report the establishment of an in vivo model using the fruit fly Drosophila melanogaster to investigate the toxic effects of L-BMAA. We found that dietary intake of BMAA reduced the lifespan as well as the neurological functions of flies. Furthermore, we have developed an HPLC method to reliably detect both free and protein-bound BMAA in fly tissue extracts.

  11. Cancer in Drosophila

    DEFF Research Database (Denmark)

    Herranz, Héctor; Eichenlaub, Teresa; Cohen, Stephen M

    2016-01-01

    Cancer genomics has greatly increased our understanding of the complexity of the genetic and epigenetic changes found in human tumors. Understanding the functional relationships among these elements calls for the use of flexible genetic models. We discuss the use of Drosophila models to study...

  12. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  13. Isoform-Selective Disruption of AKAP-Localized PKA Using Hydrocarbon Stapled Peptides

    Science.gov (United States)

    2015-01-01

    A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. PMID:24422448

  14. Sustained exposure to catecholamines affects cAMP/PKA compartmentalised signalling in adult rat ventricular myocytes.

    Science.gov (United States)

    Fields, Laura A; Koschinski, Andreas; Zaccolo, Manuela

    2016-07-01

    In the heart compartmentalisation of cAMP/protein kinase A (PKA) signalling is necessary to achieve a specific functional outcome in response to different hormonal stimuli. Chronic exposure to catecholamines is known to be detrimental to the heart and disrupted compartmentalisation of cAMP signalling has been associated to heart disease. However, in most cases it remains unclear whether altered local cAMP signalling is an adaptive response, a consequence of the disease or whether it contributes to the pathogenetic process. We have previously demonstrated that isoforms of PKA expressed in cardiac myocytes, PKA-I and PKA-II, localise to different subcellular compartments and are selectively activated by spatially confined pools of cAMP, resulting in phosphorylation of distinct downstream targets. Here we investigate cAMP signalling in an in vitro model of hypertrophy in primary adult rat ventricular myocytes. By using a real time imaging approach and targeted reporters we find that that sustained exposure to catecholamines can directly affect cAMP/PKA compartmentalisation. This appears to involve a complex mechanism including both changes in the subcellular localisation of individual phosphodiesterase (PDE) isoforms as well as the relocalisation of PKA isoforms. As a result, the preferential coupling of PKA subsets with different PDEs is altered resulting in a significant difference in the level of cAMP the kinase is exposed to, with potential impact on phosphorylation of downstream targets. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Mitochondrial cAMP-PKA signaling: What do we really know?

    Science.gov (United States)

    Ould Amer, Yasmine; Hebert-Chatelain, Etienne

    2018-04-23

    Mitochondria are key organelles for cellular homeostasis. They generate the most part of ATP that is used by cells through oxidative phosphorylation. They also produce reactive oxygen species, neurotransmitters and other signaling molecules. They are important for calcium homeostasis and apoptosis. Considering the role of this organelle, it is not surprising that most mitochondrial dysfunctions are linked to the development of pathologies. Various mechanisms adjust mitochondrial activity according to physiological needs. The cAMP-PKA signaling emerged in recent years as a direct and powerful mean to regulate mitochondrial functions. Multiple evidence demonstrates that such pathway can be triggered from cytosol or directly within mitochondria. Notably, specific anchor proteins target PKA to mitochondria whereas enzymes necessary for generation and degradation of cAMP are found directly in these organelles. Mitochondrial PKA targets proteins localized in different compartments of mitochondria, and related to various functions. Alterations of mitochondrial cAMP-PKA signaling affect the development of several physiopathological conditions, including neurodegenerative diseases. It is however difficult to discriminate between the effects of cAMP-PKA signaling triggered from cytosol or directly in mitochondria. The specific roles of PKA localized in different mitochondrial compartments are also not completely understood. The aim of this work is to review the role of cAMP-PKA signaling in mitochondrial (patho)physiology. Copyright © 2018. Published by Elsevier B.V.

  16. Curcumin Protects Neurons from Glutamate-Induced Excitotoxicity by Membrane Anchored AKAP79-PKA Interaction Network

    Directory of Open Access Journals (Sweden)

    Kui Chen

    2015-01-01

    Full Text Available Now stimulation of AMPA receptor as well as its downstream pathways is considered as potential central mediators in antidepressant mechanisms. As a signal integrator which binds to AMPA receptor, A-kinase anchoring protein 79-(AKAP79- PKA complex is regarded as a potential drug target to exert neuroprotective effects. A well-tolerated and multitarget drug curcumin has been confirmed to exert antidepressant-like effects. To explore whether AKAP79-PKA complex is involved in curcumin-mediated antiexcitotoxicity, we detected calcium signaling, subcellular location of AKAP79-PKA complex, phosphorylation of glutamate receptor, and ERK and AKT cascades. In this study, we found that curcumin protected neurons from glutamate insult by reducing Ca2+ influx and blocking the translocation of AKAP79 from cytomembrane to cytoplasm. In parallel, curcumin enhanced the phosphorylation of AMPA receptor and its downstream pathways in PKA-dependent manner. If we pretreated cells with PKA anchoring inhibitor Ht31 to disassociate PKA from AKAP79, no neuroprotective effects were observed. In conclusion, our results show that AKAP79-anchored PKA facilitated the signal relay from AMPA receptor to AKT and ERK cascades, which may be crucial for curcumin-mediated antiexcitotoxicity.

  17. Mitochondrial mislocalization underlies Abeta42-induced neuronal dysfunction in a Drosophila model of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Kanae Iijima-Ando

    2009-12-01

    Full Text Available The amyloid-beta 42 (Abeta42 is thought to play a central role in the pathogenesis of Alzheimer's disease (AD. However, the molecular mechanisms by which Abeta42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Abeta42 induces mitochondrial mislocalization, which contributes to Abeta42-induced neuronal dysfunction in a transgenic Drosophila model. In the Abeta42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Abeta42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Abeta42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Abeta42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Abeta42 in vivo.

  18. PKA controls calcium influx into motor neurons during a rhythmic behavior.

    Directory of Open Access Journals (Sweden)

    Han Wang

    Full Text Available Cyclic adenosine monophosphate (cAMP has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

  19. PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

    Science.gov (United States)

    Wang, Han; Sieburth, Derek

    2013-01-01

    Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels. PMID:24086161

  20. Drosophila VAMP7 regulates Wingless intracellular trafficking.

    Science.gov (United States)

    Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

    2017-01-01

    Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

  1. Motor Control of Drosophila Courtship Song

    Directory of Open Access Journals (Sweden)

    Troy R. Shirangi

    2013-11-01

    Full Text Available Many animals utilize acoustic signals—or songs—to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D. melanogaster (hg1 that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a “modular” anatomical substrate for the evolution of diverse songs.

  2. Differential PKA activation and AKAP association determines cell fate in cancer cells

    Science.gov (United States)

    2013-01-01

    Background The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms. Methods Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects. Results We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation. Conclusions We had identified 2

  3. Effect of methylation on the side-chain pKa value of arginine.

    Science.gov (United States)

    Evich, Marina; Stroeva, Ekaterina; Zheng, Yujun George; Germann, Markus W

    2016-02-01

    Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition. © 2015 The Protein Society.

  4. Modeling Human Cancers in Drosophila.

    Science.gov (United States)

    Sonoshita, M; Cagan, R L

    2017-01-01

    Cancer is a complex disease that affects multiple organs. Whole-body animal models provide important insights into oncology that can lead to clinical impact. Here, we review novel concepts that Drosophila studies have established for cancer biology, drug discovery, and patient therapy. Genetic studies using Drosophila have explored the roles of oncogenes and tumor-suppressor genes that when dysregulated promote cancer formation, making Drosophila a useful model to study multiple aspects of transformation. Not limited to mechanism analyses, Drosophila has recently been showing its value in facilitating drug development. Flies offer rapid, efficient platforms by which novel classes of drugs can be identified as candidate anticancer leads. Further, we discuss the use of Drosophila as a platform to develop therapies for individual patients by modeling the tumor's genetic complexity. Drosophila provides both a classical and a novel tool to identify new therapeutics, complementing other more traditional cancer tools. © 2017 Elsevier Inc. All rights reserved.

  5. PDF and cAMP enhance PER stability in Drosophila clock neurons

    Science.gov (United States)

    Li, Yue; Guo, Fang; Shen, James; Rosbash, Michael

    2014-01-01

    The neuropeptide PDF is important for Drosophila circadian rhythms: pdf01 (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light–dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. We discovered that the mutant period gene perS ameliorates the phenotypes of pdf-null flies. The period protein (PER) is a well-studied repressor of clock gene transcription, and the perS protein (PERS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing PER stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes PER, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent PER degradation in pdf01 circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance PER stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. We further suggest that the more rapid degradation of PERS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF. PMID:24707054

  6. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    Science.gov (United States)

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

  7. Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

    NARCIS (Netherlands)

    de Vries, HI; Uyetake, L; Lemstra, W; Brunsting, JF; Su, TT; Kampinga, HH; Sibon, OCM

    2005-01-01

    Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and

  8. Fear memory consolidation in sleep requires protein kinase A.

    Science.gov (United States)

    Cho, Jiyeon; Sypniewski, Krzysztof A; Arai, Shoko; Yamada, Kazuo; Ogawa, Sonoko; Pavlides, Constantine

    2018-05-01

    It is well established that protein kinase A (PKA) is involved in hippocampal dependent memory consolidation. Sleep is also known to play an important role in this process. However, whether sleep-dependent memory consolidation involves PKA activation has not been clearly determined. Using behavioral observation, animals were categorized into sleep and awake groups. We show that intrahippocampal injections of the PKA inhibitor Rp-cAMPs in post-contextual fear conditioning sleep produced a suppression of long-term fear memory, while injections of Rp-cAMPs during an awake state, at a similar time point, had no effect. In contrast, injections of the PKA activator Sp-cAMPs in awake state, rescued sleep deprivation-induced memory impairments. These results suggest that following learning, PKA activation specifically in sleep is required for the consolidation of long-term memory. © 2018 Cho et al.; Published by Cold Spring Harbor Laboratory Press.

  9. The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for Drosophila Wnt2 Required for Male Fertility

    OpenAIRE

    Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

    2014-01-01

    Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-recept...

  10. Role of AC-cAMP-PKA Cascade in Antidepressant Action of Electroacupuncture Treatment in Rats

    Directory of Open Access Journals (Sweden)

    Jian-hua Liu

    2012-01-01

    Full Text Available Adenylyl cyclase (AC-cyclic adenosine monophosphate (cAMP-cAMP-dependent protein kinase A (PKA cascade is considered to be associated with the pathogenesis and treatment of depression. The present study was conducted to explore the role of the cAMP cascade in antidepressant action of electroacupuncture (EA treatment for chronic mild stress (CMS-induced depression model rats. The results showed that EA improved significantly behavior symptoms in depression and dysfunction of AC-cAMP-PKA signal transduction pathway induced by CMS, which was as effective as fluoxetine. Moreover, the antidepressant effects of EA rather than Fluoxetine were completely abolished by H89, a specific PKA inhibitor. Consequently, EA has a significant antidepressant treatment in CMS-induced depression model rats, and AC-cAMP-PKA signal transduction pathway is crucial for it.

  11. Computational chemical analysis of unconjugated bilirubin anions and insights into pKa values clarification

    Science.gov (United States)

    Vega-Hissi, Esteban G.; Estrada, Mario R.; Lavecchia, Martín J.; Pis Diez, Reinaldo

    2013-01-01

    The pKa, the negative logarithm of the acid dissociation equilibrium constant, of the carboxylic acid groups of unconjugated bilirubin in water is a discussed issue because there are quite different experimental values reported. Using quantum mechanical calculations we have studied the conformational behavior of unconjugated bilirubin species (in gas phase and in solution modeled implicitly and explicitly) to provide evidence that may clarify pKa values because of its pathophysiological relevance. Our results show that rotation of carboxylate group, which is not restricted, settles it in a suitable place to establish stronger interactions that stabilizes the monoanion and the dianion to be properly solvated, demonstrating that the rationalization used to justify the high pKa values of unconjugated bilirubin is inappropriate. Furthermore, low unconjugated bilirubin (UCB) pKa values were estimated from a linear regression analysis.

  12. Prediction Of pKa From Chemical Structure Using Free And Open-Source Tools

    Science.gov (United States)

    The ionization state of a chemical, reflected in pKa values, affects lipophilicity, solubility, protein binding and the ability of a chemical to cross the plasma membrane. These properties govern the pharmacokinetic parameters such as absorption, distribution, metabolism, excreti...

  13. Determination of the pKa of Benzophenones in Ethanol-Water

    Directory of Open Access Journals (Sweden)

    G. T. Castro

    2000-03-01

    Full Text Available The pKa of monohydroxylated benzophenones was determined by UV spectroscopy. The values obtained are coherent with the resonant forms and hydrogen bond intramolecular of the analyzed compounds.

  14. The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins.

    Science.gov (United States)

    Nielsen, Jens E; Gunner, M R; García-Moreno, Bertrand E

    2011-12-01

    The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise. Copyright © 2011 Wiley-Liss, Inc.

  15. Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

    Science.gov (United States)

    Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2014-12-01

    The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Tolerance in Drosophila

    OpenAIRE

    Atkinson, Nigel S.

    2009-01-01

    The set of genes that underlie ethanol tolerance (inducible resistance) are likely to overlap with the set of genes responsible for ethanol addiction. Whereas addiction is difficult to recognize in simple model systems, behavioral tolerance is readily identifiable and can be induced in large populations of animals. Thus, tolerance lends itself to analysis in model systems with powerful genetics. Drosophila melanogaster has been used by a variety of laboratories for the identification of genes...

  17. Calibration of PKA meters against ion chambers of two geometries

    International Nuclear Information System (INIS)

    Almeida Junior, Jose N.; Terini, Ricardo A.; Pereira, Marco A.G.; Herdade, Silvio B.

    2011-01-01

    Kerma-area product (KAP or PKA) is a quantity that is independent of the distance to the X-ray tube focal spot and that can be used in radiological exams to assess the effective dose in patients. Clinical KAP meters are generally fixed in tube output and they are usually calibrated on-site by measuring the air kerma with an ion chamber and by evaluating the irradiated area by means of a radiographic image. Recently, a device was marketed (PDC, Patient Dose Calibrator, Radcal Co.), which was designed for calibrating clinical KAP meters with traceability to a standard laboratory. This paper presents a metrological evaluation of two methods that can be used in standard laboratories for the calibration of this device, namely, against a reference 30 cc ionization chamber or a reference parallel plates monitor chamber. Lower energy dependence was also obtained when the PDC calibration was made with the monitor chamber. Results are also shown of applying the PDC in hospital environment to the cross calibration of a clinical KAP meter from a radiology equipment. Results confirm lower energy dependence of the PDC relatively to the tested clinical meter. (author)

  18. Tet protein function during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available The TET (Ten-eleven translocation 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.

  19. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    International Nuclear Information System (INIS)

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-01-01

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

  20. Dependence of pKa on solute cavity for diprotic and triprotic acids.

    Science.gov (United States)

    Lee, Tae Bum; McKee, Michael L

    2011-06-07

    A systematic study of ΔG(aq)/pK(a) for monoprotic, diprotic, and triprotic acids has been carried out based on DFT/aug-cc-pVTZ combined with CPCM and SMD solvation modeling. All DFT/cavity set combinations considered showed similar accuracy for ΔG(aq)(1)/pK(a1) (70% within ±2.5 kcal mol(-1) of experiment) while only the M05-2X/Pauling cavity combination gave reasonable results for ΔG(aq)(2)/pK(a2) when both pK(a) values are separated by more than three units (70% within ±5.0 kcal mol(-1) of experiment). The choice of experimental data is critical to the interpretation of the calculated accuracy especially for several inorganic acids. For the calculation of ΔG(aq)(3)/pK(a3), the larger experimental uncertainty and an unrealistic orbital population of diffuse function for trianions in the gas phase hinders an evaluation of the predictive performance. We find the M05-2X functional with the Pauling cavity set is the best choice for ΔG(aq)(2)/pK(a2) prediction in aqueous media while all DFT/cavity sets considered were competitive for ΔG(aq)(1)/pK(a1).

  1. PKA-induced internalization of slack KNa channels produces dorsal root ganglion neuron hyperexcitability.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2010-10-20

    Inflammatory mediators through the activation of the protein kinase A (PKA) pathway sensitize primary afferent nociceptors to mechanical, thermal, and osmotic stimuli. However, it is unclear which ion conductances are responsible for PKA-induced nociceptor hyperexcitability. We have previously shown the abundant expression of Slack sodium-activated potassium (K(Na)) channels in nociceptive dorsal root ganglion (DRG) neurons. Here we show using cultured DRG neurons, that of the total potassium current, I(K), the K(Na) current is predominantly inhibited by PKA. We demonstrate that PKA modulation of K(Na) channels does not happen at the level of channel gating but arises from the internal trafficking of Slack channels from DRG membranes. Furthermore, we found that knocking down the Slack subunit by RNA interference causes a loss of firing accommodation analogous to that observed during PKA activation. Our data suggest that the change in nociceptive firing occurring during inflammation is the result of PKA-induced Slack channel trafficking.

  2. MD simulations to evaluate effects of applied tensile strain on irradiation-induced defect production at various PKA energies

    International Nuclear Information System (INIS)

    Miyashiro, S.; Fujita, S.; Okita, T.; Okuda, H.

    2012-01-01

    Highlights: ► Strain effects on defect formation were evaluated at various PKA energies by MD. ► Radiation-induced defects were increased numerically by external strain. ► Enhanced formation of larger clusters causes the numerical increase of defects. ► Strain influence on the number of defects was greatest at about 20 keV PKA. ► Cluster size, which is mostly affected by strain, was greater with higher PKA energy. - Abstract: Molecular Dynamics (MD) simulations were conducted to investigate the influence of applied tensile strain on defect production during cascade damages at various Primary Knock-on Atom (PKA) energies of 1–30 keV. When 1% strain was applied, the number of surviving defects increased at PKA energies higher than 5 keV, although they did not increase at 1 keV. The rate of increase by strain application was higher with higher PKA energy, and attained the maximum at 20 keV PKA energy with a subsequent gradual decrease at 30 keV PKA energy The cluster size, mostly affected by strain, was larger with higher PKA energy, although clusters with fewer than seven interstitials did not increase in number at any PKA energy.

  3. PKA tightly bound to human placental mitochondria participates in steroidogenesis and is not modified by cAMP.

    Science.gov (United States)

    Gomez-Chang, E; Espinosa-Garcia, M T; Olvera-Sanchez, S; Flores-Herrera, O; Martinez, F

    2014-09-01

    Protein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work. Placental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of (32)P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography. The PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas βII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme. The lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated. The phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila.

    Science.gov (United States)

    Choudhury, Saumitra Dey; Mushtaq, Zeeshan; Reddy-Alla, Suneel; Balakrishnan, Sruthi S; Thakur, Rajan S; Krishnan, Kozhalmannom S; Raghu, Padinjat; Ramaswami, Mani; Kumar, Vimlesh

    2016-05-01

    The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling. Copyright © 2016 by the Genetics Society of America.

  5. Behavioral Teratogenesis in Drosophila melanogaster.

    Science.gov (United States)

    Mishra, Monalisa; Barik, Bedanta Kumar

    2018-01-01

    Developmental biology is a fascinating branch of science which helps us to understand the mechanism of development, thus the findings are used in various therapeutic approach. Drosophila melanogaster served as a model to find the key molecules that initiate and regulate the mechanism of development. Various genes, transcription factors, and signaling pathways helping in development are identified in Drosophila. Many toxic compounds, which can affect the development, are also recognized using Drosophila model. These compounds, which can affect the development, are named as a teratogen. Many teratogens identified using Drosophila may also act as a teratogen for a human being since 75% of conservation exist between the disease genes present in Drosophila and human. There are certain teratogens, which do not cause developmental defect if exposed during pregnancy, however; behavioral defect appears in later part of development. Such compounds are named as a behavioral teratogen. Thus, it is worthy to identify the potential behavioral teratogen using Drosophila model. Drosophila behavior is well studied in various developmental stages. This chapter describes various methods which can be employed to test behavioral teratogenesis in Drosophila.

  6. The Drosophila melanogaster host model

    Science.gov (United States)

    Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

  7. The Drosophila melanogaster host model

    Directory of Open Access Journals (Sweden)

    Christina O. Igboin

    2012-02-01

    Full Text Available The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

  8. The Drosophila melanogaster host model.

    Science.gov (United States)

    Igboin, Christina O; Griffen, Ann L; Leys, Eugene J

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen-host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial-host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis-host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

  9. Calcium and Egg Activation in Drosophila

    Science.gov (United States)

    Sartain, Caroline V.; Wolfner, Mariana F.

    2012-01-01

    Summary In many animals, a rise in intracellular calcium levels is the trigger for egg activation, the process by which an arrested mature oocyte transitions to prepare for embryogenesis. In nearly all animals studied to date, this calcium rise, and thus egg activation, is triggered by the fertilizing sperm. However in the insects that have been examined, fertilization is not necessary to activate their oocytes. Rather, these insects’ eggs activate as they transit through the female’s reproductive tract, regardless of male contribution. Recent studies in Drosophila have shown that egg activation nevertheless requires calcium and that the downstream events and molecules of egg activation are also conserved, despite the difference in initial trigger. Genetic studies have uncovered essential roles for the calcium-dependent enzyme calcineurin and its regulator calcipressin, and have hinted at roles for calmodulin, in Drosophila egg activation. Physiological and in vitro studies have led to a model in which mechanical forces that impact the Drosophila oocyte as it moves through the reproductive tract triggers the influx of calcium from the external environment, thereby initiating egg activation. Future research will aim to test this model, as well as to determine the spatiotemporal dynamics of cytoplasmic calcium flux and mode of signal propagation in this unique system. PMID:23218670

  10. An automated paradigm for Drosophila visual psychophysics.

    Directory of Open Access Journals (Sweden)

    Oliver Evans

    Full Text Available BACKGROUND: Mutations that cause learning and memory defects in Drosophila melanogaster have been found to also compromise visual responsiveness and attention. A better understanding of attention-like defects in such Drosophila mutants therefore requires a more detailed characterization of visual responsiveness across a range of visual parameters. METHODOLOGY/PRINCIPAL FINDINGS: We designed an automated behavioral paradigm for efficiently dissecting visual responsiveness in Drosophila. Populations of flies walk through multiplexed serial choice mazes while being exposed to moving visuals displayed on computer monitors, and infra-red fly counters at the end of each maze automatically score the responsiveness of a strain. To test our new design, we performed a detailed comparison between wild-type flies and a learning and memory mutant, dunce(1. We first confirmed that the learning mutant dunce(1 displays increased responsiveness to a black/green moving grating compared to wild type in this new design. We then extended this result to explore responses to a wide range of psychophysical parameters for moving gratings (e.g., luminosity, contrast, spatial frequency, velocity as well as to a different stimulus, moving dots. Finally, we combined these visuals (gratings versus dots in competition to investigate how dunce(1 and wild-type flies respond to more complex and conflicting motion effects. CONCLUSIONS/SIGNIFICANCE: We found that dunce(1 responds more strongly than wild type to high contrast and highly structured motion. This effect was found for simple gratings, dots, and combinations of both stimuli presented in competition.

  11. PKA RIα/A-kinase anchoring proteins 10 signaling pathway and the prognosis of colorectal cancer.

    Science.gov (United States)

    Wang, Mojin; Li, Yuan; Wang, Rui; Wang, Ziqiang; Chen, Keling; Zhou, Bin; Zhou, Zongguang; Sun, Xiaofeng

    2015-03-01

    Previously study showed that the loss of the control of cAMP-dependent protein kinase A RIα (PKA RIα)/ A-kinase anchoring proteins 10 (AKAP10) signaling pathway initiate dysregulation of cellular healthy physiology leading to tumorigenesis. The aim of this study was to investigate the role of PKA RIα/AKAP10 signaling pathway in colorectal cancer (CRC). The AKAP10 expression at the mRNA and protein level have been analyzed in colon cancer cell lines, primary CRCs and matched normal mucosa samples, and compared in accordance with specific clinicopathological features of CRC. The correlation between expression of AKAP10 and PKA RIα were also analyzed. Compared with HCT116 and SW480 cells, the AKAP10 was significantly upregulated in the colon cell line KM12C and its metastatic counterparts, KM12SM and KM12L4A. Moreover, the KM12SM and KM12L4A having high metastatic potentials displayed the elevated levels of AKAP10 compared with KM12C having poor metastatic potential. A notably higher level of AKAP10 expression was found in CRC tissues at both mRNA and protein levels. Increased expression of AKAP10 in CRC patients was positively associated with the depth of invasion and the grade of differentiation. Univariate survival analysis showed that the increased expression of AKAP10 was related to poorer survival. Cox multivariate regression analysis confirmed that AKAP10 was an independent predictor of the overall survival of CRC patients. PKA RIα mRNA was also expressed at high levels in CRC. The correlation coefficient between mRNA expression of AKAP10 and PKA RIα in CRC was 0.417. AKAP10 mRNA overexpression was correlated significantly with PKA RIα. Our data indicated that PKA RIα/AKAP10 signaling pathway is associated with the progression and prognosis of CRC. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  12. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Cross-talk between PKA-Cβ and p65 mediates synergistic induction of PDE4B by roflumilast and NTHi

    Science.gov (United States)

    Susuki-Miyata, Seiko; Miyata, Masanori; Lee, Byung-Cheol; Xu, Haidong; Kai, Hirofumi; Yan, Chen; Li, Jian-Dong

    2015-01-01

    Phosphodiesterase 4B (PDE4B) plays a key role in regulating inflammation. Roflumilast, a phosphodiesterase (PDE)4-selective inhibitor, has recently been approved for treating severe chronic obstructive pulmonary disease (COPD) patients with exacerbation. However, there is also clinical evidence suggesting the development of tachyphylaxis or tolerance on repeated dosing of roflumilast and the possible contribution of PDE4B up-regulation, which could be counterproductive for suppressing inflammation. Thus, understanding how PDE4B is up-regulated in the context of the complex pathogenesis and medications of COPD may help improve the efficacy and possibly ameliorate the tolerance of roflumilast. Here we show that roflumilast synergizes with nontypeable Haemophilus influenzae (NTHi), a major bacterial cause of COPD exacerbation, to up-regulate PDE4B2 expression in human airway epithelial cells in vitro and in vivo. Up-regulated PDE4B2 contributes to the induction of certain important chemokines in both enzymatic activity-dependent and activity-independent manners. We also found that protein kinase A catalytic subunit β (PKA-Cβ) and nuclear factor-κB (NF-κB) p65 subunit were required for the synergistic induction of PDE4B2. PKA-Cβ phosphorylates p65 in a cAMP-dependent manner. Moreover, Ser276 of p65 is critical for mediating the PKA-Cβ–induced p65 phosphorylation and the synergistic induction of PDE4B2. Collectively, our data unveil a previously unidentified mechanism underlying synergistic up-regulation of PDE4B2 via a cross-talk between PKA-Cβ and p65 and may help develop new therapeutic strategies to improve the efficacy of PDE4 inhibitor. PMID:25831493

  14. Comparative study of Monte Carlo particle transport code PHITS and nuclear data processing code NJOY for PKA energy spectra and heating number under neutron irradiation

    International Nuclear Information System (INIS)

    Iwamoto, Y.; Ogawa, T.

    2016-01-01

    The modelling of the damage in materials irradiated by neutrons is needed for understanding the mechanism of radiation damage in fission and fusion reactor facilities. The molecular dynamics simulations of damage cascades with full atomic interactions require information about the energy distribution of the Primary Knock on Atoms (PKAs). The most common process to calculate PKA energy spectra under low-energy neutron irradiation is to use the nuclear data processing code NJOY2012. It calculates group-to-group recoil cross section matrices using nuclear data libraries in ENDF data format, which is energy and angular recoil distributions for many reactions. After the NJOY2012 process, SPKA6C is employed to produce PKA energy spectra combining recoil cross section matrices with an incident neutron energy spectrum. However, intercomparison with different processes and nuclear data libraries has not been studied yet. Especially, the higher energy (~5 MeV) of the incident neutrons, compared to fission, leads to many reaction channels, which produces a complex distribution of PKAs in energy and type. Recently, we have developed the event generator mode (EGM) in the Particle and Heavy Ion Transport code System PHITS for neutron incident reactions in the energy region below 20 MeV. The main feature of EGM is to produce PKA with keeping energy and momentum conservation in a reaction. It is used for event-by-event analysis in application fields such as soft error analysis in semiconductors, micro dosimetry in human body, and estimation of Displacement per Atoms (DPA) value in metals and so on. The purpose of this work is to specify differences of PKA spectra and heating number related with kerma between different calculation method using PHITS-EGM and NJOY2012+SPKA6C with different libraries TENDL-2015, ENDF/B-VII.1 and JENDL-4.0 for fusion relevant materials

  15. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    Science.gov (United States)

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Drosophila melanogaster as a Versatile Model Organism in Food and Nutrition Research.

    Science.gov (United States)

    Staats, Stefanie; Lüersen, Kai; Wagner, Anika E; Rimbach, Gerald

    2018-04-18

    Drosophila melanogaster has been widely used in the biological sciences as a model organism. Drosophila has a relatively short life span of 60-80 days, which makes it attractive for life span studies. Moreover, approximately 60% of the fruit fly genes are orthologs to mammals. Thus, metabolic and signal transduction pathways are highly conserved. Maintenance and reproduction of Drosophila do not require sophisticated equipment and are rather cheap. Furthermore, there are fewer ethical issues involved in experimental Drosophila research compared with studies in laboratory rodents, such as rats and mice. Drosophila is increasingly recognized as a model organism in food and nutrition research. Drosophila is often fed complex solid diets based on yeast, corn, and agar. There are also so-called holidic diets available that are defined in terms of their amino acid, fatty acid, carbohydrate, vitamin, mineral, and trace element compositions. Feed intake, body composition, locomotor activity, intestinal barrier function, microbiota, cognition, fertility, aging, and life span can be systematically determined in Drosophila in response to dietary factors. Furthermore, diet-induced pathophysiological mechanisms including inflammation and stress responses may be evaluated in the fly under defined experimental conditions. Here, we critically evaluate Drosophila melanogaster as a versatile model organism in experimental food and nutrition research, review the corresponding data in the literature, and make suggestions for future directions of research.

  17. Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

  18. [Effect of PKA Gene on Acute Lymphoblastic Leukemia in Children and Its Mechanism].

    Science.gov (United States)

    Wang, Chao-Jie; Wang, Li-Juan; Zhao, Ding

    2018-02-01

    To explore the effect of PKA gene on acute T lymphocyte leukemia cells in children and its mechanism. Jurkat and Sup-T1 cells were divided into 2 group: control group (Jurkat and Sup-T1 cells treated with non-specific siRNA) and transfected group (Jurkat and Sup-T1 cells transfected with PKA siRNA). The effects of down-regulating the expression of PKA gene on the viability, proliferotion, migration and cell cycle distribution of Jurkat and Sup-T1 cells in 2 groups were analyzed by CCK-8 assay, transwell experiment, cell colony-formation test and flow cytometry; the cyclin-related protein levels after transfection with PKA siRNA were detected by Western blot. It was revealed that the expression of PKA in Jurkat and Sup-T1 cells decreased to different degree after siRNA transfection(PPKA gene expression can decrease the proliferation and migration of tumor cells, and also can restrict the cell proliferation through related cell cycle proteins.

  19. Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal.

    Science.gov (United States)

    Kim, Hyungsup; Kim, Hyesu; Lee, Jesun; Lee, Byeongjun; Kim, Hee-Ryang; Jung, Jooyoung; Lee, Mi-Ock; Oh, Uhtaek

    2018-05-01

    Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca 2+ . Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function. Copyright © 2017. Published by Elsevier Ltd.

  20. PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells.

    Science.gov (United States)

    Gozal, Evelyne; Metz, Cynthia J; Dematteis, Maurice; Sachleben, Leroy R; Schurr, Avital; Rane, Madhavi J

    2017-09-05

    Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Hedgehog-PKA signaling and gnrh3 regulate the development of zebrafish gnrh3 neurons.

    Directory of Open Access Journals (Sweden)

    Ming-Wei Kuo

    Full Text Available GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA, by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA* and dominant negative regulatory subunit (PKI into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.

  2. PTPIP51: A New Interaction Partner of the Insulin Receptor and PKA in Adipose Tissue

    Directory of Open Access Journals (Sweden)

    M. A. Bobrich

    2013-01-01

    Full Text Available Aims. Our previous experiments revealed an association of PTPIP51 (protein tyrosine phosphatase interacting protein 51 with the insulin signalling pathway through PTP1B and 14-3-3beta. We aimed to clarify the role of PTPIP51 in adipocyte metabolism. Methods. Four groups of ten C57Bl/6 mice each were used. Two groups were fed a standard diet; two groups were fed a high-fat diet. Two groups (one high-fat diet and one standard diet were submitted to endurance training, while the remaining two groups served as untrained control groups. After ten weeks, we measured glucose tolerance of the mice. Adipose tissue samples were analyzed by immunofluorescence and Duolink proximity ligation assay to quantify interactions of PTPIP51 with either insulin receptor (IR or PKA. Results. PTPIP51 and the IR and PTPIP51 and PKA, respectively, were colocalized in all groups. Standard diet animals that were submitted to endurance training showed low PTPIP51-IR and PTPIP51-PKA interactions. The interaction levels of both the IR and PKA differed between the feeding and training groups. Conclusion. PTPIP51 might serve as a linking protein in adipocyte metabolism by connecting the IR-triggered lipogenesis with the PKA-dependent lipolysis. PTPIP51 interacts with both proteins, therefore being a potential gateway for the cooperation of both pathways.

  3. big bang gene modulates gut immune tolerance in Drosophila.

    Science.gov (United States)

    Bonnay, François; Cohen-Berros, Eva; Hoffmann, Martine; Kim, Sabrina Y; Boulianne, Gabrielle L; Hoffmann, Jules A; Matt, Nicolas; Reichhart, Jean-Marc

    2013-02-19

    Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

  4. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  5. Myoblast fusion in Drosophila

    International Nuclear Information System (INIS)

    Haralalka, Shruti; Abmayr, Susan M.

    2010-01-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  6. SUMOylation in Drosophila Development

    Directory of Open Access Journals (Sweden)

    Albert J. Courey

    2012-07-01

    Full Text Available Small ubiquitin-related modifier (SUMO, an ~90 amino acid ubiquitin-like protein, is highly conserved throughout the eukaryotic domain. Like ubiquitin, SUMO is covalently attached to lysine side chains in a large number of target proteins. In contrast to ubiquitin, SUMO does not have a direct role in targeting proteins for proteasomal degradation. However, like ubiquitin, SUMO does modulate protein function in a variety of other ways. This includes effects on protein conformation, subcellular localization, and protein–protein interactions. Significant insight into the in vivo role of SUMOylation has been provided by studies in Drosophila that combine genetic manipulation, proteomic, and biochemical analysis. Such studies have revealed that the SUMO conjugation pathway regulates a wide variety of critical cellular and developmental processes, including chromatin/chromosome function, eggshell patterning, embryonic pattern formation, metamorphosis, larval and pupal development, neurogenesis, development of the innate immune system, and apoptosis. This review discusses our current understanding of the diverse roles for SUMO in Drosophila development.

  7. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Science.gov (United States)

    Mulakkal, Nitha C.; Nagy, Peter; Takats, Szabolcs; Tusco, Radu; Juhász, Gábor; Nezis, Ioannis P.

    2014-01-01

    The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy. PMID:24949430

  8. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Directory of Open Access Journals (Sweden)

    Nitha C. Mulakkal

    2014-01-01

    Full Text Available The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy.

  9. Neural correlates of water reward in thirsty Drosophila

    OpenAIRE

    Lin, Suewei; Owald, David; Chandra, Vikram; Talbot, Clifford; Huetteroth, Wolf; Waddell, Scott

    2014-01-01

    Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and

  10. Development of Methods for the Determination of pKa Values

    Science.gov (United States)

    Reijenga, Jetse; van Hoof, Arno; van Loon, Antonie; Teunissen, Bram

    2013-01-01

    The acid dissociation constant (pKa) is among the most frequently used physicochemical parameters, and its determination is of interest to a wide range of research fields. We present a brief introduction on the conceptual development of pKa as a physical parameter and its relationship to the concept of the pH of a solution. This is followed by a general summary of the historical development and current state of the techniques of pKa determination and an attempt to develop insight into future developments. Fourteen methods of determining the acid dissociation constant are placed in context and are critically evaluated to make a fair comparison and to determine their applications in modern chemistry. Additionally, we have studied these techniques in light of present trends in science and technology and attempt to determine how these trends might affect future developments in the field. PMID:23997574

  11. pK(a) Values of Titrable Amino Acids at the Water/Membrane Interface.

    Science.gov (United States)

    Teixeira, Vitor H; Vila-Viçosa, Diogo; Reis, Pedro B P S; Machuqueiro, Miguel

    2016-03-08

    Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the pK(a) shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pK(a) values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pK(a) values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations.

  12. Evolutionary genetics: the Drosophila model

    Indian Academy of Sciences (India)

    Unknown

    Evolutionary genetics straddles the two fundamental processes of life, ... of the genus Drosophila have been used extensively as model systems in experimental ... issue will prove interesting, informative and thought-provoking for both estab-.

  13. SF-1 (NR5A1) expression is stimulated by the PKA pathway and is essential for the PKA-induced activation of LIPE expression in Y-1 cells.

    Science.gov (United States)

    Kulcenty, K; Holysz, M; Trzeciak, W H

    2015-10-01

    In the adrenal cortex, corticotropin induces the expression of several genes encoding proteins involved in the synthesis and intracellular transport of steroid hormones via the protein kinase A (PKA) signalling pathway, and this process is mediated by steroidogenic factor-1 (SF-1). This study was designed to elucidate the influence of the PKA and PKC pathways on the expression of the SF-1 gene in mouse adrenocortical cells, line Y-1. It has also been attempted to answer the question whether or not SF-1 plays a role in the PKA-induced expression of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase, which supplies cholesterol for steroid hormone synthesis. In this study, we found that stimulation of the PKA pathway caused a significant increase in SF-1 expression, and that this effect was abolished by the PKA inhibitor, H89. Decreased SF-1 gene transcript levels were seen with the simultaneous activation of PKA and PKC, suggesting a possible interaction between the PKA and PKC pathways. It was also observed that SF-1 increased the transcriptional activity of the LIPE gene by interacting with the SF-1 response element located in promoter A. Moreover, transient silencing of SF-1 expression with specific siRNAs abolished PKA-stimulated transcription of the LIPE gene, indicating that SF-1 is an important regulator of LIPE expression in Y-1 cells and thus could play a role in the regulation of the cholesterol supply for adrenal steroidogenesis.

  14. Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

    Science.gov (United States)

    Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

    2015-11-13

    Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. pKa values in proteins determined by electrostatics applied to molecular dynamics trajectories.

    Science.gov (United States)

    Meyer, Tim; Knapp, Ernst-Walter

    2015-06-09

    For a benchmark set of 194 measured pKa values in 13 proteins, electrostatic energy computations are performed in which pKa values are computed by solving the Poisson-Boltzmann equation. In contrast to the previous approach of Karlsberg(+) (KB(+)) that essentially used protein crystal structures with variations in their side chain conformations, the present approach (KB2(+)MD) uses protein conformations from four molecular dynamics (MD) simulations of 10 ns each. These MD simulations are performed with different specific but fixed protonation patterns, selected to sample the conformational space for the different protonation patterns faithfully. The root-mean-square deviation between computed and measured pKa values (pKa RMSD) is shown to be reduced from 1.17 pH units using KB(+) to 0.96 pH units using KB2(+)MD. The pKa RMSD can be further reduced to 0.79 pH units, if each conformation is energy-minimized with a dielectric constant of εmin = 4 prior to calculating the electrostatic energy. The electrostatic energy expressions upon which the computations are based have been reformulated such that they do not involve terms that mix protein and solvent environment contributions and no thermodynamic cycle is needed. As a consequence, conformations of the titratable residues can be treated independently in the protein and solvent environments. In addition, the energy terms used here avoid the so-called intrinsic pKa and can therefore be interpreted without reference to arbitrary protonation states and conformations.

  16. Differential Roles of the Fan-Shaped Body and the Ellipsoid Body in "Drosophila" Visual Pattern Memory

    Science.gov (United States)

    Pan, Yufeng; Zhou, Yanqiong; Guo, Chao; Gong, Haiyun; Gong, Zhefeng; Liu, Li

    2009-01-01

    The central complex is a prominent structure in the "Drosophila" brain. Visual learning experiments in the flight simulator, with flies with genetically altered brains, revealed that two groups of horizontal neurons in one of its substructures, the fan-shaped body, were required for "Drosophila" visual pattern memory. However,…

  17. Origin of the pKa shift of the catalytic lysine in acetoacetate decarboxylase.

    OpenAIRE

    Ishikita, Hiroshi

    2010-01-01

    The pKa value of Lys115, the catalytic residue in acetoacetate decarboxylate, was calculated using atomic coordinates of the X-ray crystal structure with consideration of the protonation states of all titratable sites in the protein. The calculated pKa value of Lys115 (pKa(Lys115)) was unusually low (approximately 6) in agreement with the experimentally measured value. Although charged residues impact pKa(Lys115) considerably in the native protein, the significant pKa(Lys115) downshift in the...

  18. The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

    Science.gov (United States)

    Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

    2014-01-01

    The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells. PMID:25485503

  19. Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

    Science.gov (United States)

    Selvaraj, Poonguzhali; Tham, Hong Fai; Ramanujam, Ravikrishna; Naqvi, Naweed I

    2017-08-01

    The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast. © 2017 John Wiley & Sons Ltd.

  20. The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation.

    Science.gov (United States)

    Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

    2014-01-01

    The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

  1. cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

    Science.gov (United States)

    Nedvetsky, Pavel I; Zhao, Xiaocheng; Mathivet, Thomas; Aspalter, Irene M; Stanchi, Fabio; Metzger, Ross J; Mostov, Keith E; Gerhardt, Holger

    2016-10-01

    cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior. © 2016. Published by The Company of Biologists Ltd.

  2. A Drosophila gene encoding a protein resembling the human β-amyloid protein precursor

    International Nuclear Information System (INIS)

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K.

    1989-01-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human β-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human β-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development

  3. Hermann Muller and Mutations in Drosophila

    Science.gov (United States)

    dropdown arrow Site Map A-Z Index Menu Synopsis Hermann Muller and Mutations in Drosophila Resources with University of Texas. In Austin his experiments on fruit flies (Drosophila) first showed that exposure to September to spend a year at the only Drosophila laboratory in Europe which was doing parallel work

  4. Use of Drosophila to study DNA repair

    International Nuclear Information System (INIS)

    Boyd, J.B.; Harris, P.V.; Sakaguchi, K.

    1988-01-01

    This paper discusses Drosophila, the premier metazoan organism for analyzing many fundamental features of eukaryotic gene regulation. The authors present adaptations of several approaches for studying DNA repair to an analysis of repair-defective mutants in Drosophila. A current understanding of Drosophila DNA repair is described

  5. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

    Science.gov (United States)

    Marroquin-Guzman, Margarita; Wilson, Richard A

    2015-04-01

    Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  6. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

    Directory of Open Access Journals (Sweden)

    Margarita Marroquin-Guzman

    2015-04-01

    Full Text Available Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  7. Evolutionary Paths of the cAMP-Dependent Protein Kinase (PKA) Catalytic Subunits

    Science.gov (United States)

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S.; Laerdahl, Jon K.

    2013-01-01

    3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  8. Structure of smAKAP and its regulation by PKA-mediated phosphorylation

    Science.gov (United States)

    Burgers, Pepijn P.; Bruystens, Jessica; Burnley, Rebecca J.; Nikolaev, Viacheslav O.; Keshwani, Malik; Wu, Jian; Janssen, Bert J. C.; Taylor, Susan S.; Heck, Albert J. R.; Scholten, Arjen

    2016-01-01

    The A-kinase anchoring protein (AKAP) smAKAP has three extraordinary features; it is very small, it is anchored directly to membranes by acyl motifs, and it interacts almost exclusively with the type I regulatory subunits (RI) of cAMP-dependent kinase (PKA). Here, we determined the crystal structure of smAKAP’s A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking (D/D) domain of RIα which reveals an extended hydrophobic interface with unique interaction pockets that drive smAKAP’s high specificity for RI subunits. We also identify a conserved PKA phosphorylation site at Ser66 in the AKB domain which we predict would cause steric clashes and disrupt binding. This correlates with in vivo colocalization and fluorescence polarization studies, where Ser66 AKB phosphorylation ablates RI binding. Hydrogen/deuterium exchange studies confirm that the AKB helix is accessible and dynamic. Furthermore, full-length smAKAP as well as the unbound AKB is predicted to contain a break at the phosphorylation site, and circular dichroism measurements confirm that the AKB domain loses its helicity following phosphorylation. As the active site of PKA’s catalytic subunit does not accommodate α-helices, we predict that the inherent flexibility of the AKB domain enables its phosphorylation by PKA. This represents a novel mechanism, whereby activation of anchored PKA can terminate its binding to smAKAP affecting the regulation of localized cAMP signaling events. PMID:27028580

  9. Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy

    NARCIS (Netherlands)

    Sarma, Satyam; Li, Na; van Oort, Ralph J.; Reynolds, Corey; Skapura, Darlene G.; Wehrens, Xander H. T.

    2010-01-01

    Aberrant intracellular Ca(2+) regulation is believed to contribute to the development of cardiomyopathy in Duchenne muscular dystrophy. Here, we tested whether inhibition of protein kinase A (PKA) phosphorylation of ryanodine receptor type 2 (RyR2) prevents dystrophic cardiomyopathy by reducing SR

  10. pKa Determination of water-soluble calix[4]arenes

    NARCIS (Netherlands)

    Shinkai, Seiji; Araki, Koji; Grootenhuis, P.D.J.; Reinhoudt, David

    1991-01-01

    Neutral, water-soluble 5,11,17,23-tetrakis[bis-(2-hydroxyethyl)aminosulphonyl]calix[4]arene-25,26,27,28-tetraol and 5,11,17,23-tetranitrocalix[4]arene-25,26,27,28-tetraol have been synthesized and the pKa values of the OH groups determined in an aqueous system.

  11. Status and evaluation methods of JENDL fusion file and JENDL PKA/KERMA file

    International Nuclear Information System (INIS)

    Chiba, S.; Fukahori, T.; Shibata, K.; Yu Baosheng; Kosako, K.

    1997-01-01

    The status of evaluated nuclear data in the JENDL fusion file and PKA/KERMA file is presented. The JENDL fusion file was prepared in order to improve the quality of the JENDL-3.1 data especially on the double-differential cross sections (DDXs) of secondary neutrons and gamma-ray production cross sections, and to provide DDXs of secondary charged particles (p, d, t, 3 He and α-particle) for the calculation of PKA and KERMA factors. The JENDL fusion file contains evaluated data of 26 elements ranging from Li to Bi. The data in JENDL fusion file reproduce the measured data on neutron and charged-particle DDXs and also on gamma-ray production cross sections. Recoil spectra in PKA/KERMA file were calculated from secondary neutron and charged-particle DDXs contained in the fusion file with two-body reaction kinematics. The data in the JENDL fusion file and PKA/KERMA file were compiled in ENDF-6 format with an MF=6 option to store the DDX data. (orig.)

  12. Succinate modulates Ca(2+) transient and cardiomyocyte viability through PKA-dependent pathway.

    Science.gov (United States)

    Aguiar, Carla J; Andrade, Vanessa L; Gomes, Enéas R M; Alves, Márcia N M; Ladeira, Marina S; Pinheiro, Ana Cristina N; Gomes, Dawidson A; Almeida, Alvair P; Goes, Alfredo M; Resende, Rodrigo R; Guatimosim, Silvia; Leite, M Fatima

    2010-01-01

    GPR91 is an orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate, a citric acid cycle intermediate, in several tissues. In the heart, the role of succinate is unknown. We now report that rat ventricular cardiomyocytes express GPR91. We found that succinate, through GPR91, increases the amplitude and the rate of decline of global Ca(2+) transient, by increasing the phosphorylation levels of ryanodine receptor and phospholamban, two well known Ca(2+) handling proteins. The effects of succinate on Ca(2+) transient were abolished by pre-treatment with adenylyl cyclase and cAMP-dependent protein kinase (PKA) inhibitors. Direct PKA activation by succinate was further confirmed using a FRET-based A-kinase activity reporter. Additionally, succinate decreases cardiomyocyte viability through a caspase-3 activation pathway, effect also prevented by PKA inhibition. Taken together, these observations show that succinate acts as a signaling molecule in cardiomyocytes, modulating global Ca(2+) transient and cell viability through a PKA-dependent pathway. 2009 Elsevier Ltd. All rights reserved.

  13. The significance of the pilot conditioning plant (PKA) for spent fuel management

    International Nuclear Information System (INIS)

    Willax, H.O.

    1996-01-01

    The pilot conditioning plant (PKA) is intended as a multi-purpose facility and thus may serve various purposes involved in the conditioning or disposal of spent fuel elements or radwaste. Its design as a pilot plant permits development and trial of various methods and processes for fuel element conditioning, as well as for radwaste conditioning. (orig./DG) [de

  14. Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

    Science.gov (United States)

    Castro, Liliana R V; Guiot, Elvire; Polito, Marina; Paupardin-Tritsch, Daniéle; Vincent, Pierre

    2014-02-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation.

    Science.gov (United States)

    Bruystens, Jessica Gh; Wu, Jian; Fortezzo, Audrey; Del Rio, Jason; Nielsen, Cole; Blumenthal, Donald K; Rock, Ruth; Stefan, Eduard; Taylor, Susan S

    2016-12-04

    Most disease-related mutations that impair cAMP protein kinase A (PKA) signaling are present within the regulatory (R) PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex (CNC) disease and, more recently, to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP-binding (CNB) domains (CNB-B), and here, we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the catalytic (C)-subunit reveals the dysfunctional regions of the RIα subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure, and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted, thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB domains is necessary for the functional regulation of PKA activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Sex differences in behavioral and PKA cascade responses to repeated cocaine administration.

    Science.gov (United States)

    Zhou, Luyi; Sun, Wei-Lun; Weierstall, Karen; Minerly, Ana Christina; Weiner, Jan; Jenab, Shirzad; Quinones-Jenab, Vanya

    2016-10-01

    Previous studies have shown sex different patterns in behavioral responses to cocaine. Here, we used between-subject experiment design to study whether sex differences exist in the development of behavioral sensitization and tolerance to repeated cocaine, as well as the role of protein kinase A (PKA) signaling cascade in this process. Ambulatory and rearing responses were recorded in male and female rats after 1 to 14 days of administration of saline or cocaine (15 mg/kg; ip). Correspondent PKA-associated signaling in the nucleus accumbens (NAc) and caudate-putamen (CPu) was measured at each time point. Our results showed that females exhibited higher cocaine-induced behavioral responses and developed behavioral sensitization and tolerance faster than males. Whereas females developed behavioral sensitization to cocaine after 2 days and tolerance after 14 days, male rats developed sensitization after 5 days. In addition, cocaine induced a sexual dimorphic pattern in the progression of neuronal adaptations on the PKA cascade signaling in region (NAc vs. CPu) and time (days of cocaine administration)-dependent manners. In general, more PKA signaling cascade changes were found in the NAc of males on day 5 and in the CPu of females with repeated cocaine injection. In addition, in females, behavioral activities positively correlated with FosB levels in the NAc and CPu and negatively correlated with Cdk5 and p35 in the CPu, while no correlation was observed in males. Our studies suggest that repeated cocaine administration induced different patterns of behavioral and molecular responses in the PKA cascade in male and female rats.

  17. Primary radiation damage characterization of α-iron under irradiation temperature for various PKA energies

    Science.gov (United States)

    Sahi, Qurat-ul-ain; Kim, Yong-Soo

    2018-04-01

    The understanding of radiation-induced microstructural defects in body-centered cubic (BCC) iron is of major interest to those using advanced steel under extreme conditions in nuclear reactors. In this study, molecular dynamics (MD) simulations were implemented to examine the primary radiation damage in BCC iron with displacement cascades of energy 1, 5, 10, 20, and 30 keV at temperatures ranging from 100 to 1000 K. Statistical analysis of eight MD simulations of collision cascades were carried out along each [110], [112], [111] and a high index [135] direction and the temperature dependence of the surviving number of point defects and the in-cascade clustering of vacancies and interstitials were studied. The peak time and the corresponding number of defects increase with increasing irradiation temperature and primary knock-on atom (PKA) energy. However, the final number of surviving point defects decreases with increasing lattice temperature. This is associated with the increase of thermal spike at high PKA energy and its long timespan at higher temperatures. Defect production efficiency (i.e., surviving MD defects, per Norgett-Robinson-Torrens displacements) also showed a continuous decrease with the increasing irradiation temperature and PKA energy. The number of interstitial clusters increases with both irradiation temperature and PKA energy. However, the increase in the number of vacancy clusters with PKA energy is minimal-to-constant and decreases as the irradiation temperature increases. Similarly, the probability and cluster size distribution for larger interstitials increase with temperature, whereas only smaller size vacancy clusters were observed at higher temperatures.

  18. PKA increases in the olfactory bulb act as unconditioned stimuli and provide evidence for parallel memory systems: pairing odor with increased PKA creates intermediate- and long-term, but not short-term, memories.

    Science.gov (United States)

    Grimes, Matthew T; Harley, Carolyn W; Darby-King, Andrea; McLean, John H

    2012-02-21

    Neonatal odor-preference memory in rat pups is a well-defined associative mammalian memory model dependent on cAMP. Previous work from this laboratory demonstrates three phases of neonatal odor-preference memory: short-term (translation-independent), intermediate-term (translation-dependent), and long-term (transcription- and translation-dependent). Here, we use neonatal odor-preference learning to explore the role of olfactory bulb PKA in these three phases of mammalian memory. PKA activity increased normally in learning animals 10 min after a single training trial. Inhibition of PKA by Rp-cAMPs blocked intermediate-term and long-term memory, with no effect on short-term memory. PKA inhibition also prevented learning-associated CREB phosphorylation, a transcription factor implicated in long-term memory. When long-term memory was rescued through increased β-adrenoceptor activation, CREB phosphorylation was restored. Intermediate-term and long-term, but not short-term odor-preference memories were generated by pairing odor with direct PKA activation using intrabulbar Sp-cAMPs, which bypasses β-adrenoceptor activation. Higher levels of Sp-cAMPs enhanced memory by extending normal 24-h retention to 48-72 h. These results suggest that increased bulbar PKA is necessary and sufficient for the induction of intermediate-term and long-term odor-preference memory, and suggest that PKA activation levels also modulate memory duration. However, short-term memory appears to use molecular mechanisms other than the PKA/CREB pathway. These mechanisms, which are also recruited by β-adrenoceptor activation, must operate in parallel with PKA activation.

  19. Identification and evaluation of attractiveness of lactic acid bacteria as a bait for Drosophila suzukii Matsumura

    OpenAIRE

    Maddalena, Giuseppe

    2016-01-01

    The spotted-wing drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), native of Eastern Asia, is an invasive alien species in Europe and the Americas and is one of the main emerging pests of valuable crops, including soft fruits and wine grapes. The conventional approach to handle infestations of SWD involves the use of commercially available insecticides, but these do not seem able to ensure effective results; consequently, alternative strategies are strongly required. Ma...

  20. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project.

    OpenAIRE

    Spradling, A C; Stern, D M; Kiss, I; Roote, J; Laverty, T; Rubin, G M

    1995-01-01

    Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and a...

  1. Phylogeny of the Genus Drosophila

    Science.gov (United States)

    O’Grady, Patrick M.; DeSalle, Rob

    2018-01-01

    Understanding phylogenetic relationships among taxa is key to designing and implementing comparative analyses. The genus Drosophila, which contains over 1600 species, is one of the most important model systems in the biological sciences. For over a century, one species in this group, Drosophila melanogaster, has been key to studies of animal development and genetics, genome organization and evolution, and human disease. As whole-genome sequencing becomes more cost-effective, there is increasing interest in other members of this morphologically, ecologically, and behaviorally diverse genus. Phylogenetic relationships within Drosophila are complicated, and the goal of this paper is to provide a review of the recent taxonomic changes and phylogenetic relationships in this genus to aid in further comparative studies. PMID:29716983

  2. Biases in Drosophila melanogaster protein trap screens

    Directory of Open Access Journals (Sweden)

    Müller Ilka

    2009-05-01

    utility of P-element vectors for protein trap screens has largely been exhausted, and that approximately 2,800 genes may still be amenable using piggyBac vectors. Thus protein trap strategies based on current approaches are unlikely to offer true genome-wide coverage. We suggest that either transposons with reduced insertion bias or recombineering-based targeting techniques will be required for comprehensive genome coverage in Drosophila.

  3. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-01-01

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  4. In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

    Science.gov (United States)

    Yamauchi, Fumio; Kamioka, Yuji; Yano, Tetsuya; Matsuda, Michiyuki

    2016-09-15

    Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Predicting the pKa and stability of organic acids and bases at an oil-water interface.

    Science.gov (United States)

    Andersson, M P; Olsson, M H M; Stipp, S L S

    2014-06-10

    We have used density functional theory and the implicit solvent model, COSMO-RS, to investigate how the acidity constant, pKa, of organic acids and bases adsorbed at the organic compound-aqueous solution interface changes, compared to its value in the aqueous phase. The pKa determine the surface charge density of the molecules that accumulate at the fluid-fluid interface. We have estimated the pKa by comparing the stability of the protonated and unprotonated forms of a series of molecules in the bulk aqueous solution and at an interface where parts of each molecule reside in the hydrophobic phase and the rest remains in the hydrophilic phase. We found that the pKa for acids is shifted by ∼1 pH unit to higher values compared to the bulk water pKa, whereas they are shifted to lower values by a similar amount for bases. Because this pKa shift is similar in magnitude for each of the molecules studied, we propose that the pKa for molecules at a water-organic compound interface can easily be predicted by adding a small shift to the aqueous pKa. This shift is general and correlates with the functional group. We also found that the relative composition of molecules at the fluid-fluid interface is not the same as in the bulk. For example, species such as carboxylic acids are enriched at the interface, where they can dominate surface properties, even when they are a modest component in the bulk fluid. For high surface concentrations of carboxylic acid groups at an interface, such as a self-assembled monolayer, we have demonstrated that the pKa depends on the degree of deprotonation through direct hydrogen bonding between protonated and deprotonated acidic headgroups.

  6. Modelling Cooperative Tumorigenesis in Drosophila

    Science.gov (United States)

    2018-01-01

    The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression. PMID:29693007

  7. Modelling Cooperative Tumorigenesis in Drosophila

    Directory of Open Access Journals (Sweden)

    Helena E. Richardson

    2018-01-01

    Full Text Available The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression.

  8. Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

    Science.gov (United States)

    Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

    2017-06-01

    The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

  9. Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns.

    Directory of Open Access Journals (Sweden)

    Andrea Maesani

    2015-11-01

    Full Text Available The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs-locomotor bouts-matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

  10. Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

    Science.gov (United States)

    Chin, Stephanie; Hung, Maurita; Bear, Christine E

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

  11. Centriole Remodeling during Spermiogenesis in Drosophila.

    Science.gov (United States)

    Khire, Atul; Jo, Kyoung H; Kong, Dong; Akhshi, Tara; Blachon, Stephanie; Cekic, Anthony R; Hynek, Sarah; Ha, Andrew; Loncarek, Jadranka; Mennella, Vito; Avidor-Reiss, Tomer

    2016-12-05

    The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM) [1]. In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote [2-4]. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis [5, 6]. Here, we show that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction [7]. We found that the ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and that the PCL does not resemble a typical centriole. We also describe a new phenomenon of Poc1 enrichment of the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Revalidation and rationale for high pKa values of unconjugated bilirubin

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    Ostrow J Donald

    2007-05-01

    Full Text Available Abstract Background Our prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0. Methods We repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay. Results At pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay. Conclusion These results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b hindered solvation of the -COO- group after ionization; and c restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at

  13. High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla.

    Directory of Open Access Journals (Sweden)

    Brian Null

    Full Text Available High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.

  14. Tensin stabilizes integrin adhesive contacts in Drosophila.

    Science.gov (United States)

    Torgler, Catherine N; Narasimha, Maithreyi; Knox, Andrea L; Zervas, Christos G; Vernon, Matthew C; Brown, Nicholas H

    2004-03-01

    We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

  15. Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

    Science.gov (United States)

    Liu, S; Shapiro, J M; Saloustros, E; Stratakis, C A

    2016-11-01

    Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.

  16. Differentially regulated protein kinase A (PKA) activity in adipose tissue and liver is associated with resistance to diet-induced obesity and glucose intolerance in mice that lack PKA regulatory subunit type IIα.

    Science.gov (United States)

    London, Edra; Nesterova, Maria; Sinaii, Ninet; Szarek, Eva; Chanturiya, Tatyana; Mastroyannis, Spyridon A; Gavrilova, Oksana; Stratakis, Constantine A

    2014-09-01

    The cAMP-dependent protein kinase A (PKA) signaling system is widely expressed and has a central role in regulating cellular metabolism in all organ systems affected by obesity. PKA has four regulatory (RIα, RIIα, RIβ, RIIβ) and four catalytic (Cα, Cβ, Cγ, Prkx) subunit isoforms that have tissue-specific expression profiles. In mice, knockout (KO) of RIIβ, the primary PKA regulatory subunit in adipose tissue or knockout of the catalytic subunit Cβ resulted in a lean phenotype that resists diet-induced obesity and associated metabolic complications. Here we report that the disruption of the ubiquitously expressed PKA RIIα subunit in mice (RIIαKO) confers resistance to diet-induced obesity, glucose intolerance, and hepatic steatosis. After 2-week high-fat diet exposure, RIIαKO mice weighed less than wild-type littermates. Over time this effect was more pronounced in female mice that were also leaner than their wild-type counterparts, regardless of the diet. Decreased intake of a high-fat diet contributed to the attenuated weight gain in RIIαKO mice. Additionally, RIIα deficiency caused differential regulation of PKA in key metabolic organs: cAMP-stimulated PKA activity was decreased in liver and increased in gonadal adipose tissue. We conclude that RIIα represents a potential target for therapeutic interventions in obesity, glucose intolerance, and nonalcoholic fatty liver disease.

  17. 20180318 - Prediction Of pKa From Chemical Structure Using Free And Open-Source Tools (ACS Spring)

    Science.gov (United States)

    The ionization state of a chemical, reflected in pKa values, affects lipophilicity, solubility, protein binding and the ability of a chemical to cross the plasma membrane. These properties govern the pharmacokinetic parameters such as absorption, distribution, metabolism, excreti...

  18. pKa prediction for acidic phosphorus-containing compounds using multiple linear regression with computational descriptors.

    Science.gov (United States)

    Yu, Donghai; Du, Ruobing; Xiao, Ji-Chang

    2016-07-05

    Ninety-six acidic phosphorus-containing molecules with pKa 1.88 to 6.26 were collected and divided into training and test sets by random sampling. Structural parameters were obtained by density functional theory calculation of the molecules. The relationship between the experimental pKa values and structural parameters was obtained by multiple linear regression fitting for the training set, and tested with the test set; the R(2) values were 0.974 and 0.966 for the training and test sets, respectively. This regression equation, which quantitatively describes the influence of structural parameters on pKa , and can be used to predict pKa values of similar structures, is significant for the design of new acidic phosphorus-containing extractants. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Epigenetic telomere protection by Drosophila DNA damage response pathways.

    Science.gov (United States)

    Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

    2006-05-01

    Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

  20. Effects of hypo-O-GlcNAcylation on Drosophila development.

    Science.gov (United States)

    Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

    2018-05-11

    Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

  1. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  2. Determination of pKa values of diastereomers of phosphinic pseudopeptides by CZE

    Czech Academy of Sciences Publication Activity Database

    Koval, Dušan; Kašička, Václav; Jiráček, Jiří; Collinsová, Michaela

    2006-01-01

    Roč. 27, č. 23 (2006), s. 4648-4657 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA203/04/0098; GA ČR(CZ) GA203/05/2539 Institutional research plan: CEZ:AV0Z40550506 Keywords : diastereomer separation * phosphinic pseudopeptides * pKa determination Subject RIV: CC - Organic Chemistry Impact factor: 4.101, year: 2006

  3. Prediction of pKa values using the PM6 semiempirical method

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    Jimmy C. Kromann

    2016-08-01

    Full Text Available The PM6 semiempirical method and the dispersion and hydrogen bond-corrected PM6-D3H+ method are used together with the SMD and COSMO continuum solvation models to predict pKa values of pyridines, alcohols, phenols, benzoic acids, carboxylic acids, and phenols using isodesmic reactions and compared to published ab initio results. The pKa values of pyridines, alcohols, phenols, and benzoic acids considered in this study can generally be predicted with PM6 and ab initio methods to within the same overall accuracy, with average mean absolute differences (MADs of 0.6–0.7 pH units. For carboxylic acids, the accuracy (0.7–1.0 pH units is also comparable to ab initio results if a single outlier is removed. For primary, secondary, and tertiary amines the accuracy is, respectively, similar (0.5–0.6, slightly worse (0.5–1.0, and worse (1.0–2.5, provided that di- and tri-ethylamine are used as reference molecules for secondary and tertiary amines. When applied to a drug-like molecule where an empirical pKa predictor exhibits a large (4.9 pH unit error, we find that the errors for PM6-based predictions are roughly the same in magnitude but opposite in sign. As a result, most of the PM6-based methods predict the correct protonation state at physiological pH, while the empirical predictor does not. The computational cost is around 2–5 min per conformer per core processor, making PM6-based pKa prediction computationally efficient enough to be used for high-throughput screening using on the order of 100 core processors.

  4. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    Science.gov (United States)

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

  5. Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.

    Science.gov (United States)

    Bryant, Simon; Kimura, Tomomi E; Kong, Cherrie H T; Watson, Judy J; Chase, Anabelle; Suleiman, M Saadeh; James, Andrew F; Orchard, Clive H

    2014-03-01

    L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

    Science.gov (United States)

    Jastrzebska, Zaneta; Kaminska, Joanna; Chelstowska, Anna; Domanska, Anna; Rzepnikowska, Weronika; Sitkiewicz, Ewa; Cholbinski, Piotr; Gourlay, Campbell; Plochocka, Danuta; Zoladek, Teresa

    2015-12-01

    Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

    Science.gov (United States)

    Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

    2016-06-15

    We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

    Science.gov (United States)

    Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

    2011-11-01

    In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

  9. Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory.

    Science.gov (United States)

    Shuai, Yichun; Hirokawa, Areekul; Ai, Yulian; Zhang, Min; Li, Wanhe; Zhong, Yi

    2015-12-01

    Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β'1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.

  10. Effects of primary recoil (PKA) energy spectrum on radiation damage in fcc metals

    International Nuclear Information System (INIS)

    Iwata, Tadao; Iwase, Akihiro

    1997-10-01

    Irradiation effects by different energetic particles such as electrons, various ions and neutrons are compared in fcc metals, particularly in Cu and Ni. It is discussed on the statistical consideration that the logarithm of the so-called PKA median energy, log T 1/2 , is a good representative to characterize the primary recoil (i.e. PKA) energy spectrum with the resultant defect production. For the irradiations of electrons, various ions and neutrons to Cu and Ni, fundamental physical quantities such as the fraction of stage I recovery, the defect production cross sections and the radiation annealing cross sections can be well scaled as a function of log T 1/2 , if the effects of the electron excitation caused by irradiating ions are excluded. Namely, all data of the respective physical quantity lie on a single continuous curve as a function of log T 1/2 . This characteristic curve is utilized to predict the damage accumulation (i.e. defect concentration) as a function of dpa in Cu and Ni with the PKA median energy as a parameter. (author)

  11. Effects of primary recoil (PKA) energy spectrum on radiation damage in fcc metals

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Tadao; Iwase, Akihiro [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1997-10-01

    Irradiation effects by different energetic particles such as electrons, various ions and neutrons are compared in fcc metals, particularly in Cu and Ni. It is discussed on the statistical consideration that the logarithm of the so-called PKA median energy, log T{sub 1/2}, is a good representative to characterize the primary recoil (i.e. PKA) energy spectrum with the resultant defect production. For the irradiations of electrons, various ions and neutrons to Cu and Ni, fundamental physical quantities such as the fraction of stage I recovery, the defect production cross sections and the radiation annealing cross sections can be well scaled as a function of log T{sub 1/2}, if the effects of the electron excitation caused by irradiating ions are excluded. Namely, all data of the respective physical quantity lie on a single continuous curve as a function of log T{sub 1/2}. This characteristic curve is utilized to predict the damage accumulation (i.e. defect concentration) as a function of dpa in Cu and Ni with the PKA median energy as a parameter. (author)

  12. Computing pKa Values with a Mixing Hamiltonian Quantum Mechanical/Molecular Mechanical Approach.

    Science.gov (United States)

    Liu, Yang; Fan, Xiaoli; Jin, Yingdi; Hu, Xiangqian; Hu, Hao

    2013-09-10

    Accurate computation of the pKa value of a compound in solution is important but challenging. Here, a new mixing quantum mechanical/molecular mechanical (QM/MM) Hamiltonian method is developed to simulate the free-energy change associated with the protonation/deprotonation processes in solution. The mixing Hamiltonian method is designed for efficient quantum mechanical free-energy simulations by alchemically varying the nuclear potential, i.e., the nuclear charge of the transforming nucleus. In pKa calculation, the charge on the proton is varied in fraction between 0 and 1, corresponding to the fully deprotonated and protonated states, respectively. Inspired by the mixing potential QM/MM free energy simulation method developed previously [H. Hu and W. T. Yang, J. Chem. Phys. 2005, 123, 041102], this method succeeds many advantages of a large class of λ-coupled free-energy simulation methods and the linear combination of atomic potential approach. Theory and technique details of this method, along with the calculation results of the pKa of methanol and methanethiol molecules in aqueous solution, are reported. The results show satisfactory agreement with the experimental data.

  13. Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

    Science.gov (United States)

    Fernández Núñez, Lucas; Ocampo, Josefina; Gottlieb, Alexandra M; Rossi, Silvia; Moreno, Silvia

    2016-12-01

    Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  14. Estimation of uncertainty in pKa values determined by potentiometric titration.

    Science.gov (United States)

    Koort, Eve; Herodes, Koit; Pihl, Viljar; Leito, Ivo

    2004-06-01

    A procedure is presented for estimation of uncertainty in measurement of the pK(a) of a weak acid by potentiometric titration. The procedure is based on the ISO GUM. The core of the procedure is a mathematical model that involves 40 input parameters. A novel approach is used for taking into account the purity of the acid, the impurities are not treated as inert compounds only, their possible acidic dissociation is also taken into account. Application to an example of practical pK(a) determination is presented. Altogether 67 different sources of uncertainty are identified and quantified within the example. The relative importance of different uncertainty sources is discussed. The most important source of uncertainty (with the experimental set-up of the example) is the uncertainty of pH measurement followed by the accuracy of the burette and the uncertainty of weighing. The procedure gives uncertainty separately for each point of the titration curve. The uncertainty depends on the amount of titrant added, being lowest in the central part of the titration curve. The possibilities of reducing the uncertainty and interpreting the drift of the pK(a) values obtained from the same curve are discussed.

  15. PKA/AMPK signaling in relation to adiponectin's antiproliferative effect on multiple myeloma cells.

    Science.gov (United States)

    Medina, E A; Oberheu, K; Polusani, S R; Ortega, V; Velagaleti, G V N; Oyajobi, B O

    2014-10-01

    Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced by adipocytes, but paradoxically decreased in obesity, that has been implicated in MM progression. Herein, we evaluated how prolonged exposure to adiponectin affected the survival of MM cells as well as putative signaling mechanisms. Adiponectin activates protein kinase A (PKA), which leads to decreased AKT activity and increased AMP-activated protein kinase (AMPK) activation. AMPK, in turn, induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated, at least in part, by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC), which is essential to lipogenesis. Supplementation with palmitic acid, the preliminary end product of fatty acid synthesis, rescues MM cells from adiponectin-induced apoptosis. Furthermore, 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an ACC inhibitor, exhibited potent antiproliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus, adiponectin's ability to reduce survival of MM cells appears to be mediated through its ability to suppress lipogenesis. Our findings suggest that PKA/AMPK pathway activators, or inhibitors of ACC, may be useful adjuvants to treat MM. Moreover, the antimyeloma effect of adiponectin supports the concept that hypoadiponectinemia, as occurs in obesity, promotes MM tumor progression.

  16. A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons

    OpenAIRE

    Richhariya, Shlesha; Jayakumar, Siddharth; Abruzzi, Katharine; Rosbash, Michael; Hasan, Gaiti

    2017-01-01

    Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrated a requirement for SOCE in neurons that regulate Drosophila flight bouts. We refine this requirement temporally to the early pupal stage and use RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervou...

  17. Estimated pKa values for the environmentally relevant C1 through C8 perfluorinated sulfonic acid isomers.

    Science.gov (United States)

    Rayne, Sierra; Forest, Kaya

    2016-10-14

    In order to estimate isomer-specific acidity constants (pKa) for the perfluorinated sulfonic acid (PFSA) environmental contaminants, the parameterization method 6 (PM6) pKa prediction method was extensively validated against a wide range of carbon oxyacids and related sulfonic/sulfinic acids. Excellent pKa prediction performance was observed for the carbon oxyacids using the PM6 method, but this approach was found to have a severe positive bias for sulfonic/sulfinic acids. To overcome this obstacle, a correlation was developed between non-adjusted PM6 pKa values and the corresponding experimentally obtained/estimated acidity constants for a range of representative alkyl, aryl and halogen-substituted sulfonic acids. Application of this correction to the PM6 values allows for extension of this computational method to a new acid functional group. When used to estimate isomer-specific pKa values for the C1 through C8 PFSAs, the modified PM6 approach suggests an adjusted pKa range from -5.3 to -9.0, indicating that all members of this class of well-known environmental contaminants will be effectively completely dissociated in aquatic systems.

  18. A cost of long-term memory in Drosophila

    OpenAIRE

    Mery, Frederic; Kawecki, Tadeusz J.

    2005-01-01

    Two distinct forms of consolidated associative memory are known in Drosophila: long-term memory and so-called anesthesia-resistant memory. Long-term memory is more stable, but unlike anesthesia-resistant memory, its formation requires protein synthesis. We show that flies induced to form long-term memory become more susceptible to extreme stress (such as desiccation). In contrast, induction of anesthesia-resistant memory had no detectable effect on desiccation resistance. This finding may hel...

  19. An accurate density functional theory based estimation of pK(a) values of polar residues combined with experimental data: from amino acids to minimal proteins.

    Science.gov (United States)

    Matsui, Toru; Baba, Takeshi; Kamiya, Katsumasa; Shigeta, Yasuteru

    2012-03-28

    We report a scheme for estimating the acid dissociation constant (pK(a)) based on quantum-chemical calculations combined with a polarizable continuum model, where a parameter is determined for small reference molecules. We calculated the pK(a) values of variously sized molecules ranging from an amino acid to a protein consisting of 300 atoms. This scheme enabled us to derive a semiquantitative pK(a) value of specific chemical groups and discuss the influence of the surroundings on the pK(a) values. As applications, we have derived the pK(a) value of the side chain of an amino acid and almost reproduced the experimental value. By using our computing schemes, we showed the influence of hydrogen bonds on the pK(a) values in the case of tripeptides, which decreases the pK(a) value by 3.0 units for serine in comparison with those of the corresponding monopeptides. Finally, with some assumptions, we derived the pK(a) values of tyrosines and serines in chignolin and a tryptophan cage. We obtained quite different pK(a) values of adjacent serines in the tryptophan cage; the pK(a) value of the OH group of Ser13 exposed to bulk water is 14.69, whereas that of Ser14 not exposed to bulk water is 20.80 because of the internal hydrogen bonds.

  20. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

    Science.gov (United States)

    Shao, Xiao-Mei; Sun, Jing; Jiang, Yong-Liang; Liu, Bo-Yi; Shen, Zui; Fang, Fang; Du, Jun-Ying; Wu, Yuan-Yuan; Wang, Jia-Ling; Fang, Jian-Qiao

    2016-01-01

    Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

  1. Semi-automated quantitative Drosophila wings measurements.

    Science.gov (United States)

    Loh, Sheng Yang Michael; Ogawa, Yoshitaka; Kawana, Sara; Tamura, Koichiro; Lee, Hwee Kuan

    2017-06-28

    Drosophila melanogaster is an important organism used in many fields of biological research such as genetics and developmental biology. Drosophila wings have been widely used to study the genetics of development, morphometrics and evolution. Therefore there is much interest in quantifying wing structures of Drosophila. Advancement in technology has increased the ease in which images of Drosophila can be acquired. However such studies have been limited by the slow and tedious process of acquiring phenotypic data. We have developed a system that automatically detects and measures key points and vein segments on a Drosophila wing. Key points are detected by performing image transformations and template matching on Drosophila wing images while vein segments are detected using an Active Contour algorithm. The accuracy of our key point detection was compared against key point annotations of users. We also performed key point detection using different training data sets of Drosophila wing images. We compared our software with an existing automated image analysis system for Drosophila wings and showed that our system performs better than the state of the art. Vein segments were manually measured and compared against the measurements obtained from our system. Our system was able to detect specific key points and vein segments from Drosophila wing images with high accuracy.

  2. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  3. Studies on Drosophila radiosensitive strains

    International Nuclear Information System (INIS)

    Varentsova, E.P.; Zakharov, I.A.

    1976-01-01

    45 of radiosensitive strains of Drosophila melanogaster were isolated by Curly/Lobe technique after EMS treatment of Livadia population males. The lethality of non-Curly late larvae after gamma-irradiation (4000r) characterized radiosensitivity strains. Most of them exhibited higher frequency of the spontaneous dominant lethals (up to 69%). The males of 6 strains were semi-sterile. 5 of these strains exhibited higher frequency of X-chromosome non-disjunction

  4. Olfactory memory traces in Drosophila

    OpenAIRE

    Berry, Jacob; Krause, William C.; Davis, Ronald L.

    2008-01-01

    In Drosophila the fruit fly, coincident exposure to an odor and an aversive electric shock can produce robust behavioral memory. This behavioral memory is thought to be regulated by cellular memory traces within the central nervous system of the fly. These molecular, physiological or structural changes in neurons, induced by pairing odor and shock, regulate behavior by altering the neurons’ response to the learned environment. Recently, novel in vivo functional imaging techniques have allowed...

  5. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

    Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

  6. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

    Science.gov (United States)

    Cheung, Samantha K; Scott, Kristin

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

  7. Conserved family of glycerol kinase loci in Drosophila melanogaster

    Science.gov (United States)

    Martinez Agosto, Julian A.; McCabe, Edward R.B.

    2009-01-01

    Glycerol kinase (GK) is an enzyme that catalyzes the formation of glycerol 3-phosphate from ATP and glycerol, the rate-limiting step in glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for glycerol kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders. PMID:16545593

  8. The role of Drosophila mushroom body signaling in olfactory memory.

    Science.gov (United States)

    McGuire, S E; Le, P T; Davis, R L

    2001-08-17

    The mushroom bodies of the Drosophila brain are important for olfactory learning and memory. To investigate the requirement for mushroom body signaling during the different phases of memory processing, we transiently inactivated neurotransmission through this region of the brain by expressing a temperature-sensitive allele of the shibire dynamin guanosine triphosphatase, which is required for synaptic transmission. Inactivation of mushroom body signaling through alpha/beta neurons during different phases of memory processing revealed a requirement for mushroom body signaling during memory retrieval, but not during acquisition or consolidation.

  9. Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells

    Science.gov (United States)

    Yaniv, Yael; Ganesan, Ambhighainath; Yang, Dongmei; Ziman, Bruce D.; Lyashkov, Alexey E.; Levchenko, Andre; Zhang, Jin; Lakatta, Edward G.

    2015-01-01

    cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alter the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirous expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca2+-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate. PMID:26241846

  10. Oral treatment with methanolic extract of the root bark of Condalia buxifolia Reissek alleviates acute pain and inflammation in mice: Potential interactions with PGE2, TRPV1/ASIC and PKA signaling pathways.

    Science.gov (United States)

    Simões, Róli Rodrigues; Dos Santos Coelho, Igor; do Espírito Santo, Caroline Cunha; Morel, Ademir Farias; Zanchet, Eliane Maria; Santos, Adair Roberto Soares

    2016-06-05

    The Condalia buxifolia root bark infusion is used in traditional medicine in Brazil as antipyretic, anti-inflammatory and anti-dysentery. Previous data from our group showed that methanolic extract of Condalia buxifolia (MECb) produced a marked antinociceptive effect in animal models of acute pain. The purpose of this study was to investigate the mechanisms of MECb-induced antinociception as measured by nocifensive behavior in pain induced by endogenous (prostaglandin E2) or exogenous (TRPs and ASIC agonist, and protein kinase A and C activators) chemical stimuli, and the potential role of PKA signaling and capsaicin-sensitive central C-fiber afferents. The effect of MECb administered orally (0.1-300mg/kg, i.g.) to mice on nociception induced by capsaicin (TRPV1 agonist), cinnamaldehyde (TRPA1 agonist), menthol (TRPM8 agonist), acidified saline (ASIC agonist), PMA (protein kinase C activator), PGE2 and forskolin (protein kinase A activator) was assessed. Moreover, this study also investigated the role of C-fibers desensitizing mice with a high dose of intrathecal capsaicin. Furthermore, this study performed the western blot to PKA phosphorylated on nocifensive behavior induced by forskolin. MECb was able to reduce the nociception and paw edema induced by capsaicin, acidified saline, PMA, PGE2 and forskolin, but not by cinnamaldehyde or menthol. Western blot analyses showed that MECb reduced the levels of PKA phosphorylation induced by forskolin in hind paws. Finally, ablating central afferent C-fibers abolished MECb antinociception. In accordance with its use in traditional medicine, these findings provide new evidence indicating that Condalia buxifolia reduces the acute painful behavior of animals caused by chemical stimuli. The precise mechanism of MECb antinociceptive activity is not completely understood but the results suggest involvement of PGE2, TRPV1/ASIC and PKA signaling pathways, and require integrity of the capsaicin-sensitive central C-fiber afferents

  11. Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.

    Science.gov (United States)

    Guo, Zhihao; Batiha, Osamah; Bourouh, Mohammed; Fifield, Eric; Swan, Andrew

    2016-02-01

    Chromosome segregation in meiosis is controlled by a conserved pathway that culminates in Separase-mediated cleavage of the α-kleisin Rec8, leading to dissolution of cohesin rings. Drosophila has no gene encoding Rec8, and the absence of a known Separase target raises the question of whether Separase and its regulator Securin (Pim in Drosophila) are important in Drosophila meiosis. Here, we investigate the role of Securin, Separase and the cohesin complex in female meiosis using fluorescence in situ hybridization against centromeric and arm-specific sequences to monitor cohesion. We show that Securin destruction and Separase activity are required for timely release of arm cohesion in anaphase I and centromere-proximal cohesion in anaphase II. They are also required for release of arm cohesion on polar body chromosomes. Cohesion on polar body chromosomes depends on the cohesin components SMC3 and the mitotic α-kleisin Rad21 (also called Vtd in Drosophila). We provide cytological evidence that SMC3 is required for arm cohesion in female meiosis, whereas Rad21, in agreement with recent findings, is not. We conclude that in Drosophila meiosis, cohesion is regulated by a conserved Securin-Separase pathway that targets a diverged Separase target, possibly within the cohesin complex. © 2016. Published by The Company of Biologists Ltd.

  12. Drosophila: Retrotransposons Making up Telomeres.

    Science.gov (United States)

    Casacuberta, Elena

    2017-07-19

    Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.

  13. Optogenetic pacing in Drosophila melanogaster

    Science.gov (United States)

    Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

  14. Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift

    Directory of Open Access Journals (Sweden)

    Douglas Mark Ruden

    2012-03-01

    Full Text Available This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms (SNPs, multiple nucleotide polymorphisms (MNPs, insertions and deletions (InDels in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently-isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of PCR-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate premature stop codon mutation in each of the two allelic mutants whereas the other 4 candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically-mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic

  15. The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

    Science.gov (United States)

    Sealover, Natalie R; Felts, Bruce; Kuntz, Charles P; Jarrard, Rachel E; Hockerman, Gregory H; Lamb, Patrick W; Barker, Eric L; Henry, L Keith

    2016-11-15

    The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Determination of pKa values of benzoxa-, benzothia- and benzoselena-zolinone derivatives by capillary electrophoresis. Comparison with potentiometric titration and spectrometric data.

    Science.gov (United States)

    Foulon, C; Duhal, N; Lacroix-Callens, B; Vaccher, C; Bonte, J P; Goossens, J F

    2007-07-01

    Acidity constants of benzoxa-, benzothia- and benzoselena-zolinone derivatives were determined by capillary electrophoresis, potentiometry and spectrophotometry experiments. These three analytical techniques gave pK(a) results that were in good agreement. A convenient, accurate and precise method for the determination of pK(a) was developed to measure changes in acidity constants induced by heteroatom or 6-benzoyl substituted derivatives. pK(a) values were determined simultaneously for two compounds characterized by different electrophoretic mobility (micro(e)) and pK(a) value and in the presence of an analogous neutral marker.

  17. Radioresistance and radiosensitivity in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Reguly, M.L.

    1983-01-01

    Studying the mechanisms controlling radioresistant in Drosophila the sensibility of four strains of Drosophila melanogaster to sex-linked recessive lethal mutations induced by 5kR Cobalt-60 gamma radiation and 0,006 M EMS or 0,25% of caffeine was determined. (M.A.C.) [pt

  18. The Drosophila melanogaster circadian pacemaker circuit

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Keywords. circadian rhythm; neuronal network; ion channel; behaviour; neurotransmitter; electrophysiology; Drosophila. Abstract. As an experimental model system, the fruit fly Drosophila melanogaster has been seminal in shaping our understanding of the circadian clockwork. The wealth of genetic tools ...

  19. Unique properties of Drosophila spermatocyte primary cilia

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Riparbelli

    2013-09-01

    The primary cilium is an essential organelle required for animal development and adult homeostasis that is found on most animal cells. The primary cilium contains a microtubule-based axoneme cytoskeleton that typically grows from the mother centriole in G0/G1 phase of the cell cycle as a membrane-bound compartment that protrudes from the cell surface. A unique system of bidirectional transport, intraflagellar transport (IFT, maintains the structure and function of cilia. While the axoneme is dynamic, growing and shrinking at its tip, at the same time it is very stable to the effects of microtubule-targeting drugs. The primary cilia found on Drosophila spermatocytes diverge from the general rules of primary cilium biology in several respects. Among these unique attributes, spermatocyte cilia assemble from all four centrioles in an IFT-independent manner in G2 phase, and persist continuously through two cell divisions. Here, we show that Drosophila spermatocyte primary cilia are extremely sensitive to microtubule-targeting drugs, unlike their mammalian counterparts. Spermatocyte cilia and their axonemes fail to assemble or be maintained upon nocodazole treatment, while centriole replication appears unperturbed. On the other hand, paclitaxel (Taxol, a microtubule-stabilizing drug, disrupted transition zone assembly and anchoring to the plasma membrane while causing spermatocyte primary cilia to grow extensively long during the assembly/elongation phase, but did not overtly affect the centrioles. However, once assembled to their mature length, spermatocyte cilia appeared unaffected by Taxol. The effects of these drugs on axoneme dynamics further demonstrate that spermatocyte primary cilia are endowed with unique assembly properties.

  20. Measurement of lifespan in Drosophila melanogaster.

    Science.gov (United States)

    Linford, Nancy J; Bilgir, Ceyda; Ro, Jennifer; Pletcher, Scott D

    2013-01-07

    Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals. The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts(1-4). In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (http://sitemaker.umich.edu/pletcherlab/software). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying

  1. Transcriptional regulation of Drosophila gonad formation.

    Science.gov (United States)

    Tripathy, Ratna; Kunwar, Prabhat S; Sano, Hiroko; Renault, Andrew D

    2014-08-15

    The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation we identified a SGP cell autonomous role for Midline (Mid) and Longitudinals lacking (Lola). These transcriptional factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms and we have identified an isoform specific requirement for lola in the gonad which is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. Our findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling.

    Science.gov (United States)

    Monterisi, Stefania; Lobo, Miguel J; Livie, Craig; Castle, John C; Weinberger, Michael; Baillie, George; Surdo, Nicoletta C; Musheshe, Nshunge; Stangherlin, Alessandra; Gottlieb, Eyal; Maizels, Rory; Bortolozzi, Mario; Micaroni, Massimo; Zaccolo, Manuela

    2017-05-02

    cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.

  3. Predicting the pKa and stability of organic acids and bases at an oil-water interface

    DEFF Research Database (Denmark)

    Andersson, Martin Peter; Olsson, Mats Henrik Mikael; Stipp, Susan Louise Svane

    2014-01-01

    We have used density functional theory and the implicit solvent model, COSMO-RS, to investigate how the acidity constant, pKa, of organic acids and bases adsorbed at the organic compound-aqueous solution interface changes, compared to its value in the aqueous phase. The pKa determine the surface...... phase and the rest remains in the hydrophilic phase. We found that the pKa for acids is shifted by ∼1 pH unit to higher values compared to the bulk water pKa, whereas they are shifted to lower values by a similar amount for bases. Because this pKa shift is similar in magnitude for each of the molecules...... is not the same as in the bulk. For example, species such as carboxylic acids are enriched at the interface, where they can dominate surface properties, even when they are a modest component in the bulk fluid. For high surface concentrations of carboxylic acid groups at an interface, such as a self...

  4. The IUPAC aqueous and non-aqueous experimental pKa data repositories of organic acids and bases.

    Science.gov (United States)

    Slater, Anthony Michael

    2014-10-01

    Accurate and well-curated experimental pKa data of organic acids and bases in both aqueous and non-aqueous media are invaluable in many areas of chemical research, including pharmaceutical, agrochemical, specialty chemical and property prediction research. In pharmaceutical research, pKa data are relevant in ligand design, protein binding, absorption, distribution, metabolism, elimination as well as solubility and dissolution rate. The pKa data compilations of the International Union of Pure and Applied Chemistry, originally in book form, have been carefully converted into computer-readable form, with value being added in the process, in the form of ionisation assignments and tautomer enumeration. These compilations offer a broad range of chemistry in both aqueous and non-aqueous media and the experimental conditions and original reference for all pKa determinations are supplied. The statistics for these compilations are presented and the utility of the computer-readable form of these compilations is examined in comparison to other pKa compilations. Finally, information is provided about how to access these databases.

  5. Accurate pKa calculation of the conjugate acids of alkanolamines, alkaloids and nucleotide bases by quantum chemical methods.

    Science.gov (United States)

    Gangarapu, Satesh; Marcelis, Antonius T M; Zuilhof, Han

    2013-04-02

    The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum model (CPCM) and universal solvation models (SMD, SM8). G3, SCS-MP2 and M11-L methods coupled with SMD and SM8 solvation models perform well for alkanolamines with mean unsigned errors below 0.20 pKa units, in all cases. Extending this method to the pKa calculation of 35 nitrogen-containing compounds spanning 12 pKa units showed an excellent correlation between experimental and computational pKa values of these 35 amines with the computationally low-cost SM8/M11-L density functional approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. TENDL-TMC for Δdpa and Δpka

    International Nuclear Information System (INIS)

    Rochman, D.; Ferroukhi, H.; Koning, A.J.; Sjostrand, H.; Helgesson, P.; Gilbert, M.; Sublet, J.C.

    2016-01-01

    Full text: The TENDL library (Talys Evaluated Nuclear Data Library) contains the necessary information (e.g. recoil spectra, double differential data) to calculate quantities of interest for the material damage. Additionally, it is one of the most complete libraries in terms of number of isotopes and format-wise: 2800 isotopes (ground states and isomers) and all ENDF-6 sections from MF1 to MF40. It can then be naturally used for the estimation of “DPA” and “PKA”, given the correct NJOY processing. Details of the library, its production, formatting and processing are given during the technical meeting. Comparison with other libraries indicated the importance of including all the “MT” sections for the correct processing with NJOY, but also it showed the difference obtained depending of the format chosen to store the decay data. Regarding the uncertainties on DPA and PKA, the TMC method seems to be one of the most convenient methods. As presented during the meeting, the uncertainty propagation using random ENDF-6 files produced from variations of model parameters leads to non-Gaussian distributions for the damage quantities. As a function of the incident neutron energy, the skewness of such distributions can strongly vary and be far from 0. This indicates that the standard deviation alone cannot represent, well enough, the dispersion of the calculated data. A viable alternative is the production of so-called random ENDF-6 files based on given covariance information. This method is limited by the available information given in the covariance files, but can help to capture part of the uncertainties for the DPA and PKA quantities. For TENDL-2016, the covariance format MF32 will be less used and efforts will be devoted to produce MF33, which will facilitate the production of random ENDF-6 files with SCK codes such as SANDY. A PSI internal project to link the nuclear data with the atomistic simulation of damage formation and microstructure evolution was also

  7. Human Intellectual Disability Genes Form Conserved Functional Modules in Drosophila

    Science.gov (United States)

    Oortveld, Merel A. W.; Keerthikumar, Shivakumar; Oti, Martin; Nijhof, Bonnie; Fernandes, Ana Clara; Kochinke, Korinna; Castells-Nobau, Anna; van Engelen, Eva; Ellenkamp, Thijs; Eshuis, Lilian; Galy, Anne; van Bokhoven, Hans; Habermann, Bianca; Brunner, Han G.; Zweier, Christiane; Verstreken, Patrik; Huynen, Martijn A.; Schenck, Annette

    2013-01-01

    Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules. PMID:24204314

  8. A Genetic Toolkit for Dissecting Dopamine Circuit Function in Drosophila

    Directory of Open Access Journals (Sweden)

    Tingting Xie

    2018-04-01

    Full Text Available Summary: The neuromodulator dopamine (DA plays a key role in motor control, motivated behaviors, and higher-order cognitive processes. Dissecting how these DA neural networks tune the activity of local neural circuits to regulate behavior requires tools for manipulating small groups of DA neurons. To address this need, we assembled a genetic toolkit that allows for an exquisite level of control over the DA neural network in Drosophila. To further refine targeting of specific DA neurons, we also created reagents that allow for the conversion of any existing GAL4 line into Split GAL4 or GAL80 lines. We demonstrated how this toolkit can be used with recently developed computational methods to rapidly generate additional reagents for manipulating small subsets or individual DA neurons. Finally, we used the toolkit to reveal a dynamic interaction between a small subset of DA neurons and rearing conditions in a social space behavioral assay. : The rapid analysis of how dopaminergic circuits regulate behavior is limited by the genetic tools available to target and manipulate small numbers of these neurons. Xie et al. present genetic tools in Drosophila that allow rational targeting of sparse dopaminergic neuronal subsets and selective knockdown of dopamine signaling. Keywords: dopamine, genetics, behavior, neural circuits, neuromodulation, Drosophila

  9. Preference for and learning of amino acids in larval Drosophila

    Directory of Open Access Journals (Sweden)

    Nana Kudow

    2017-03-01

    Full Text Available Relative to other nutrients, less is known about how animals sense amino acids and how behaviour is organized accordingly. This is a significant gap in our knowledge because amino acids are required for protein synthesis − and hence for life as we know it. Choosing Drosophila larvae as a case study, we provide the first systematic analysis of both the preference behaviour for, and the learning of, all 20 canonical amino acids in Drosophila. We report that preference for individual amino acids differs according to the kind of amino acid, both in first-instar and in third-instar larvae. Our data suggest that this preference profile changes across larval instars, and that starvation during the third instar also alters this profile. Only aspartic acid turns out to be robustly attractive across all our experiments. The essentiality of amino acids does not appear to be a determinant of preference. Interestingly, although amino acids thus differ in their innate attractiveness, we find that all amino acids are equally rewarding. Similar discrepancies between innate attractiveness and reinforcing effect have previously been reported for other tastants, including sugars, bitter substances and salt. The present analyses will facilitate the ongoing search for the receptors, sensory neurons, and internal, homeostatic amino acid sensors in Drosophila.

  10. Drosophila cell cycle under arrest: uncapped telomeres plead guilty.

    Science.gov (United States)

    Cenci, Giovanni

    2009-04-01

    Telomeres are specialized structures that protect chromosome ends from degradation and fusion events. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences preventing chromosome ends from being recognized as DNA double strand breaks (DSBs). Telomeres that lose their cap activate the DNA damage response (DDR) likewise DSBs and, if inappropriately repaired, generate telomeric fusions, which eventually lead to genome instability. In Drosophila there is not telomerase, and telomere length is maintained by transposition of three specialized retroelements. However, fly telomeres are protected by multi protein complexes like their yeast and vertebrate counterparts; these complexes bind chromosome ends in a sequence-independent fashion and are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres elicit a DDR just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. BubR1 accumulations at chromosome ends trigger the SAC that inhibits the metaphase-to-anaphase transition. These findings, reviewed here, highlight an intriguing and unsuspected connection between telomeres and cell cycle regulation, providing a clue to understand human telomere function.

  11. FMRFamide signaling promotes stress-induced sleep in Drosophila.

    Science.gov (United States)

    Lenz, Olivia; Xiong, Jianmei; Nelson, Matthew D; Raizen, David M; Williams, Julie A

    2015-07-01

    Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Hox gene regulation in the central nervous system of Drosophila

    Directory of Open Access Journals (Sweden)

    Maheshwar eGummalla

    2014-04-01

    Full Text Available Hox genes specify the structures that form along the anteroposterior (AP axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called posterior dominance, states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. Here we review a noticeable exception to this rule where the more-posteriorly expressed Abd-B hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS. While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92kb long non-coding RNA (lncRNA encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA. Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The 1st mechanism is mediated by a microRNA (mir-iab-8 encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila hox complexes

  13. Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

    Science.gov (United States)

    Fusi, Fabio; Trezza, Alfonso; Spiga, Ottavia; Sgaragli, Giampietro; Bova, Sergio

    2017-09-15

    To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca 2+ current through Ca v 1.2 channels [I Ca1.2 ], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the I Ca1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both I Ca1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited I Ca1.2 with a pIC 50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of I Ca1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Ca v 1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α 1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K + - and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular I Ca1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  15. Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

    Science.gov (United States)

    He, Shuai; Kah, James C. Y.

    2017-04-01

    Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

  16. Prolonged fasting reduces IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and reverse immunosuppression.

    Science.gov (United States)

    Cheng, Chia-Wei; Adams, Gregor B; Perin, Laura; Wei, Min; Zhou, Xiaoying; Lam, Ben S; Da Sacco, Stefano; Mirisola, Mario; Quinn, David I; Dorff, Tanya B; Kopchick, John J; Longo, Valter D

    2014-06-05

    Immune system defects are at the center of aging and a range of diseases. Here, we show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSCs) and niche cells that promote stress resistance, self-renewal, and lineage-balanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused by chemotherapy and reversed age-dependent myeloid-bias in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The proregenerative effects of fasting on stem cells were recapitulated by deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-1. These findings link the reduced levels of IGF-1 caused by fasting to PKA signaling and establish their crucial role in regulating hematopoietic stem cell protection, self-renewal, and regeneration. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Marko Kostic

    2015-10-01

    Full Text Available Mitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1 function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX, leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

  18. Caffeine taste signaling in Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Anthi A Apostolopoulou

    2016-08-01

    Full Text Available The Drosophila larva has a simple peripheral nervous system with a comparably small number of sensory neurons located externally at the head or internally along the pharynx to assess its chemical environment. It is assumed that larval taste coding occurs mainly via external organs (the dorsal, terminal and ventral organ. However, the contribution of the internal pharyngeal sensory organs has not been explored. Here we find that larvae require a single pharyngeal gustatory receptor neuron pair called D1, which is located in the dorsal pharyngeal sensilla, in order to avoid caffeine and to associate an odor with caffeine punishment. In contrast, caffeine-driven reduction in feeding in non-choice situations does not require D1. Hence, this work provides data on taste coding via different receptor neurons, depending on the behavioral context. Furthermore, we show that the larval pharyngeal system is involved in bitter tasting. Using ectopic expressions, we show that the caffeine receptor in neuron D1 requires the function of at least four receptor genes: the putative coreceptors Gr33a, Gr66a, the putative caffeine-specific receptor Gr93a, and yet unknown additional molecular component(s. This suggests that larval taste perception is more complex than previously assumed already at the sensory level. Taste information from different sensory organs located outside at the head or inside along the pharynx of the larva is assembled to trigger taste guided behaviours.

  19. Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution.

    Science.gov (United States)

    Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C

    2017-01-27

    The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca 2+ -regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution*

    Science.gov (United States)

    Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C.

    2017-01-01

    The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. PMID:27998980

  1. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

    Science.gov (United States)

    Lum, H; Jaffe, H A; Schulz, I T; Masood, A; RayChaudhury, A; Green, R D

    1999-09-01

    We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

  2. Influence of subcascade formation on displacement damage at high PKA energies

    Energy Technology Data Exchange (ETDEWEB)

    Stoller, R.E. [Oak Ridge National Lab., TN (United States); Greenwood, L.R. [Pacific Northwest National Lab., Richland, WA (United States)

    1997-08-01

    The design of first generation fusion reactors will have to be rely on radiation effects data obtained from experiments conducted in fission reactors. Two issues must be addressed to use this data with confidence. The first is differences in the neutron energy spectrum, and the second is differences in nuclear transmutation rates. Differences in the neutron energy spectra are reflected in the energy spectra of the primary knockon atoms (PKA). The issue of PKA energy effects has been addressed through the use of displacement cascade simulations using the method of molecular dynamics (MD). Although MD simulations can provide a detailed picture of the formation and evolution of displacement cascades, they impose a substantial computational burden. However, recent advances in computing equipment permit the simulation of high energy displacement events involving more than one-million atoms; the results presented here encompass MD cascade simulation energies from near the displacement threshold to as high as 40 keV. Two parameters have been extracted from the MD simulations: the number of point defects that remain after the displacement event is completed and the fraction of the surviving interstitials that are contained in clusters. The MD values have been normalized to the number of atomic displacements calculated with the secondary displacement model by Norgett, Robinson, and Torrens (NRT).

  3. Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

    Science.gov (United States)

    Malinowska, D H; Kupert, E Y; Bahinski, A; Sherry, A M; Cuppoletti, J

    1995-01-01

    cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

  4. Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Yuan Hao

    2015-08-01

    Full Text Available Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (ISC in a human bronchial epithelial cell line (16HBE14o-, and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The ISC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca2+]i and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in ISC, which was due to Cl- secretion. The increase in ISC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172, but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS, Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated ISC was also sensitive to a protein kinase A (PKA inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in ISC in a cystic fibrosis (CF cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca2+]i. Conclusion: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.

  5. L-carnitine contributes to enhancement of neurogenesis from mesenchymal stem cells through Wnt/β-catenin and PKA pathway.

    Science.gov (United States)

    Fathi, Ezzatollah; Farahzadi, Raheleh; Charoudeh, Hojjatollah Nozad

    2017-03-01

    The identification of factors capable of enhancing neurogenesis has great potential for cellular therapies in neurodegenerative diseases. Multiple studies have shown the neuroprotective effects of L-carnitine (LC). This study determined whether neuronal differentiation of rat adipose tissue-derived mesenchymal stem cells (ADSCs) can be activated by LC. In this study, protein kinase A (PKA) and Wnt/β-catenin pathways were detected to show if this activation was due to these pathways. The expression of LC-induced neurogenesis markers in ADSCs was characterized using real-time PCR. ELISA was conducted to assess the expression of cyclic adenosine monophosphate (cAMP) and PKA. The expression of β-catenin, reduced dickkopf1 (DKK1), low-density lipoprotein receptor-related protein 5 (LRP5), Wnt1, and Wnt3a genes as Wnt/β-catenin signaling members were used to detect the Wnt/β-catenin pathway. It was observed that LC could promote neurogenesis in ADSCs as well as expression of some neurogenic markers. Moreover, LC causes to increase the cAMP levels and PKA activity. Treatment of ADSCs with H-89 (dihydrochloride hydrate) as PKA inhibitor significantly inhibited the promotion of neurogenic markers, indicating that the PKA signaling pathway could be involved in neurogenesis induction. Analyses of real-time PCR data showed that the mRNA expressions of β-catenin, DKK1, LRP5c-myc, Wnt1, and Wnt3a were increased in the presence of LC. Therefore, the present study showed that LC promotes ADSCs neurogenesis and the LC-induced neurogenic markers could be due to both the PKA and Wnt/β-catenin signaling pathway. Impact statement Neural tissue has long been believed as incapable of regeneration and the identification of cell types and factors capable of neuronal differentiation has generated intense interest. Mesenchymal stem cells (MSCs) are considered as potential targets for stem cell-based therapy. L-carnitin (LC) as an antioxidant may have neuroprotective effects in

  6. Modeling of cascade and sub-cascade formation at high pka energies in irradiated fusion structural materials

    International Nuclear Information System (INIS)

    Ryazanov, A.; Metelkin, E.V.; Semenov, E.A.

    2007-01-01

    Full text of publication follows: A new theoretical model is developed for the investigations of cascade and sub-cascade formation in fusion structural materials under fast neutron irradiation at high primary knock atom (PKA) energies. Under 14 MeV neutron irradiation especially of light fusion structural materials such as Be, C, SiC materials PKA will have the energies up to 1 MeV. At such high energies it is very difficult to use the Monte Carlo or molecular dynamic simulations. The developed model is based on the analytical consideration of elastic collisions between displaced moving atoms into atomic cascades produced by a PKAs with the some kinetic energy obtained from fast neutrons. The Tomas-Fermy interaction potential is used for the describing of elastic collisions between moving atoms. The suggested model takes into account also the electronic losses for moving atoms between elastic collisions. The self consistent criterion for sub-cascade formation is suggested here which is based on the comparison of mean distance between two consequent PKA collisions and size of sub-cascade produced by PKA. The analytical relations for the most important characteristics of cascades and sub-cascade are determined including the average number of sub-cascades per one PKA in the dependence on PKA energy, the distance between sub-cascades and the average cascade and sub-cascade sizes as a function of PKA energy. The developed model allows determining the total numbers, distribution functions of cascades and sub-cascades in dependence on their sizes and generation rate of cascades and sub-cascades for different fusion neutron energy spectra. Based on the developed model the numerical calculations for main characteristics of cascades and sub-cascades in different fusion structural materials are performed using the neutron flux and PKA energy spectra for fusion reactors: ITER and DEMO. The main characteristics for cascade and sub-cascade formation are calculated here for the

  7. PKA spectral effects on subcascade structures and free defect survival ratio as estimated by cascade-annealing computer simulation

    International Nuclear Information System (INIS)

    Muroga, Takeo

    1990-01-01

    The free defect survival ratio is calculated by ''cascade-annealing'' computer simulation using the MARLOWE and modified DAIQUIRI codes in various cases of Primary Knock-on Atom (PKA) spectra. The number of subcascades is calculated by ''cut-off'' calculation using MARLOWE. The adequacy of these methods is checked by comparing the results with experiments (surface segregation measurements and Transmission Electron Microscope cascade defect observations). The correlation using the weighted average recoil energy as a parameter shows that the saturation of the free defect survival ratio at high PKA energies has a close relation to the cascade splitting into subcascades. (author)

  8. Beta-Adrenergic Receptor Activation during Distinct Patterns of Stimulation Critically Modulates the PKA-Dependence of LTP in the Mouse Hippocampus

    Science.gov (United States)

    Gelinas, Jennifer N.; Tenorio, Gustavo; Lemon, Neal; Abel, Ted; Nguyen, Peter V.

    2008-01-01

    Activation of Beta-adrenergic receptors (Beta-ARs) enhances hippocampal memory consolidation and long-term potentiation (LTP), a likely mechanism for memory storage. One signaling pathway linked to Beta-AR activation is the cAMP-PKA pathway. PKA is critical for the consolidation of hippocampal long-term memory and for the expression of some forms…

  9. PKA-RIIB Deficiency Induces Brown Fatlike Adipocytes in Inguinal WAT and Promotes Energy Expenditure in Male FVB/NJ Mice.

    Science.gov (United States)

    Su, Jing; Wu, Wei; Huang, Shan; Xue, Ruidan; Wang, Yi; Wan, Yun; Zhang, Lv; Qin, Lang; Zhang, Qiongyue; Zhu, Xiaoming; Zhang, Zhaoyun; Ye, Hongying; Wu, Xiaohui; Li, Yiming

    2017-03-01

    Obesity has become the most common metabolic disorder worldwide. Promoting brown adipose tissue (BAT) and beige adipose tissue formation, and therefore, a functional increase in energy expenditure, may counteract obesity. Mice lacking type IIβ regulatory subunit of adenosine 3',5' cyclic monophosphate (cAMP)-dependent protein kinase A (PKA-RIIB) display reduced adiposity and resistance to diet-induced obesity. PKA-RIIB, encoded by the Prkar2b gene, is most abundant in BAT and white adipose tissue (WAT) and in the brain. In this study, we show that mice lacking PKA-RIIB have increased energy expenditure, limited weight gain, and improved glucose metabolism. PKA-RIIB deficiency induces brownlike adipocyte in inguinal WAT (iWAT). PKA-RIIB deficiency also increases the expression of uncoupling protein 1 and other thermogenic genes in iWAT and primary preadipocytes from iWAT through a mechanism involving increased PKA activity, which is represented by increased phosphorylation of PKA substrate, cAMP response element binding protein, and P38 mitogen-activated protein kinase. Our study provides evidence for the role of PKA-RIIB deficiency in regulating thermogenesis in WAT, which may potentially have therapeutic implications for the treatment of obesity and related metabolic disorders. Copyright © 2017 by the Endocrine Society.

  10. Conservation and divergence of the cyclic adenosine monophosphate–protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Science.gov (United States)

    The importance of cAMP signaling in fungal development and pathogenesis has been well documented in many fungal species including several phytopathogenic Fusarium spp. Two key components of the cAMP-PKA pathway, adenylate cyclase (AC) and catalytic subunit of PKA (CPKA), have been functionally chara...

  11. Adaptive genic evolution in the Drosophila genomes

    DEFF Research Database (Denmark)

    Shapiro, Joshua A; Huang, Wei; Zhang, Chenhui

    2007-01-01

    and stable population. In this study, we sequenced 419 genes from 24 lines of Drosophila melanogaster and its close relatives. Together with data from Drosophila simulans, these data reveal the following. (i) Approximately 10% of the loci in regions of normal recombination are much less polymorphic at silent...... sites than expected, hinting at the action of selective sweeps. (ii) The level of polymorphism is negatively correlated with the rate of nonsynonymous divergence across loci. Thus, even under strict neutrality, the ratio of amino acid to silent nucleotide changes (A:S) between Drosophila species...

  12. The AKAP Cypher/Zasp contributes to β-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels.

    Science.gov (United States)

    Yu, Haijie; Yuan, Can; Westenbroek, Ruth E; Catterall, William A

    2018-06-04

    Stimulation of the L-type Ca 2+ current conducted by Ca V 1.2 channels in cardiac myocytes by the β-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the Ca V 1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in β-adrenergic regulation of Ca V 1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of Ca V 1.2, PKA, and calcineurin. In contrast, the cell surface density of Ca V 1.2 channels and the basal Ca 2+ current conducted by Ca V 1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. β-adrenergic regulation of these L-type Ca 2+ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca 2+ current. At 100 nM isoproterenol, the net β-adrenergic-Ca 2+ current conducted by Ca V 1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for β-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for β-adrenergic regulation of cardiac Ca V 1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of β-adrenergic regulation of Ca V 1.2 channels observed in vivo. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

  13. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology.

    Science.gov (United States)

    Castells-Nobau, Anna; Nijhof, Bonnie; Eidhof, Ilse; Wolf, Louis; Scheffer-de Gooyert, Jolanda M; Monedero, Ignacio; Torroja, Laura; van der Laak, Jeroen A W M; Schenck, Annette

    2017-05-03

    Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

  14. Development and validation of a FIA/UV-vis method for pK(a) determination of oxime based acetylcholinesterase reactivators.

    Science.gov (United States)

    Musil, Karel; Florianova, Veronika; Bucek, Pavel; Dohnal, Vlastimil; Kuca, Kamil; Musilek, Kamil

    2016-01-05

    Acetylcholinesterase reactivators (oximes) are compounds used for antidotal treatment in case of organophosphorus poisoning. The dissociation constants (pK(a1)) of ten standard or promising acetylcholinesterase reactivators were determined by ultraviolet absorption spectrometry. Two methods of spectra measurement (UV-vis spectrometry, FIA/UV-vis) were applied and compared. The soft and hard models for calculation of pK(a1) values were performed. The pK(a1) values were recommended in the range 7.00-8.35, where at least 10% of oximate anion is available for organophosphate reactivation. All tested oximes were found to have pK(a1) in this range. The FIA/UV-vis method provided rapid sample throughput, low sample consumption, high sensitivity and precision compared to standard UV-vis method. The hard calculation model was proposed as more accurate for pK(a1) calculation. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Determination of pKa values of alendronate sodium in aqueous solution by piecewise linear regression based on acid-base potentiometric titration.

    Science.gov (United States)

    Ke, Jing; Dou, Hanfei; Zhang, Ximin; Uhagaze, Dushimabararezi Serge; Ding, Xiali; Dong, Yuming

    2016-12-01

    As a mono-sodium salt form of alendronic acid, alendronate sodium presents multi-level ionization for the dissociation of its four hydroxyl groups. The dissociation constants of alendronate sodium were determined in this work by studying the piecewise linear relationship between volume of titrant and pH value based on acid-base potentiometric titration reaction. The distribution curves of alendronate sodium were drawn according to the determined pKa values. There were 4 dissociation constants (pKa 1 =2.43, pKa 2 =7.55, pKa 3 =10.80, pKa 4 =11.99, respectively) of alendronate sodium, and 12 existing forms, of which 4 could be ignored, existing in different pH environments.

  16. Studies on Drosophila radiosensitivity strains

    International Nuclear Information System (INIS)

    Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.U.

    1985-01-01

    Fertility of radiosensitive mutant drosophila female strain rad (2) 201 61 after irradiation and frequency of dominant lethal mutations (DLM), induced by γ-radiation for 0-5 h and 5-7 days, are investigated. It is shown, that oocytes of the mutant strain are more radiosensitive as compared with cells of mongrel flies as to criterion of DLM appearance over the period of maturing. Early oocytes of stages 2-7 are the most sensitive, i.e. at the stages, corresponding to the manifestation of previously established recombination-defective properties of mutations rad (2) 201 61 . It is also sown, that doses of γ-rays, exceeding 10 Gy produce a strong sterilizing effect on mutant females due to destruction and resorption of egg chambers, irradiated at the stages of previtellogenetic growth of oocytes. In females, carrying mutation of radiosensitivity there is no direct correlation betwen sensitivity of oocytes proper to DLM induction and sensitivity of egg folleicles to resorbing effect of γ-rays. The ways of possible involvement of mutant locus studied into genetic processes in various specialized cells of drosophila

  17. Human muscle-specific A-kinase anchoring protein (mAKAP) polymorphisms modulate the susceptibility to cardiovascular diseases by altering cAMP/ PKA signaling.

    Science.gov (United States)

    Suryavanshi, Santosh V; Jadhav, Shweta M; Anderson, Kody L; Katsonis, Panagiotis; Lichtarge, Olivier; McConnell, Bradley K

    2018-03-30

    One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction which is regulated by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase4D3 (PDE4D3) among other proteins and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Especially, single nucleotide polymorphisms (SNPs) in various proteins have been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter cAMP/PKA pathway influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease related patients with highest predicted deleterious effects, Ser(S) 1653 Arg(R) and Glu(E) 2124 Gly(G). Co-immunoprecipitation data in HEK293T cells showed that S1653R SNP, present in the PDE4D3 binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity and cytosolic PDE activity when compared with the wild-type (WT) before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be up-regulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions.

  18. Editor's Highlight: Genetic Targets of Acute Toluene Inhalation in Drosophila melanogaster

    Science.gov (United States)

    Interpretation and use of data from high-throughput assays for chemical toxicity require links between effects at molecular targets and adverse outcomes in whole animals. The well-characterized genome of Drosophila melanogaster provides a potential model system by which phenotypi...

  19. Altered Gene Regulation and Synaptic Morphology in "Drosophila" Learning and Memory Mutants

    Science.gov (United States)

    Guan, Zhuo; Buhl, Lauren K.; Quinn, William G.; Littleton, J. Troy

    2011-01-01

    Genetic studies in "Drosophila" have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in "radish" ("rsh") mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways…

  20. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  1. Functional Analysis of Drosophila NF1

    National Research Council Canada - National Science Library

    Bernards, Andre

    2005-01-01

    ...) for Ras, yet homozygous loss of a highly conserved Drosophila NF1 ortholog results in several phenotypes that are insensitive to manipulating Ras signal transduction, but rescued by increasing...

  2. The Drosophila bipectinata species complex: phylogenetic ...

    Indian Academy of Sciences (India)

    PARUL BANERJEE

    c Indian Academy of Sciences. RESEARCH ARTICLE. The Drosophila bipectinata species complex: phylogenetic relationship among different members based on chromosomal variations. PARUL BANERJEE and BASHISTH N. SINGH. ∗. Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi ...

  3. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  4. Ecdysteroid receptors in Drosophila melanogaster adult females

    Science.gov (United States)

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  5. The arouser EPS8L3 gene is critical for normal memory in Drosophila.

    Directory of Open Access Journals (Sweden)

    Holly LaFerriere

    Full Text Available The genetic mechanisms that influence memory formation and sensitivity to the effects of ethanol on behavior in Drosophila have some common elements. So far, these have centered on the cAMP/PKA signaling pathway, synapsin and fas2-dependent processes, pumilio-dependent regulators of translation, and a few other genes. However, there are several genes that are important for one or the other behaviors, suggesting that there is an incomplete overlap in the mechanisms that support memory and ethanol sensitive behaviors. The basis for this overlap is far from understood. We therefore examined memory in arouser (aru mutant flies, which have recently been identified as having ethanol sensitivity deficits. The aru mutant flies showed memory deficits in both short-term place memory and olfactory memory tests. Flies with a revertant aru allele had wild-type levels of memory performance, arguing that the aru gene, encoding an EPS8L3 product, has a role in Drosophila memory formation. Furthermore, and interestingly, flies with the aru(8-128 insertion allele had deficits in only one of two genetic backgrounds in place and olfactory memory tests. Flies with an aru imprecise excision allele had deficits in tests of olfactory memory. Quantitative measurements of aru EPS8L3 mRNA expression levels correlate decreased expression with deficits in olfactory memory while over expression is correlated with place memory deficits. Thus, mutations of the aru EPS8L3 gene interact with the alleles of a particular genetic background to regulate arouser expression and reveals a role of this gene in memory.

  6. Mapping of gene mutations in drosophila melanogaster

    OpenAIRE

    Halvorsen, Charlotte Marie

    2004-01-01

    In this experiment, mutant genes of a given unknown mutant strain of Drosophila melanogaster were mapped to specific chromosomes. Drosophila melanogaster, commonly known as the fruit fly, was the appropriate choice for the organism to use in this specific experiment because of its relatively rapid life cycle of 10-14 days and because of the small amount of space and food neccessary for maintaining thousands of flies. The D. Melanogaster unknown strain specifically used in this experiment wa...

  7. Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

    Science.gov (United States)

    Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

    2014-04-01

    Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

  8. [Low-frequency pulsed electromagnetic fields promotes rat osteoblast differentiation in vitro through cAMP/PKA signal pathway].

    Science.gov (United States)

    Fang, Qing-Qing; Li, Zhi-Zhong; Zhou, Jian; Shi, Wen-Gui; Yan, Juan-Li; Xie, Yan-Fang; Chen, Ke-Ming

    2016-11-20

    To study whether low-frequency pulsed electromagnetic fields promotes the differentiation of cultured rat osteoblasts through the cAMP/PKA signal pathway. Rat calvarial osteoblasts isolated by enzyme digestion were exposed to 50 Hz 0.6 mT low-frequency pulsed electromagnetic field for varying lengths of time, and the concentration of cAMP and levels of phosphorylated PKA in the cells were assayed. In cells treated with DDA to inhibit the activity of adenylate cyclase, the changes of ALP activity and transcription of osteogenic gene were detected after exposure to low-frequency pulsed electromagnetic field. The changes of osteogenic gene transcription and protein expression were tested in the osteoblasts pretreated with KT5720 in response to low-frequency pulsed electromagnetic field exposure. The intracellular cAMP concentration in the cells increased significantly at 20 min during exposure to low-frequency pulsed electromagnetic field, began to decrease at 40 min during the exposure, and increased again after a 2-h exposure; the same pattern of variation was also observed in p-PKA level. Application of DDA and KT5720 pretreatment both suppressed the increase in ALP activity and osteogenic gene transcription induced by electromagnetic field exposure. Low- frequency pulsed electromagnetic field exposure improves the differentiation of cultured rat osteoblasts by activating cAMP/PKA signal pathway.

  9. Accurate pKa Calculation of the Conjugate Acids of Alkanolamines, Alkaloids and Nucleotide Bases by Quantum Chemical Methods

    NARCIS (Netherlands)

    Gangarapu, S.; Marcelis, A.T.M.; Zuilhof, H.

    2013-01-01

    The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum

  10. The oncogenic RAS2 val19 mutation locks respiration, independently of PKA, in a mode prone to generate ROS

    Czech Academy of Sciences Publication Activity Database

    Hlavatá, Lydie; Aguilaniu, H.; Pichová, Alena; Nystrom, T.

    2003-01-01

    Roč. 22, č. 13 (2003), s. 3337-3345 ISSN 0261-4189 R&D Projects: GA ČR GA301/03/0289 Institutional research plan: CEZ:AV0Z5020903 Keywords : pka/ras2/ * ucp1 * respiratory state Subject RIV: EE - Microbiology, Virology Impact factor: 10.456, year: 2003

  11. Osthole pretreatment alleviates TNBS-induced colitis in mice via both cAMP/PKA-dependent and independent pathways.

    Science.gov (United States)

    Sun, Wu; Cai, Yun; Zhang, Xin-Xin; Chen, Hao; Lin, Yan-Die; Li, Hao

    2017-08-01

    Osthole, a natural coumarin found in traditional Chinese medicinal plants, has shown multiple biological activities. In the present study, we investigated the preventive effects of osthole on inflammatory bowel disease (IBD). Colitis was induced in mice by infusing TNBS into the colonic lumen. Before TNBS treatment, the mice received osthole (100 mg·kg -1 ·d -1 , ip) for 3 d. Pretreatment with osthole significantly ameliorated the clinical scores, colon length shortening, colonic histopathological changes and the expression of inflammatory mediators in TNBS-induced colitis. Pretreatment with osthole elevated serum cAMP levels; but treatment with the PKA inhibitor H89 (10 mg·kg -1 ·d -1 , ip) did not abolish the beneficial effects of osthole on TNBS-induced colitis. In mouse peritoneal macrophages, pretreatment with osthole (50 μmol/L) significantly attenuated the LPS-induced elevation of cytokines at the mRNA level; inhibition of PKA completely reversed the inhibitory effects of osthole on IL-1β, IL-6, COX2, and MCP-1 but not on TNFα. In Raw264.7 cells, the p38 inhibitor SB203580 markedly suppressed LPS-induced upregulation of the cytokines, whereas the PKA inhibitors H89 or KT5720 did not abolish the inhibitory effects of SB203580. Moreover, in LPS-stimulated mouse peritoneal macrophages, SB203580 strongly inhibited the restored expression of IL-1β, IL-6, COX2, and MCP-1, which was achieved by abolishing the suppressive effects of osthole with the PKA inhibitors. Western blot analysis showed that osthole significantly suppressed the phosphorylation of p38, which was induced by TNBS in mice or by LPS in Raw264.7 cells. Inhibition of PKA partially reversed the suppressive effects of osthole on p38 phosphorylation in LPS-stimulated cells. Collectively, our results suggest that osthole is effective in the prevention of TNBS-induced colitis by reducing the expression of inflammatory mediators and attenuating p38 phosphorylation via both cAMP/PKA-dependent and

  12. Metabolome analysis of Drosophila melanogaster during embryogenesis.

    Science.gov (United States)

    An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

    2014-01-01

    The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos' metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.

  13. Characterization of Autophagic Responses in Drosophila melanogaster.

    Science.gov (United States)

    Xu, T; Kumar, S; Denton, D

    2017-01-01

    Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed. © 2017 Elsevier Inc. All rights reserved.

  14. PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Halayko Andrew J

    2009-09-01

    Full Text Available Abstract Background Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8. IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP effectors protein kinase A (PKA and exchange proteins directly activated by cAMP (Epac1 and Epac2 in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. Methods IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases, U0126 (extracellular signal-regulated kinases ERK1/2 and Rp-8-CPT-cAMPS (PKA. The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. Results The β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP

  15. Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii

    Science.gov (United States)

    In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially toxic to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly ...

  16. New record for the invasive Spotted Wing Drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) in Anillaco, Argentina

    Science.gov (United States)

    The invasive Spotted Wing Drosophila (SWD), Drosophila suzukii Matsumura, is reported for the first time in La Rioja, Argentina. This represents a major range expansion for this species. The natural enemies of SWD, Leptopilina clavipes and Ganaspis hookeri were also collected with the SWD at the s...

  17. PKA, PKC, and AKAP localization in and around the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Newton Alexandra

    2001-10-01

    Full Text Available Abstract Background One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA and protein kinase C (PKC. A-kinase anchoring proteins (AKAPs can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIα in addition to the RII subunits. Results Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIα at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIα and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIα and RIIβ are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCβ appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. Conclusions The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIα and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIα in these regions. Likewise, gravin may be an anchor of PKCβ at the NMJ.

  18. Melatonin regulates CRE-dependent gene transcription underlying osteoblast proliferation by activating Src and PKA in parallel.

    Science.gov (United States)

    Tao, Lin; Zhu, Yue

    2018-01-01

    Several studies have indicated a relationship between melatonin and idiopathic scoliosis, including our previous work which demonstrated that melatonin can inhibit osteoblast proliferation; however, the mechanism remains unclear. Here, we utilized a MTT assay to show that melatonin significantly reduces osteoblast proliferation in a concentration-and time-dependent manner. Through a combination of techniques, including real-time PCR, MTT assays, immunofluorescence, and luciferase assays, we confirmed that melatonin-induced changes in phosphorylated cAMP response element-binding protein (CREB) reduced transcriptional activity in a melatonin receptor-dependent manner. Surprisingly, treatment of osteoblasts with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 up-regulated other cascades upstream of CREB. We next treated cells with PKA and Src inhibitors and observed that melatonin can also activate the protein kinase A (PKA) and Src pathways. To examine whether Src is upstream from the cAMP-PKA pathway, we measured cAMP levels in response to melatonin with and without a Src inhibitor (PP2) and found that PP2 had no additional effect. Therefore, the transcription-dependent mechanisms involved in CREB phosphorylation, along with melatonin, activated Src via a parallel signaling pathway that was separate from that of PKA. Finally, we transfected osteoblasts with lentiviral CREB short hairpin (sh) RNAs and found a decrease in the expression of proliferating cell nuclear antigen (PCNA) and osteoblast proliferation. These results suggest that CREB and PCNA are downstream targets of melatonin signaling, and that the down-regulation of CREB, which is regulated via PKA and Src pathways, contributes to the melatonin-induced inhibition of osteoblast proliferation.

  19. Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

    Science.gov (United States)

    Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

    2009-01-01

    A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

  20. [The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

    Science.gov (United States)

    Toneatto, Judith; Charó, Nancy L; Susperreguy, Sebastián; Piwien-Pilipuk, Graciela

    2013-01-01

    Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

  1. Conditioning of spent fuel for interim and final storage in the pilot conditioning plant (PKA) at Gorleben

    International Nuclear Information System (INIS)

    Lahr, H.; Willax, H.O.; Spilker, H.

    1999-01-01

    In 1994, due to the change of the nuclear law in Germany, the concept of direct final disposal for spent fuel was developed as an equivalent alternative to the waste management with reprocessing. Since 1979, tests for the direct final disposal of spent fuel have been conducted in Germany. In 1985, the State and the utilities came to an agreement to develop this concept of waste management to technical maturity. Gesellschaft fuer Nuklear-Service (GNS) was commissioned by the utilities with the following tasks: to develop and test components with regard to conditioning technology, to construct and operate the pilot conditioning plant (PKA), and to develop casks suitable for final disposal. Since 1990, the construction of the PKA has taken place at the Brennelementlager Gorleben site. The PKA has been designed as a multipurpose facility and can thus fulfil various tasks within the framework of the conditioning and management of spent fuel assemblies and radioactive waste. The pilot character of the plant allows for development and testing in the field of spent fuel assembly conditioning. The objectives of the PKA may be summarized as follows: to condition spent fuel assemblies, to reload spent fuel assemblies and waste packages, to condition radioactive waste, and to do maintenance work on transport and storage casks as well as on waste packages. Currently, the buildings of the PKA are constructed and the technical facilities are installed. The plant will be ready for service in the middle of 1999. It is the first plant of its kind in the world. (author)

  2. Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

    DEFF Research Database (Denmark)

    Lenz, C; Williamson, M; Hansen, G N

    2001-01-01

    The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown...... to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr...... weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional...

  3. Effect of Hawthorn on Drosophila Melanogaster Antioxidant-Related ...

    African Journals Online (AJOL)

    Results: The results indicate that hawthorn extract prolonged the life span of Drosophila, with 50 % survival time of 0.8 ... Drosophila's aging gene is highly similar to humans [4,5]. ..... reduces lipid peroxidation in senescence-accelerated mice .

  4. Gustatory Processing in Drosophila melanogaster.

    Science.gov (United States)

    Scott, Kristin

    2018-01-07

    The ability to identify nutrient-rich food and avoid toxic substances is essential for an animal's survival. Although olfaction and vision contribute to food detection, the gustatory system acts as a final checkpoint control for food acceptance or rejection. The vinegar fly Drosophila melanogaster tastes many of the same stimuli as mammals and provides an excellent model system for comparative studies of taste detection. The relative simplicity of the fly brain and behaviors, along with the molecular genetic and functional approaches available in this system, allow the examination of gustatory neural circuits from sensory input to motor output. This review discusses the molecules and cells that detect taste compounds in the periphery and the circuits that process taste information in the brain. These studies are providing insight into how the detection of taste compounds regulates feeding decisions.

  5. Microtubules are organized independently of the centrosome in Drosophila neurons

    Directory of Open Access Journals (Sweden)

    Nguyen Michelle M

    2011-12-01

    Full Text Available Abstract Background The best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons. Results In developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment. Conclusion We conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells.

  6. [TRPM8 mediates PC-12 neuronal cell apoptosis induced by oxygen-glucose deprivation through cAMP-PKA/UCP4 signaling].

    Science.gov (United States)

    Li, Hong-Wei; Zhou, Bin; Zhang, Hai-Hong

    2016-08-20

    To explore the molecular mechanism responsible for apoptosis of PC-12 neuronal cells induced by oxygen-glucose deprivation (OGD). PC12 cells were exposed to OGD for 24 h to simulate ischemia-reperfusion injury. Flow cytometry was employed detect the cell apoptosis, and the expresions of TRPM8, UCP4, cAMP and PKA in the exposed cells were detected with RT-PCR and Western blotting. The changes in the expressions of Bax, Bcl-2, cAMP, PKA and UCP4 proteins were detected in the exposed cells in resposne to inhibition of TRPM8 and cAMP-PKA signal or over-expression of UCP4. OGD for 24 induced obvious apoptosis in PC-12 cells and caused TRPM8 over-expression and inhibition of UCP4 and cAMP-PKA signaling. Inhibiting TRPM8 expression reduced the cell apoptosis and up-regulated cAMP, p-PKA and UCP4 in the cells exposed to OGD. In cells exposed to OGD, inhibition of TRPM8 and cAMP-PKA signaling suppressed the expressio of UCP4 and increased the cell apoptosis. TRPM8 mediates OGD-induced PC12 cell apoptosis through cAMP-PKA/UCP4 signaling.

  7. Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

    Science.gov (United States)

    Nader, Nancy; Courjaret, Raphael; Dib, Maya; Kulkarni, Rashmi P; Machaca, Khaled

    2016-06-01

    Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway. © 2016. Published by The Company of Biologists Ltd.

  8. The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

    Science.gov (United States)

    Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

    2015-01-01

    The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

  9. Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: involvement of PKA pathway signaling.

    Science.gov (United States)

    França, F D; Ferreira, A F; Lara, R C; Rossoni, J V; Costa, D C; Moraes, K C M; Tagliati, C A; Chaves, M M

    2014-12-01

    Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B. Copyright © 2013 John Wiley & Sons, Ltd.

  10. Single Nucleotide Polymorphism Markers for Genetic Mapping in Drosophila melanogaster

    OpenAIRE

    Hoskins, Roger A.; Phan, Alexander C.; Naeemuddin, Mohammed; Mapa, Felipa A.; Ruddy, David A.; Ryan, Jessica J.; Young, Lynn M.; Wells, Trent; Kopczynski, Casey; Ellis, Michael C.

    2001-01-01

    For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that recently have revolutionized human, mouse, and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila by using a sequence tagged site-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that sp...

  11. The role of carcinine in signaling at the Drosophila photoreceptor synapse.

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    Brendan A Gavin

    2007-12-01

    Full Text Available The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H(3 receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H(3 receptor.

  12. The Role of Carcinine in Signaling at the Drosophila Photoreceptor Synapse

    Science.gov (United States)

    Gavin, Brendan A; Arruda, Susan E; Dolph, Patrick J

    2007-01-01

    The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H3 receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor. PMID:18069895

  13. Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning.

    Science.gov (United States)

    Yamaguchi, Takashi; Goto, Akihiro; Nakahara, Ichiro; Yawata, Satoshi; Hikida, Takatoshi; Matsuda, Michiyuki; Funabiki, Kazuo; Nakanishi, Shigetada

    2015-09-08

    The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Förster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention.

  14. Global identification of bursicon-regulated genes in Drosophila melanogaster

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    Beerntsen Brenda

    2008-09-01

    Full Text Available Abstract Background Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation. Results We used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster that received recombinant bursicon (r-bursicon. Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes. Analysis of these genes by inference from the fly database http://flybase.bio.indiana.edu revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data. Conclusion Using r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that

  15. Functions of the nonsense-mediated mRNA decay pathway in Drosophila development.

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    Mark M Metzstein

    2006-12-01

    Full Text Available Nonsense-mediated mRNA decay (NMD is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

  16. Drosophila growth cones: a genetically tractable platform for the analysis of axonal growth dynamics.

    Science.gov (United States)

    Sánchez-Soriano, Natalia; Gonçalves-Pimentel, Catarina; Beaven, Robin; Haessler, Ulrike; Ofner-Ziegenfuss, Lisa; Ballestrem, Christoph; Prokop, Andreas

    2010-01-01

    The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F-actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross-talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F-actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin-MT linking factor Short stop, thus identifying an essential molecular player in this context.

  17. Analysis of Thioester-Containing Proteins during the Innate Immune Response of Drosophila melanogaster

    Science.gov (United States)

    Bou Aoun, Richard; Hetru, Charles; Troxler, Laurent; Doucet, Daniel; Ferrandon, Dominique; Matt, Nicolas

    2010-01-01

    Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1–Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila. PMID:21063077

  18. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    Science.gov (United States)

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22.

    Science.gov (United States)

    Cheng, Fan; Ramos da Silva, Suzane; Huang, I-Chueh; Jung, Jae U; Gao, Shou-Jiang

    2018-02-15

    The recent outbreak of Zika virus (ZIKV), a reemerging flavivirus, and its associated neurological disorders, such as Guillain-Barré (GB) syndrome and microcephaly, have generated an urgent need to develop effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors of ZIKV replication. Because several pathways, including the AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors and agonists of these pathways for their effects on ZIKV replication. We identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both the African and Asian/American lineages with a high efficiency and minimal cytotoxicity. While ZIKV infection does not induce PKA activation, endogenous PKA activity is essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited another unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the postentry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication which could be further explored for future therapeutic application. IMPORTANCE There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a reemerging flavivirus associated with neurological disorders, including Guillain-Barré (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. We show that PKI effectively suppresses the replication of all ZIKV

  20. Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel.

    Science.gov (United States)

    Sherry, A M; Stroffekova, K; Knapp, L M; Kupert, E Y; Cuppoletti, J; Malinowska, D H

    1997-08-01

    A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

  1. A novel assay reveals hygrotactic behavior in Drosophila.

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    Feiteng Ji

    Full Text Available Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. Here, we established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, we found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while arista was required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in arista which is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, we demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.

  2. Mutational Analysis of Drosophila Basigin Function in the Visual System

    Science.gov (United States)

    Munro, Michelle; Akkam, Yazan; Curtin, Kathryn D.

    2009-01-01

    Drosophila basigin is a cell-surface glycoprotein of the Ig superfamily and a member of a protein family that includes mammalian EMMPRIN/CD147/basigin, neuroplastin, and embigin. Our previous work on Drosophila basigin has shown that it is required for normal photoreceptor cell structure and normal neuron-glia interaction in the fly visual system. Specifically, the photoreceptor neurons of mosaic animals that are mutant in the eye for basigin show altered cell structure with nuclei, mitochondria and rER misplaced and variable axon diameter compared to wild-type. In addition, glia cells in the optic lamina that contact photoreceptor axons are misplaced and show altered structure. All these defects are rescued by expression of either transgenic fly basigin or transgenic mouse basigin in the photoreceptors demonstrating that mouse basigin can functionally replace fly basigin. To determine what regions of the basigin protein are required for each of these functions, we have created mutant basigin transgenes coding for proteins that are altered in conserved residues, introduced these into the fly genome, and tested them for their ability to rescue both photoreceptor cell structure defects and neuron-glia interaction defects of basigin. The results suggest that the highly conserved transmembrane domain and the extracellular domains are crucial for basigin function in the visual system while the short intracellular tail may not play a role in these functions. PMID:19782733

  3. Gut-associated microbes of Drosophila melanogaster

    Science.gov (United States)

    Broderick, Nichole; Lemaitre, Bruno

    2012-01-01

    There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1 PMID:22572876

  4. Drosophila Courtship Conditioning As a Measure of Learning and Memory.

    Science.gov (United States)

    Koemans, Tom S; Oppitz, Cornelia; Donders, Rogier A T; van Bokhoven, Hans; Schenck, Annette; Keleman, Krystyna; Kramer, Jamie M

    2017-06-05

    Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular

  5. The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

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    Miguel J Xavier

    Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

  6. The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis

    OpenAIRE

    Horner, Michael A.; Pardee, Keith; Liu, Suya; King-Jones, Kirst; Lajoie, Gilles; Edwards, Aled; Krause, Henry M.; Thummel, Carl S.

    2009-01-01

    Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-choleste...

  7. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica

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    Andrew T Kempsell

    2015-09-01

    Full Text Available Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature versus advanced age Aplysia. Glutamate- (L-Glu- evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT in sensory neurons (SN isolated from mature but not aged animals. Activation of PKA and PKC signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems

  8. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica.

    Science.gov (United States)

    Kempsell, Andrew T; Fieber, Lynne A

    2015-01-01

    Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature vs. advanced age Aplysia californica (Aplysia). L-Glutamate- (L-Glu-) evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT) in sensory neurons (SN) isolated from mature but not aged animals. Activation of protein kinase A (PKA) and protein kinase C (PKC) signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems.

  9. Viruses and Antiviral Immunity in Drosophila

    Science.gov (United States)

    Xu, Jie; Cherry, Sara

    2013-01-01

    Viral pathogens present many challenges to organisms, driving the evolution of a myriad of antiviral strategies to combat infections. A wide variety of viruses infect invertebrates, including both natural pathogens that are insect-restricted, and viruses that are transmitted to vertebrates. Studies using the powerful tools available in the model organism Drosophila have expanded our understanding of antiviral defenses against diverse viruses. In this review, we will cover three major areas. First, we will describe the tools used to study viruses in Drosophila. Second, we will survey the major viruses that have been studied in Drosophila. And lastly, we will discuss the well-characterized mechanisms that are active against these diverse pathogens, focusing on non-RNAi mediated antiviral mechanisms. Antiviral RNAi is discussed in another paper in this issue. PMID:23680639

  10. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  11. Apoptosis in Drosophila: which role for mitochondria?

    Science.gov (United States)

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  12. Nutrient-Dependent Impact of Microbes on Drosophila suzukii Development.

    Science.gov (United States)

    Bing, XiaoLi; Gerlach, Joseph; Loeb, Gregory; Buchon, Nicolas

    2018-03-20

    Drosophila suzukii Matsumura is an invasive species of vinegar fly that has become a prominent pest of berries and other soft-skinned fruits. Unlike most other Drosophila species, female D. suzukii flies lay their eggs in ripening and ripe fruits and larvae develop within the fruit. To understand how D. suzukii larvae utilize ripe and ripening fruits, which usually have low levels of protein, we investigated the microbiota of field-captured and laboratory-reared D. suzukii flies and further examined the combined influence of diet and microbes on host fitness. Field-captured flies were associated with diverse microbiota, which varied significantly with sampling location and season. In contrast, laboratory-reared flies possessed strikingly lower bacterial abundance and diversity. A comparison of conventionally reared (CR) and germ-free (GF) flies revealed that the microbiota of D. suzukii does not alter its development significantly but decreases its life span under conditions of a nutrient-sufficient diet. However, the microbiota is essential for D. suzukii development on strawberry-based or blueberry-based fruit diets. This developmental failure could be rescued by reassociation with single bacterial or fungal species or by the addition of a high quantity of heat-killed microbes. In addition, we found that proteins are limiting with respect to fly development on fruit-based diets and that GF flies show signs of protein starvation. Taken together, our study results demonstrate that the microbiota provides key proteins required for the development of D. suzukii reared on fresh fruit. Our work shows that the impact of microbes on fly fitness depends strongly on nutritional conditions. IMPORTANCE Animals are commonly associated with specific microbes, which play important roles in host development and fitness. However, little information about the function of microbes has been available for the important invasive pest Drosophila suzukii , also known as Spotted

  13. Identification of a Novel TGFβ/PKA Signaling Transduceome in Mediating Control of Cell Survival and Metastasis in Colon Cancer

    Science.gov (United States)

    Rajput, Ashwani; Teggart, Carol A.; Brattain, Lisa E.; Weber, Hannah R.; Chowdhury, Aparajita; Brattain, Michael G.

    2011-01-01

    Background Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown. Methodology/Principal Findings Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival. Conclusion/Significance This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors. PMID:21559296

  14. Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA.

    Science.gov (United States)

    Deckelbaum, Ron A; Chan, George; Miao, Dengshun; Goltzman, David; Karaplis, Andrew C

    2002-07-15

    Indian Hedgehog (Ihh), a member of the hedgehog (HH) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) are key regulators of cartilage cell (chondrocyte) differentiation. We have investigated, in vitro, the actions of HH signalling and its possible interplay with PTHrP using rat CFK-2 chondrocytic cells. Markers of chondrocyte differentiation [alkaline phosphatase (ALP) activity, and type II (Col2a1) and type X collagen (Col10a1) expression] were enhanced by overexpression of Ihh or its N-terminal domain (N-Ihh), effects mimicked by exogenous administration of recombinant N-terminal HH peptide. Moreover, a missense mutation mapping to the N-terminal domain of Ihh (W160G) reduces the capacity of N-Ihh to induce differentiation. Prolonged exposure of CFK-2 cells to exogenous N-Shh (5x10(-9) M) in the presence of PTHrP (10(-8) M) or forskolin (10(-7) M) resulted in perturbation of HH-mediated differentiation. In addition, overexpression of a constitutively active form of the PTHrP receptor (PTHR1 H223R) inhibited Ihh-mediated differentiation, implicating activation of protein kinase A (PKA) by PTHR1 as a probable mediator of the antagonistic effects of PTHrP. Conversely, overexpression of Ihh/N-Ihh or exogenous treatment with N-Shh led to dampening of PTHrP-mediated activation of PKA. Taken together, our data suggest that Ihh harbors the capacity to induce rather than inhibit chondrogenic differentiation, that PTHrP antagonizes HH-mediated differentiation through a PKA-dependent mechanism and that HH signalling, in turn, modulates PTHrP action through functional inhibition of signalling by PTHR1 to PKA.

  15. Identification of a novel TGFβ/PKA signaling transduceome in mediating control of cell survival and metastasis in colon cancer.

    Directory of Open Access Journals (Sweden)

    Sanjib Chowdhury

    2011-05-01

    Full Text Available Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.

  16. Microwave effects in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Dardalhon, M.; Averbeck, D.; Berteaud, A.J.

    1979-01-01

    Experiments were set up to investigate the effects of open space microwave irradiation of the millimeter (73 GHz) and the centimeter (17 GHz) range in Drosophila melanogaster. We used the wild type strain Paris and the strain delta carrying melanitic tumors in the 3rd larval stage, in the pupae and the adults. The power densities were up to 100mW.cm -2 for 73 GHz and about 60 mW.cm -2 for microwaves at 17 GHz. After 2h exposure to microwaves of 17 GHz or 73 GHz the hatching of the irradiated eggs and their development were normal. In a few cases there was a tendency towards a diminution of the survival of eggs treated at different stages, of larvae treated in the stages 1, 2 and 3 and of treated pupae. However, this was not always statistically significant. The microwave treatment did not induce teratological changes in the adults. A statistical analysis brought about slight diminutions in the incidence and multiplicity of tumors in adult flies. When wild type females were exposed to microwaves of 17 GHz for 16 or 21 h and crossed with untreated males we observed a marked increase in fertility as compared to untreated samples. The viability and tumor incidence in the offspring was not affected. Similar results were obtained when microwaves treated males were crossed with untreated females

  17. Olfactory memory traces in Drosophila.

    Science.gov (United States)

    Berry, Jacob; Krause, William C; Davis, Ronald L

    2008-01-01

    In Drosophila, the fruit fly, coincident exposure to an odor and an aversive electric shock can produce robust behavioral memory. This behavioral memory is thought to be regulated by cellular memory traces within the central nervous system of the fly. These molecular, physiological, or structural changes in neurons, induced by pairing odor and shock, regulate behavior by altering the neurons' response to the learned environment. Recently, novel in vivo functional imaging techniques have allowed researchers to observe cellular memory traces in intact animals. These investigations have revealed interesting temporal and spatial dynamics of cellular memory traces. First, a short-term cellular memory trace was discovered that exists in the antennal lobe, an early site of olfactory processing. This trace represents the recruitment of new synaptic activity into the odor representation and forms for only a short period of time just after training. Second, an intermediate-term cellular memory trace was found in the dorsal paired medial neuron, a neuron thought to play a role in stabilizing olfactory memories. Finally, a long-term protein synthesis-dependent cellular memory trace was discovered in the mushroom bodies, a structure long implicated in olfactory learning and memory. Therefore, it appears that aversive olfactory associations are encoded by multiple cellular memory traces that occur in different regions of the brain with different temporal domains.

  18. Participation of Antidiuretic Hormone (ADH) in Asthma Exacerbations Induced by Psychological Stress via PKA/PKC Signal Pathway in Airway-Related Vagal Preganglionic Neurons (AVPNs).

    Science.gov (United States)

    Hou, Lili; Zhu, Lei; Zhang, Min; Zhang, Xingyi; Zhang, Guoqing; Liu, Zhenwei; Li, Qiang; Zhou, Xin

    2017-01-01

    Present study was performed to examine whether ADH was implicated in psychological stress asthma and to explore the underlying molecular mechanism. We not only examined ADH levels in the cerebrospinal fluid (CSF) via radioimmunoassay, but also measured ADH receptor (ADHR) expression in airway-related vagal preganglionic neurons (AVPNs) through real-time PCR in all experimental mice. Western blotting was performed to evaluate the relationship between ADH and PKA/PKC in psychological stress asthma. Finally, the role of PKA/PKC in psychological stress asthma was analyzed. Marked asthma exacerbations were noted owing to significantly elevated levels of ADH and ADHR after psychological stress induction as compared to OVA alone (asthma group). ADHR antagonists (SR-49095 or SR-121463A) dramatically lowered higher protein levels of PKAα and PKCα induced by psychological stress as compared to OVA alone, suggesting the correlation between ADH and PKA/PKC in psychological stress asthma. KT-5720 (PKA inhibitor) and Go-7874 (PKC inhibitor) further directly revealed the involvement of PKA/PKC in psychological stress asthma. Some notable changes were also noted after employing PKA and PKC inhibitors in psychological stress asthma, including reduced asthmatic inflammation (lower eosinophil peroxidase (EPO) activity, myeloperoxidase (MPO) activity, immunoglobulin E (IgE) level, and histamine release), substantial decrements in inflammatory cell counts (eosinophils and lymphocytes), and decreased cytokine secretion (IL-6, IL-10, and IFN-γ), indicating the involvement of PKA/PKC in asthma exacerbations induced by psychological stress. Our results strongly suggested that ADH participated in psychological stress-induced asthma exacerbations via PKA/PKC signal pathway in AVPNs. © 2017 The Author(s)Published by S. Karger AG, Basel.

  19. The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae.

    Science.gov (United States)

    Amigoni, Loredana; Colombo, Sonia; Belotti, Fiorella; Alberghina, Lilia; Martegani, Enzo

    2015-08-03

    To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

  20. Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01. The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81 with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.

  1. Effect of sevoflurane on the ATPase activity of hippocampal neurons in a rat model of cerebral ischemia-reperfusion injury via the cAMP-PKA signaling pathway.

    Science.gov (United States)

    Liu, Tie-Jun; Zhang, Jin-Cun; Gao, Xiao-Zeng; Tan, Zhi-Bin; Wang, Jian-Jun; Zhang, Pan-Pan; Cheng, Ai-Bin; Zhang, Shu-Bo

    2018-01-01

    We aim to investigate the effects of sevoflurane on the ATPase activity of the hippocampal neurons in rats with cerebral ischemia-reperfusion injury (IRI) via the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signaling pathway. Sixty rats were assigned into the normal, model and sevoflurane groups (n = 20, the latter two groups were established as focal cerebral IRI models). The ATPase activity was detected using an ultramicro Na (+)-K (+)-ATP enzyme kit. Immunohistochemical staining was used to detect the positive protein expression of cAMP and PKA. The hippocampal neurons were assigned to the normal, IRI, IRI + sevoflurane, IRI + forskolin, IRI + H89 and IRI + sevoflurane + H89 groups. qRT-PCR and Western blotting were performed for the expressions of cAMP, PKA, cAMP-responsive element-binding protein (CREB) and brain derived neurotrophic factor (BDNF). The normal and sevoflurane groups exhibited a greater positive protein expression of cAMP and PKA than the model group. Compared with the normal group, the expressions of cAMP, PKA, CREB and BDNF all reduced in the IRI, model and IRI + H89 groups. The sevoflurane group showed higher cAMP, PKA, CREB and BDNF expressions than the model group. Compared with the IRI group, ATPase activity and expressions of cAMP, PKA, CREB and BDNF all increased in the normal, IRI + sevoflurane and IRI + forskolin groups but decreased in the IRI + H89 group. It suggests that sevoflurane could enhance ATPase activity in hippocampal neurons of cerebral IRI rats through activating cAMP-PKA signaling pathway. Copyright © 2017. Published by Elsevier Taiwan.

  2. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

    Science.gov (United States)

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

  3. Drosophila Studies on Autism Spectrum Disorders

    Institute of Scientific and Technical Information of China (English)

    Yao Tian; Zi Chao Zhang; Junhai Han

    2017-01-01

    In the past decade,numerous genes associated with autism spectrum disorders (ASDs) have been identified.These genes encode key regulators of synaptogenesis,synaptic function,and synaptic plasticity.Drosophila is a prominent model system for ASD studies to define novel genes linked to ASDs and decipher their molecular roles in synaptogenesis,synaptic function,synaptic plasticity,and neural circuit assembly and consolidation.Here,we review Drosophila studies on ASD genes that regulate synaptogenesis,synaptic function,and synaptic plasticity through modulating chromatin remodeling,transcription,protein synthesis and degradation,cytoskeleton dynamics,and synaptic scaffolding.

  4. REDfly: a Regulatory Element Database for Drosophila.

    Science.gov (United States)

    Gallo, Steven M; Li, Long; Hu, Zihua; Halfon, Marc S

    2006-02-01

    Bioinformatics studies of transcriptional regulation in the metazoa are significantly hindered by the absence of readily available data on large numbers of transcriptional cis-regulatory modules (CRMs). Even the richly annotated Drosophila melanogaster genome lacks extensive CRM information. We therefore present here a database of Drosophila CRMs curated from the literature complete with both DNA sequence and a searchable description of the gene expression pattern regulated by each CRM. This resource should greatly facilitate the development of computational approaches to CRM discovery as well as bioinformatics analyses of regulatory sequence properties and evolution.

  5. Longevity and the stress response in Drosophila

    DEFF Research Database (Denmark)

    Vermeulen, Corneel J.; Loeschcke, Volker

    2007-01-01

    briefly review the state of the art of research on ageing and longevity in the model organism Drosophila, with focus on the role of the general stress response. We will conclude by contemplating some of the implications of the findings in this research and will suggest several directions for future...... research. Keywords: Ageing; Stress response; Hsp; Drosophila; Stress......The concept that lifespan is a function of the capacity to withstand extrinsic stress is very old. In concordance with this, long-lived individuals often have increased resistance against a variety of stresses throughout life. Genes underlying the stress response may therefore have the ability...

  6. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    Directory of Open Access Journals (Sweden)

    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

  7. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway.

    Science.gov (United States)

    Zeng, Qi-bing; Xu, Yu-yan; Yu, Xian; Yang, Jun; Hong, Feng; Zhang, Ai-hua

    2014-01-01

    Chronic exposure to combined fluoride and arsenic continues to be a major public health problem worldwide, affecting thousands of people. In recent years, more and more researchers began to focus on the interaction between the fluorine and the arsenic. In this study, the selected investigation site was located in China. The study group was selected from people living in fluoride-arsenic polluted areas due to burning coal. The total number of participants was 196; including the fluoride-arsenic anomaly group (130) and the fluoride-arsenic normal group (63). By observing the changes in gene and protein expression of PTH/PKA/AP1 signaling pathway, the results show that fluoride can increase the expression levels of PTH, PKA, and AP1, but arsenic can only affect the expression of AP1; fluoride and arsenic have an interaction on the expression of AP1. Further study found that fluoride and arsenic can affect the mRNA expression level of c-fos gene (AP1 family members), and have an interaction on the expression of c-fos, but not c-jun. The results indicate that PTH/PKA/AP1 signaling pathway may play an important role in bone toxicity of fluoride. Arsenic can affect the expression of c-fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity. Copyright © 2013. Published by Elsevier B.V.

  8. PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

    Science.gov (United States)

    Witt, P S; Pietrowski, D; Keck, C

    2004-02-01

    New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

  9. Theoretical prediction of pKa in methanol: testing SM8 and SMD models for carboxylic acids, phenols, and amines.

    Science.gov (United States)

    Miguel, Elizabeth L M; Silva, Poliana L; Pliego, Josefredo R

    2014-05-29

    Methanol is a widely used solvent for chemical reactions and has solvation properties similar to those of water. However, the performance of continuum solvation models in this solvent has not been tested yet. In this report, we have investigated the performance of the SM8 and SMD models for pKa prediction of 26 carboxylic acids, 24 phenols, and 23 amines in methanol. The gas phase contribution was included at the X3LYP/TZVPP+diff//X3LYP/DZV+P(d) level. Using the proton exchange reaction with acetic acid, phenol, and ammonia as reference species leads to RMS error in the range of 1.4 to 3.6 pKa units. This finding suggests that the performance of the continuum models for methanol is similar to that found for aqueous solvent. Application of simple empirical correction through a linear equation leads to accurate pKa prediction, with uncertainty less than 0.8 units with the SM8 method. Testing with the less expensive PBE1PBE/6-311+G** method results in a slight improvement in the results.

  10. Determination of pKa and the corresponding structures of quinclorac using combined experimental and theoretical approaches

    Science.gov (United States)

    Song, Dean; Sun, Huiqing; Jiang, Xiaohua; Kong, Fanyu; Qiang, Zhimin; Zhang, Aiqian; Liu, Huijuan; Qu, Jiuhui

    2018-01-01

    As an emerging environmental contaminant, the herbicide quinclorac has attracted much attention in recent years. However, a very fundamental issue, the acid dissociation of quinclorac has not yet to be studied in detail. Herein, the pKa value and the corresponding structures of quinclorac were systematically investigated using combined experimental and theoretical approaches. The experimental pKa of quinclorac was determined by the spectrophotometric method to be 2.65 at 25 °C with ionic strength of 0.05 M, and was corrected to be 2.56 at ionic strength of zero. The molecular structures of quinclorac were then located by employing the DFT calculation. The anionic quinclorac was directly located with the carboxylic group perpendicular to the aromatic ring, while neutral quinclorac was found to be the equivalent twin structures. The result was further confirmed by analyzing the UV/Vis and MS-MS2 spectra from both experimental and theoretical viewpoints. By employing the QSPR approach, the theoretical pKa of QCR was determined to be 2.50, which is excellent agreement with the experimental result obtained herein. The protonation of QCR at the carboxylic group instead of the quinoline structure was attributed to the weak electronegative property of nitrogen atom induced by the electron-withdrawing groups. It is anticipated that this work could not only help in gaining a deep insight into the acid dissociation of quinclorac but also offering the key information on its reaction and interaction with others.

  11. Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways.

    Science.gov (United States)

    Chu, Wen-Ting; Chu, Xiakun; Wang, Jin

    2017-09-19

    The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding.

  12. The role of PKA in the translational response to heat stress in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Carla E Barraza

    Full Text Available Cellular responses to stress stem from a variety of different mechanisms, including translation arrest and relocation of the translationally repressed mRNAs to ribonucleoprotein particles like stress granules (SGs and processing bodies (PBs. Here, we examine the role of PKA in the S. cerevisiae heat shock response. Under mild heat stress Tpk3 aggregates and promotes aggregation of eIF4G, Pab1 and eIF4E, whereas severe heat stress leads to the formation of PBs and SGs that contain both Tpk2 and Tpk3 and a larger 48S translation initiation complex. Deletion of TPK2 or TPK3 impacts upon the translational response to heat stress of several mRNAs including CYC1, HSP42, HSP30 and ENO2. TPK2 deletion leads to a robust translational arrest, an increase in SGs/PBs aggregation and translational hypersensitivity to heat stress, whereas TPK3 deletion represses SGs/PBs formation, translational arrest and response for the analyzed mRNAs. Therefore, this work provides evidence indicating that Tpk2 and Tpk3 have opposing roles in translational adaptation during heat stress, and highlight how the same signaling pathway can be regulated to generate strikingly distinct physiological outputs.

  13. Secretin Modulates the Postnatal Development of Mouse Cerebellar Cortex Via PKA- and ERK-dependent Pathways

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2017-11-01

    Full Text Available Postnatal development of the cerebellum is critical for its intact function such as motor coordination and has been implicated in the pathogenesis of psychiatric disorders. We previously reported that deprivation of secretin (SCT from cerebellar Purkinje neurons impaired motor coordination and motor learning function, while leaving the potential role of SCT in cerebellar development to be determined. SCT and its receptor (SCTR were constitutively expressed in the postnatal cerebellum in a temporal and cell-specific manner. Using a SCT knockout mouse model, we provided direct evidence showing altered developmental patterns of Purkinje cells (PCs and granular cells (GCs. SCT deprivation reduced the PC density, impaired the PC dendritic formation, induced accelerated GC migration and potentiated cerebellar apoptosis. Furthermore, our results indicated the involvement of protein kinase A (PKA and extracellular signal regulated kinase (ERK signaling pathways in SCT-mediated protective effects against neuronal apoptosis. Results of this study illustrated a novel function of SCT in the postnatal development of cerebellum, emphasizing the necessary role of SCT in cerebellar-related functions.

  14. A compartmental model of the cAMP/PKA/MAPK pathway in Bio-PEPA

    Directory of Open Access Journals (Sweden)

    Federica Ciocchetta

    2009-11-01

    Full Text Available The vast majority of biochemical systems involve the exchange of information between different compartments, either in the form of transportation or via the intervention of membrane proteins which are able to transmit stimuli between bordering compartments. The correct quantitative handling of compartments is, therefore, extremely important when modelling real biochemical systems. The Bio-PEPA process algebra is equipped with the capability of explicitly defining quantitative information such as compartment volumes and membrane surface areas. Furthermore, the recent development of the Bio-PEPA Eclipse Plug-in allows us to perform a correct stochastic simulation of multi-compartmental models. Here we present a Bio-PEPA compartmental model of the cAMP/PKA/MAPK pathway. We analyse the system using the Bio-PEPA Eclipse Plug-in and we show the correctness of our model by comparison with an existing ODE model. Furthermore, we perform computational experiments in order to investigate certain properties of the pathway. Specifically, we focus on the system response to the inhibition and strengthening of feedback loops and to the variation in the activity of key pathway reactions and we observe how these modifications affect the behaviour of the pathway. These experiments are useful to understand the control and regulatory mechanisms of the system.

  15. Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA pathway.

    Science.gov (United States)

    Wang, M-D; Huang, Y; Zhang, G-P; Mao, L; Xia, Y-P; Mei, Y-W; Hu, B

    2012-12-13

    Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8 μg/ml (Pglucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (Pneurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3h after ODG (Pneurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and may serve as a novel therapeutic agent for stroke. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Loss of Centrobin Enables Daughter Centrioles to Form Sensory Cilia in Drosophila.

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    Gottardo, Marco; Pollarolo, Giulia; Llamazares, Salud; Reina, Jose; Riparbelli, Maria G; Callaini, Giuliano; Gonzalez, Cayetano

    2015-08-31

    Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    Directory of Open Access Journals (Sweden)

    Shilpi Verghese

    2016-09-01

    Full Text Available Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1 and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.

  18. Drosophila melanogaster Models of Metal-Related Human Diseases and Metal Toxicity.

    Science.gov (United States)

    Calap-Quintana, Pablo; González-Fernández, Javier; Sebastiá-Ortega, Noelia; Llorens, José Vicente; Moltó, María Dolores

    2017-07-06

    Iron, copper and zinc are transition metals essential for life because they are required in a multitude of biological processes. Organisms have evolved to acquire metals from nutrition and to maintain adequate levels of each metal to avoid damaging effects associated with its deficiency, excess or misplacement. Interestingly, the main components of metal homeostatic pathways are conserved, with many orthologues of the human metal-related genes having been identified and characterized in Drosophila melanogaster . Drosophila has gained appreciation as a useful model for studying human diseases, including those caused by mutations in pathways controlling cellular metal homeostasis. Flies have many advantages in the laboratory, such as a short life cycle, easy handling and inexpensive maintenance. Furthermore, they can be raised in a large number. In addition, flies are greatly appreciated because they offer a considerable number of genetic tools to address some of the unresolved questions concerning disease pathology, which in turn could contribute to our understanding of the metal metabolism and homeostasis. This review recapitulates the metabolism of the principal transition metals, namely iron, zinc and copper, in Drosophila and the utility of this organism as an experimental model to explore the role of metal dyshomeostasis in different human diseases. F